The C. elegans class A synthetic multivulva genes inhibit ectopic Ras-mediated
vulval development by tightly restricting expression of lin-3 EGF
by
Adam M. Saffer
B.S., Biology and Biochemistry
Brandeis University, 2002
SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
AT THE
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
FEBRUARY 2011
© 2011 Adam M. Saffer. All rights reserved.
The author hereby grants to MIT permission to reproduce and to distribute publicly
paper and electronic copies of this thesis document in whole or in part in any medium
now known or hereafter created.
Signature of Author:______________________________________________________
Department of Biology
Certified by_____________________________________________________________
H. Robert Horvitz
Professor of Biology
Thesis Supervisor
Accepted by:___________________________________________________________
Stephen P. Bell
Professor of Biology
Chairman of the Graduate Committee
1
The C. elegans class A synthetic multivulva genes inhibit ectopic Ras-mediated
vulval development by tightly restricting expression of lin-3 EGF
by
Adam M. Saffer
Submitted to the Department of Biology in Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
Abstract
The class A and B synthetic multivulva (synMuv) genes of C. elegans
redundantly antagonize an EGF/Ras pathway to prevent ectopic vulval induction. The
class B synMuv genes encode many proteins known to remodel chromatin and repress
transcription. The class A synMuv genes likely also function in transcription, although
their specific molecular functions are unknown.
We have identified a class A synMuv mutation in the promoter of lin-3 EGF,
revealing that lin-3 is the key biological target of the class A synMuv genes in vulval
development. Using FISH with single mRNA molecule resolution, we found that class
AB synMuv double mutants exhibit widespread ectopic lin-3 expression. Our results
show that lin-3 EGF is normally expressed in the germline, and many class B synMuv
genes have previously been implicated in inhibiting germline fates in somatic cells. We
propose that the class A synMuv genes specifically repress ectopic lin-3 EGF
expression through a site in the lin-3 promoter and the class B synMuv genes either
directly or indirectly repress lin-3 as a consequence of their role in regulating the
germline/soma distinction.
The class A and B synMuv genes had previously been thought of as two parallel
pathways, but we have found that each of those pathways is actually composed of
multiple parallel pathways. While class AB synMuv double mutants have a strong Muv
phenotype, most class AA synMuv double mutants exhibit a weak Muv phenotype, and
most pairs of class B synMuv mutants can enhance each other in sensitized
backgrounds, indicating that most genes within a class can function in parallel. We also
found that some pairs of synMuv genes cannot act in parallel, indicating that they
function together to repress ectopic lin-3 expression.
We also report the molecular characterization of the class A synMuv gene lin-38
and the identification of mcd-1 as a class A synMuv gene. lin-38 and mcd-1 encode
paralogous zinc-finger proteins. Unlike previously studied class A synMuv genes that
function specifically in vulval development by repressing lin-3, both lin-38 and mcd-1
control multiple aspects of development by regulating target genes other than lin-3.
Thesis Supervisor: H. Robert Horvitz
Title: Professor of Biology
2
Acknowledgments
First, I would like to thank my advisor, Bob Horvitz, for giving me the independence to
pursue my interests, while also providing advice and encouragement when needed. I
would also like to thank my committee members, Chris Kaiser, Richard Hynes, Dennis
Kim, Peter Reddien, and Susan Mango.
Iʼd like to thank everyone I have overlapped with in the Horvitz lab (I will not name them
individually for fear of accidentally omitting someone). Their advice and assistance over
the years has been invaluable. I had the pleasure of working with Erik Andersen and
Melissa Harrison in our quest to discover everything there is to know about the C.
elegans vulva. My experience in graduate school would not have been the same
without my baymate Niels Ringstad. His helpful advice and enthusiastic encouragement
easily outweighed the inconvenience of losing a few pens a week. I was especially
fortunate to have Dave Harris and Erik Andersen as friends and colleagues.
All of the people who accompanied me on the daily trips to Annaʼs, including Melissa
Harrison, Johanna Varner, Dan Omura, Dan Denning, Nick Paquin, Mike Hurwitz, and of
course Dave Harris, made lunchtimes very enjoyable. I also thank Dan Omura and
everyone else I played squash with.
I am grateful for the friendship of many members of Biograd2002, and especially my
longtime roommates Rami Rahal, Mauro Calabrese, Lucas Dennis, and Mark Gill. Their
friendship made graduate school much more enjoyable.
Finally, I want to thank my parents, Jeff and Paula, and my sister Amy, for their support
and all that they have taught me.
3
Table of Contents
Abstract ............................................................................................................................2
Acknowledgments ............................................................................................................3
Table of Contents .............................................................................................................4
Chapter One: Introduction.............................................................................................9
The mammalian EGF signaling pathway........................................................................10
EGF signaling in mammalian development ....................................................................12
EGF signaling in cancer .................................................................................................15
C. elegans vulval development.......................................................................................16
EGF/Ras signaling induces vulval fates .........................................................................17
Wnt and Notch signaling are also required for normal vulval development....................20
Additional roles of EGF signaling in C. elegans .............................................................21
The synMuv genes antagonize EGF signaling ...............................................................24
The class A synMuv genes encode putative transcription factors..................................25
The class B synMuv genes encode transcriptional repressors ......................................25
synMuv protein complexes .............................................................................................27
The site of action of the synMuv genes ..........................................................................28
Relationship between EGF signaling and synMuv genes ..............................................29
The synMuv genes have numerous functions ................................................................29
Conclusion......................................................................................................................32
Acknowledgments ..........................................................................................................32
Chapter Two: The C. elegans synthetic multivulva genes prevent Ras
pathway activation by tightly repressing ectopic expression of lin-3 EGF..........33
Summary ........................................................................................................................34
Introduction.....................................................................................................................35
Materials and Methods ...................................................................................................38
Results............................................................................................................................40
n4441 causes a dominant class A synMuv phenotype...............................................40
n4441 is an allele of lin-3 ............................................................................................41
lin-3(n4441) specifically prevents repression of lin-3 ..................................................43
Expression pattern of lin-3 in wild-type animals and synMuv mutants .......................44
Discussion ......................................................................................................................47
lin-3 is the major target of the class A synMuv genes in vulval development .............47
The class A and B synMuv genes repress lin-3 by two distinct mechanisms.............48
The synMuv genes repress lin-3 throughout the animal .............................................49
Conclusion ..................................................................................................................50
Acknowledgments ..........................................................................................................51
Table 1: lin-3(n4441) causes a dominant class A synMuv phenotype ...........................52
Table 2: lin-3 overexpression is enhanced more strongly by a class A synMuv
mutation than by a class B synMuv mutation ...............................................................53
Figure 1: n4441 is an allele of lin-3.................................................................................54
Figure 2: The lin-3(n4441) mutation specifically prevents repression of lin-3 ...............56
Figure 3: The synMuv genes prevent widespread ectopic expression of lin-3 mRNA. .58
4
Figure 4: The lin-3(n4441) mutation causes widespread ectopic expression of
lin-3 mRNA. ..................................................................................................................60
Supplemental Table 1: Oligonucleotides in the lin-3 in situ hybridization probe.............62
Chapter Three: Multiple levels of redundant processes inhibit C. elegans
vulval cell fates...........................................................................................................63
Summary ........................................................................................................................64
Introduction.....................................................................................................................65
Materials and Methods ...................................................................................................68
Results............................................................................................................................70
Temperature sensitizes the vulval phenotype.............................................................70
Partial loss-of-function mutations in class A or class B synMuv genes can
sensitize the vulval phenotype ..................................................................................71
Most class A-A double mutants have Muv phenotypes ..............................................71
lin-15A and lin-56 act in the same process to inhibit the specification of vulval
cell fates....................................................................................................................72
Most class B-B double mutants do not have Muv phenotypes ...................................73
Most class B synMuv genes act redundantly with genes of the same class
to inhibit vulval cell fates ...........................................................................................74
Some synMuv genes within the same class function non-redundantly to inhibit
vulval cell fates..........................................................................................................75
The penetrances of synMuv vulval phenotypes correlate with the level of lin-3
mRNA expression .....................................................................................................77
Discussion ......................................................................................................................79
Genetic enhancement tests can distinguish processes that act in series or
in parallel...................................................................................................................79
The synMuv genes define two distinct classes ...........................................................80
Lack of genetic enhancement can identify proteins that function in a complex
or a process ..............................................................................................................81
Genetic enhancement tests can identify functionally related groups of
evolutionarily conserved but uncharacterized genes ................................................82
Acknowledgments ..........................................................................................................83
Table 1: synMuv alleles used in this study. ....................................................................84
Table 2: Most class A synMuv double mutants have multivulva phenotypes.................85
Table 3: Mutations in most class A synMuv genes enhance the Muv phenotypes
caused by mutations in other class A synMuv genes in a class B synMuv
mutant background.......................................................................................................86
Table 4: Class B synMuv single and class B-B double mutants do not have
appreciable Muv phenotypes........................................................................................87
Table 5: Mutations of most class B and C genes enhance the Muv phenotypes
caused by mutations in other class B genes in a class A synMuv mutant
background...................................................................................................................89
Table 6: Mutations in most class B synMuv genes enhance the Muv phenotypes
caused by mutations of other class B synMuv genes in a class A synMuv
mutant background.......................................................................................................91
5
Figure 1: Regulation of lin-3 transcription by the synMuv genes....................................93
Figure 2: Model: The synMuv classes function in two redundant pathways
or processes, each composed of separate molecular pathways or processes,
to mediate the inhibition of vulval cell fates. .................................................................95
Supplemental Figure 1: Correlation between lin-3 overexpression and the
synMuv phenotype .......................................................................................................97
Chapter Four: lin-38 and mcd-1 antagonize the class A synMuv pathway to
promote C. elegans vulval development .................................................................98
Summary ........................................................................................................................99
Introduction...................................................................................................................100
Materials and Methods .................................................................................................103
Results..........................................................................................................................108
lin-38 encodes a zinc-finger protein ..........................................................................108
Characterization of lin-38 class A synMuv alleles.....................................................109
Loss of lin-38 causes larval lethality and suppresses the class A synMuv
phenotype of lin-38(n751) ......................................................................................110
Some mcd-1 mutations cause a class A synMuv phenotype....................................112
mcd-1 and lin-38 class A synMuv alleles exhibit intergenic non-complementation ..113
mcd-1 encodes two distinct transcripts .....................................................................114
A subset of mcd-1 mutations cause cell-death and synthetic lethality defects .........116
mcd-1 null phenotype................................................................................................117
Discussion ....................................................................................................................119
LIN-38 and MCD-1 are putative transcription factors................................................119
lin-38 is required for viability and likely functions as a synMuv suppressor ..............120
mcd-1 has two opposing functions in vulval development ........................................121
mcd-1 controls multiple distinct aspects of development..........................................122
Conclusions ..............................................................................................................123
Acknowledgments ........................................................................................................123
Table 1: lin-38 class A synMuv mutations ....................................................................124
Table 2: RNAi of lin-38 causes lethality........................................................................125
Table 3: Only some mcd-1 mutations cause a class A synMuv phenotype .................126
Table 4: Non-complementation between lin-38 and mcd-1 alleles...............................128
Table 5: Recombination between lin-38 and mcd-1 alleles ..........................................129
Figure 1: Y48E1B.7 is lin-38.........................................................................................130
Figure 2: LIN-38 is a zinc-finger protein .......................................................................132
Figure 3: LIN-38 and MCD-1 are paralogs ...................................................................134
Figure 4: mcd-1 gene structure ....................................................................................136
Figure 5: mcd-1(n4418) does not block programmed cell death ..................................138
Figure 6: Loss of mcd-1 suppresses the synMuv phenotype .......................................140
Figure 7: Summary of mcd-1 defects and model for mcd-1 function ............................142
Chapter Five: Two putative C. elegans transcription factors, LIN-15A
and LIN-56, interact and function redundantly with an Rb pathway to
regulate vulval development...................................................................................144
Summary ......................................................................................................................145
6
Introduction...................................................................................................................146
Materials and Methods .................................................................................................149
Results..........................................................................................................................153
lin-56 encodes a putative transcription factor containing a THAP-like domain .........153
LIN-56 is a ubiquitous nuclear protein ......................................................................155
LIN-56 protein, but not lin-56 mRNA, is reduced in lin-15A(lf) mutants ....................156
LIN-15A protein, but not lin-15A RNA, is reduced in lin-56(lf) mutants .....................157
Overexpression of lin-56 does not rescue the lin-15AB(lf) synMuv phenotype,
nor does overexpression of lin-15A rescue the lin-56(lf) synMuv phenotype .........157
LIN-56 and LIN-15A physically interact.....................................................................158
Discussion ....................................................................................................................160
LIN-56 and LIN-15A might function as a complex in vivo .........................................160
The class A synMuv genes likely directly regulate gene expression ........................160
Class A synMuv genes might have functions beyond those in vulval development .162
Implications for mammalian tumorigenesis...............................................................162
Acknowledgments ........................................................................................................163
Table 1: lin-56 overexpression rescues the lin-56(lf); lin-15B(lf) but not the
lin-15AB(lf) synMuv phenotype...................................................................................164
Table 2: lin-15A overexpression rescues the lin-36(lf); lin-15A(lf) but not the
lin-56(lf); lin-36(lf) synMuv phenotype. .......................................................................165
Figure 1: Cloning of lin-56. ...........................................................................................166
Figure 2: LIN-56 is broadly expressed and localized to nuclei. ....................................169
Figure 3: LIN-56 protein but not lin-56 mRNA levels are greatly reduced in
lin-15A(lf) mutants. .....................................................................................................173
Figure 4: LIN-15A protein but not lin-15A RNA levels are reduced in lin-56(lf)
mutants.......................................................................................................................176
Figure 5: LIN-15A and LIN-56 interact with each other in the yeast two-hybrid
system. .......................................................................................................................179
Supplemental Figure 1: Expression of LIN-56 driven by heat-shock promoters ..........181
Chapter Six: Future Directions .................................................................................182
Where is lin-3 ectopically expressed in each synMuv mutant? ....................................183
Do any synMuv suppressors promote germline lin-3 expression? ...............................184
Why is the synMuv phenotype temperature-sensitive? ................................................185
What transcriptional targets are responsible for the lin-38(null) lethality? ....................186
What are the cis-acting synMuv elements in the lin-3 promoter?.................................187
What proteins physically interact with class A synMuv proteins?.................................188
What are the molecular functions of the class A synMuv proteins? .............................189
What proteins are present at the lin-3 promoter? .........................................................190
Concluding remarks......................................................................................................192
Acknowledgments ........................................................................................................193
Appendix One: Identification of new class A synMuv mutations..........................194
Materials and Methods .................................................................................................195
Results..........................................................................................................................196
Table 1: Previous genetic screens for class A synMuv mutations................................198
7
Table 2: Class A synMuv screen isolates.....................................................................199
Appendix Two: The class A synMuv genes lin-15A and lin-56 function
in multiple tissues ....................................................................................................200
Introduction...................................................................................................................201
Materials and Methods .................................................................................................203
Results..........................................................................................................................204
Activity of the dpy-7 and lin-31 promoters.................................................................204
lin-15A and lin-56 might function in the Pn.p cells, hyp7, or both .............................205
Discussion ....................................................................................................................206
Acknowledgments ........................................................................................................207
Table 1: Expression of let-60 cDNA under the control of either dpy-7p or
lin-31p rescues the vulvaless phenotype of let-60(n1876) .........................................208
Table 2: Expression of lin-15A under the control of either dpy-7p or lin-31p
rescues the synMuv phenotype..................................................................................209
Table 3: Expression of lin-56 under the control of either dpy-7p or lin-31p
rescues the synMuv phenotype..................................................................................210
Appendix Three: Progress towards identifying proteins that bind to the
lin-3 promoter ...........................................................................................................211
Introduction...................................................................................................................212
Materials and Methods .................................................................................................214
Results..........................................................................................................................218
The effect of the lin-3(4441) mutation on vulval development is not replicated
with a repetitive extrachromosomal array ...............................................................218
EMSA experiments detect a sequence-specific, lin-3-promoter-binding protein ......219
Affinity purification of lin-3p binding proteins.............................................................221
EMSA experiments to test direct interactions between the lin-3 promoter
and class A synMuv proteins ..................................................................................223
Discussion ....................................................................................................................224
Acknowledgments ........................................................................................................227
Table 1: lin-3(+) and lin-3(n4441) affect vulval development similarly when
present on a repetitive extrachromosomal array ........................................................228
Table 2: Deletion of F13E9.13 does not cause or suppress the synMuv phenotype ...229
Figure 1: Identification of a sequence-specific lin-3p-binding protein...........................230
Figure 2: Purification of sequence-specific lin-3p binding protein. ...............................232
Figure 3: EMSA with in vitro transcribed and translated class A synMuv proteins.......234
References..................................................................................................................236!
8
Chapter One
Introduction
Chapter One: Introduction
9
The Caenorhabditis elegans vulva is an excellent system for studying signal
transduction, cell-fate determination, and the role of cell-cell interactions in spatial
patterning and development. C. elegans vulval development can be divided into three
phases: First, a set of precursor cells with equivalent developmental potential are
generated (Kimble, 1981; Sulston and Horvitz, 1977; Sulston and White, 1980).
Second, cell-cell interactions specify a subset of those precursor cells to adopt vulval
cell fates (Kimble, 1981; Sulston and White, 1980). Finally, the vulval cells divide and
their descendants undergo morphogenesis to form the vulva (Sulston and Horvitz,
1977). Extensive screens and genetic analysis have identified a large number of genes
that control the different phases of vulval development. Despite containing only 22
nuclei, proper development of the vulva requires a Hox gene, an epidermal growth
factor (EGF) and Ras pathway, Notch signaling, and Wnt signaling (Sternberg, 2005).
Studies of the C. elegans vulva have also produced insights into cancer biology,
because carcinogenesis involves the misregulation of normal developmental processes.
Homologs of many genes required for proper vulval cell-fate specification play a role in
cancer, including both oncogenes and tumor suppressor genes. For example, studies
of the C. elegans vulva helped elucidate the EGF/Ras signaling pathway (Sternberg,
2006), which is a frequent target of mutations in human cancers (Bos, 1989; Normanno
et al., 2006). Because overactivation of the EGF/Ras pathway can promote cancerous
growth, it is important that mechanisms exist to negatively regulate EGF/Ras signaling.
Therefore, my investigations have focused on the negative regulation of EGF signaling
in C. elegans by the synthetic multivulva (synMuv) genes.
The mammalian EGF signaling pathway
In mammals, signaling by EGF-like ligands controls many aspects of
development and cell-proliferation. EGF is the founding member of the EGF-like family
of peptide growth factors (Cohen, 1962; Cohen and Elliott, 1963). Many other EGF-like
growth factors have since been identified, including transforming growth factor ! (TGF!), amphiregulin, heparin-binding EGF (HB-EGF), betacellulin, epiregulin, epigen, and
the neuregulins (Busfield et al., 1997; Carraway et al., 1997; Chang et al., 1997;
10
Derynck et al., 1984; Higashiyama et al., 1992; Holmes et al., 1992; Lee et al., 1985;
Riese et al., 1995; Shing et al., 1993; Shoyab et al., 1989; Strachan et al., 2001; Toyoda
et al., 1995). Each EGF-like growth factor has one or more EGF domains, a motif of
approximately 40 amino acids containing six cysteines with stereotypical spacing
(Schneider and Wolf, 2009). EGF-like growth factors are synthesized as large
transmembrane proteins (Derynck et al., 1984; Gray et al., 1983; Higashiyama et al.,
1992), and the membrane-bound growth factors can signal to adjacent cells in a
process known as juxtacrine signaling (Harris et al., 2003). The extracellular domains of
EGF-like growth factors are also released by proteolytic cleavage, allowing them to
signal to the cells that released them (autocrine signaling), or to diffuse and signal to
neighboring or more distant cells (paracrine signaling) (Singh and Harris, 2005).
Regulation of proteolytic cleavage of EGF-like ligands is necessary for proper signaling.
For example, mice in which HB-EGF is replaced by an uncleavable HB-EGF show heart
defects similar to loss of HB-EGF, while replacement of HB-EGF with a constitutively
soluble form of HB-EGF causes severe hyperplasia defects, perhaps as a result of
excessive HB-EGF signaling (Yamazaki et al., 2003).
EGF binds to the EGF receptor (EGFR), also called ErbB1 or HER1. There are
three other related receptors, ErbB2/Neu/HER2, ErbB3/HER3, and ErbB4/HER4.
Hereafter I refer to this receptor family as the ErbB receptors. All ErbB receptors are
transmembrane receptor tyrosine kinases (RTKs) with an extracellular ligand-binding
domain and a cytoplasmic protein tyrosine kinase domain. The binding of EGF-like
ligands to ErbB receptors leads to either homodimerization or heterodimerization of the
receptors, and possibly to oligomerization of larger numbers of receptor molecules
(Schlessinger, 2000). Some ligands, such as EGF, TGF-!, and amphiregulin bind
specifically to EGFR, while other ligands, including HB-EGF, bind to either EGFR or
ErbB4, and the neuregulin ligands bind to ErbB3 and ErbB4 (Carraway et al., 1994;
Carraway et al., 1997; Olayioye et al., 2000). There is no known ligand for ErbB2, but
ErbB2 is the preferred heterodimerization partner for the other ErbB family members,
underscoring the importance of heterodimerization (Graus-Porta et al., 1997; Tzahar et
al., 1996). Furthermore, at least eight of the ten possible homodimers and
11
heterodimers of ErbB receptors can be formed in response to ligand binding (Tzahar et
al., 1996).
Upon binding of an EGF-like ligand to an ErbB receptor, the receptor dimerizes,
activating its protein tyrosine kinase activity and autophosphorylating the receptor dimer.
The phosphorylated tyrosine residues serve as docking sites for proteins with Src
homology 2 (SH2) and phosphotyrosine binding (PTB) domains, leading to the
activation of various signal transduction pathways (Schlessinger, 2000). For example,
phosphorylated ErbB receptors activate phosolipase C gamma (PLC"), which in turn
generates the second messengers inositol triphosphate (IP3) and diacylglylcerol (DAG)
(Rhee and Bae, 1997). Perhaps the best known pathway downstream of EGF signaling
is the Ras and mitogen activated protein kinase (MAPK) signaling cascade. Ras is a
small guanine nucleotide binding protein that is active when bound to GTP, but is
inactive when bound to GDP. Ras possesses intrinsic GTPase activity that converts
GTP to GDP, inactivating the Ras protein (Sweet et al., 1984). Upon EGF receptor
activation, the Grb2 adaptor protein binds to the phosphorylated receptor and recruits
the guanine nucleotide exchange factor SOS, which switches Ras proteins from an
inactive GDP-bound state to an active GTP-bound state (Buday and Downward, 1993;
Chardin et al., 1993; Egan et al., 1993; Gale et al., 1993; Li et al., 1993; Olivier et al.,
1993; Rozakis-Adcock et al., 1993; Simon et al., 1993). Activated Ras can
subsequently activate multiple downstream targets, including the protein kinase Raf
(Vojtek et al., 1993; Warne et al., 1993; Zhang et al., 1993). Raf phosphorylates MEK
(MAPK/Erk kinase), which phosphorylates MAPKs, which then translocate to the
nucleus and phosphorylate targets including transcription factors. Ras/MAPK signaling
controls many processes and is best known for promoting cell proliferation.
EGF signaling in mammalian development
EGF and most EGF-like ligands were identified by their ability to promote growth
and proliferation in cell culture or in vivo, and mutant mice lacking various ErbB
receptors indicate that EGF signaling has many roles in normal development. The
different ErbB receptor knockouts have a combination of overlapping and distinct
12
defects, as might be expected given that the receptors function in multiple homo- and
hetero-dimeric combinations. Mice lacking EGFR grow slowly and exhibit numerous
developmental defects (Miettinen et al., 1995; Sibilia and Wagner, 1995; Threadgill et
al., 1995). The mice die at various stages depending on the background and exhibit
abnormal epithelial development, including thin skin and hair growth defects. Targeted
expression of a dominant negative EGFR, as well as transplantation experiments, also
implicate EGFR in mammary duct development (Wiesen et al., 1999; Xie et al., 1997).
Mutant mice lacking ErbB2 die as a result of cardiac defects, and animals in which the
cardiac defect is rescued show that ErbB2 also promotes the proliferation and survival
of sensory neurons, motor neurons, and Schwann cells (Lee et al., 1995; Morris et al.,
1999). ErbB3 knockout mice have abnormal development of neurons, heart, and other
organs (Erickson et al., 1997; Riethmacher et al., 1997). Mice lacking ErbB4 exhibit
lethal cardiac development defects and axon guidance defects, and a neuron-specific
ErbB4 knockout has some behavioral abnormalities (Gassmann et al., 1995; Golub et
al., 2004). Overall, the ErbB family knockouts implicate EGF signaling in promoting
proliferation and survival of many cell-types in vivo.
Mutant mice lacking EGF-like ligands exhibit far fewer abnormalities than
receptor mutants, suggesting that there is substantial redundancy amongst the EGF-like
ligands. The only known essential EGF-like ligands are HB-EGF and neuregulin-1.
Mice lacking neuregulin-1 die during embryogenesis with heart defects and the absence
of Schwann cell precursors and some neurons (Meyer and Birchmeier, 1995). Most
mice without HB-EGF also die during embryogenesis and exhibit heart defects (Iwamoto
et al., 2003; Jackson et al., 2003). Mutant mice lacking TGF-! are fertile and healthy,
although they do have wavy hair and whiskers as a result of disorganized hair follicles
as well as slight defects in neuronal proliferation (Blum, 1998; Luetteke et al., 1993;
Mann et al., 1993; Tropepe et al., 1997). EGF, betacellulin, and amphiregulin knockouts
are viable and fertile, although amphiregulin mutants have mammary duct development
defects and betacellulin mutants enhance the phenotype of HB-EGF mutants (Jackson
et al., 2003; Luetteke et al., 1999). Furthermore, a triple mutant between EGF, TGF-!,
and amphiregulin is slow-growing but viable (Luetteke et al., 1999; Troyer et al., 2001).
13
Many of the phenotypes caused by loss of EGF-like ligands are similar to defects
caused by ErbB receptors, as might be expected. The relatively mild defects caused by
loss of most EGF-like ligands suggests that multiple EGF-like ligands have overlapping
roles in vivo.
Because EGF-like ligands often act over short distances, EGF signaling can be
controlled by limiting the spatial and temporal expression of EGF-like ligands. Some
EGF-like ligands are widely expressed, while others are expressed in more restricted
patterns. For example, neuregulin-1 is expressed in many mesenchymal and neuronal
cells, neuregulin-2 is expressed specifically in the nervous system, and neuregulin-3 is
expressed highly only in the pancreas and muscle (Harari et al., 1999; Meyer and
Birchmeier, 1995; Zhang et al., 1997). Epiregulin is primarily expressed specifically in
the placenta and in certain macrophages (Toyoda et al., 1997). Perhaps the most
striking example of restricted EGF-like ligand expression is HB-EGF expression in the
uterus: HB-EGF is expressed 6-7 hours before implantation precisely at the site where
the blastocyst attaches (Das et al., 1994).
Consistent with the restricted expression of many EGF-like ligands,
overexpression, ectopic expression, or exogenous application of EGF-like ligands can
disrupt normal development. For example, EGF was first identified based on its ability to
cause premature eyelid opening and incisor eruption in newborn mice when
exogenously applied (Cohen, 1962). In contrast to the relatively normal phenotype of
mice lacking TGF-!, TGF-! overexpression causes substantial defects, including
hyperplasia in the epithelia of many organs and excessive proliferation of multiple cell
types in the pancreas (Sandgren et al., 1990). Epigen-overexpressing mice have
enlarged sebaceous glands, and transgenic mice expressing amphiregulin exhibit a
psoriasis-like phenotype, perhaps both reflecting overproliferation (Cook et al., 1997;
Dahlhoff et al., 2010). Furthermore, while betacellulin knockout mice are relatively
normal, mice overexpressing betacellulin have alterations in skull shape, multiple
defects in eye development, and severe lung abnormalities that lead to reduced lifespan
(Schneider et al., 2005). Taken together, these experiments show that controlled
availability of EGF-like ligands is essential for normal development.
14
EGF signaling in cancer
Many pathways that control development are co-opted by tumor cells, and EGF
signaling is a frequent target of misregulation in cancers. Normal cells depend on
continuously receiving growth signals such as EGF-like peptides to proliferate. By
contrast, tumor cells develop independence from external growth factor signals, allowing
them to proliferate excessively. One mechanism by which cancer cells can overcome
their dependence on external growth factor signals is by producing those growth factors
themselves and promoting their own growth via autocrine signaling. TGF-! is
expressed in many cell lines derived from tumors and was initially identified as a
substance secreted by tumor cells that was capable of transforming other cells in culture
(Todaro et al., 1980). In vivo, overexpression of TGF-! causes carcinomas (Sandgren
et al., 1990).
ErbB receptors are also misregulated in cancer cells. Mutations can allow ErbB
receptors to signal independently of ligand binding, or the receptors can be amplified so
as to respond more strongly to existing ligand concentrations (Sharma et al., 2007). As
a result, drugs that target EGFR are in clinical use. These include anti-EGFR
monoclonal antibodies that compete with EGF ligands for binding sites and small
molecule inhibitors that compete with ATP-binding and thereby inhibit ErbB receptor
tyrosine kinase activity (Ciardiello and Tortora, 2008). Tumor cells can also acquire
mutations in signaling molecules that transduce the growth signals downstream of ErbB
receptors, causing constitutively active signaling. For example, approximately 20% of
human cancers have an activating mutation in a Ras gene (Bos, 1989).
Because inappropriate activation of EGF and Ras signaling can promote
cancerous growth, genes that negatively regulate EGF/Ras signaling are likely
candidates to function as tumor suppressor genes. One such tumor suppressor is the
neurofibromatosis type 1 (NF1) gene, which encodes a GTPase activating protein and
negatively regulates Ras by promoting the intrinsic ability of Ras to inactivate itself by
hydrolyzing GTP to GDP (Weiss et al., 1999). The NF1 disorder, which is inherited in a
dominant fashion, is caused by a loss-of-function mutation of one allele of NF1. When
15
the second allele is compromised by loss of heterozygosity, malignant tumors appear,
presumably as a result of EGF/Ras pathway overactivation (Weiss et al., 1999).
C. elegans vulval development
Much of the current knowledge of EGF signaling comes from studies of vulval
development in C. elegans. The six cells P(3-8).p, also called Pn.p cells, are each
innately capable of adopting either a vulval or a non-vulval cell fate. In wild-type
animals, an inductive signal from the anchor cell of the somatic gonad causes three of
the cells, P(5-7).p, to adopt vulval cell fates at the beginning of the third larval stage.
P(5-7).p then divide several times and their 22 descendents form the vulva (Sulston and
Horvitz, 1977). The other three cells, P(3,4,8).p, do not receive sufficient inductive
signal to adopt vulval cell fates. Therefore, P(3,4,8).p adopt non-vulval cell fates, divide
once, and their progeny fuse with the hypodermis.
A series of physical perturbations showed that Pn.p cells are each capable of
adopting either vulval or non-vulval fates, and that the anchor cell induces vulval fates
through a diffusible signal. If the anchor cell is removed by laser-ablating either the
anchor cell precursors or the entire gonad, then no vulval induction occurs and all six
Pn.p cells adopt non-vulval fates (Kimble, 1981; Sulston and White, 1980). Conversely,
laser ablation of every cell in the gonad except the anchor cell does not prevent wildtype vulval development (Kimble, 1981). In wild-type animals, the anchor cell is closest
to P6.p, and P(5-7).p are induced to adopt vulval cell fates. In dig-1 mutants, the gonad,
including the anchor cell, is frequently displaced. This leads to a corresponding shift in
the specific Pn.p cells that are induced to adopt vulval cell fates. For example, when the
anchor cell is closest to P5.p, P(4-6).p adopt vulval fates and P(3,7,8).p adopt nonvulval fates (Thomas et al., 1990). In wild-type animals, only P(5-7).p adopt vulval fates,
but if P(5-7).p are ablated then P(3,4,8).p will adopt vulval fates, proving that each Pn.p
cell is capable of adopting a vulval cell fate (Sulston and White, 1980). Ablation of the
gonad and some Pn.p cells showed that the default state of Pn.p cells in the absence of
these cell-cell interactions is a non-vulval fate (Sternberg and Horvitz, 1986). Taken
together, these experiments prove that each Pn.p cell is capable of adopting either a
16
vulval or a non-vulval cell fate, and that a signal from the anchor cell induces the closest
Pn.p cells to adopt vulval cell fates.
In wild-type animals, the anchor cell is located in close proximity to P6.p along
the ventral side of the animal. In some dig-1 mutant animals, the gonad, and hence the
anchor cell, is displaced to the dorsal side of the animal (Thomas et al., 1990). Despite
being located up to 45 µm away from the nearest Pn.p cell, the anchor cell signal can
still induce vulval cell fates. Therefore, the inductive signal from the anchor cell can act
at a significant distance, and is likely to be a secreted and diffusible factor.
EGF/Ras signaling induces vulval fates
Many mutants with abnormal vulval development have been identified (e.g.
Ferguson and Horvitz, 1985). Animals in which none of the six Pn.p cells adopt a vulval
cell fate have a vulvaless (Vul) phenotype. Animals in which more than three cells
adopt vulval cell fates have a multivulva (Muv) phenotype, with the extra vulval cells
forming ectopic pseudovulvae on the ventral side of the animal. Studies of mutants with
multivulva and vulvaless phenotypes led to the discovery of an EGF/Ras pathway that
induces vulval cell fates. Loss-of-function mutations in components of the EGF/Ras
pathway prevent vulval induction, causing a vulvaless phenotype (Aroian et al., 1990;
Beitel et al., 1990; Han and Sternberg, 1990; Hill and Sternberg, 1992). Conversely,
overactivation of the EGF/Ras pathway causes a multivulva phenotype (Beitel et al.,
1990; Han and Sternberg, 1990; Hill and Sternberg, 1992; Katz et al., 1996). Studies of
EGF/Ras signaling in C. elegans vulval development, along with studies of the
Drosophila melanogastar eye and mammalian cell culture, helped elucidate the
components of this pathway and provided genetic evidence for the order of action of the
different components (Sternberg, 2006). C. elegans mutants with abnormal vulval
development have allowed the study of core component of EGF/Ras signaling as well
as multiple regulatory inputs.
lin-3 encodes a membrane-spanning protein with homology to EGF-like growth
factors, and mutations in lin-3 cause a vulvaless phenotype (Hill and Sternberg, 1992).
Unlike mammals, which possess numerous partially redundant EGF-like ligands, lin-3 is
17
the sole C. elegans EGF-like ligand (www.wormbase.org WS190). lin-3 is expressed in
the anchor cell, where it acts as the inductive signal that controls vulval development
(Hill and Sternberg, 1992). There are both membrane-bound and secreted forms of
LIN-3 (Dutt et al., 2004). During vulval development, the anchor cell is in close
proximity to P6.p, so the membrane bound form of LIN-3 might be used. The anchor
cell can also induce vulval development at a distance (Thomas et al., 1990), likely using
secreted LIN-3.
let-23 encodes an EGF receptor subfamily receptor tyrosine kinase (Aroian et al.,
1990). LIN-3 EGF expressed from the anchor cell is thought to bind to LET-23 EGFR in
the Pn.p cells, and mosaic analysis indicates that let-23 acts cell-autonomously in Pn.p
cells to specify vulval fates (Koga and Ohshima, 1995; Simske and Kim, 1995).
Mutations in lin-2, lin-7, and lin-10 cause a vulvaless phenotype as a result of the
subcellular mislocalization of LET-23. lin-2 encodes a membrane-associated guanylate
kinase (MAGUK) protein, and lin-7 and lin-10 encode PDZ domain-containing proteins
(Hoskins et al., 1996; Simske et al., 1996; Whitfield et al., 1999). LET-23 is specifically
localized to the basolateral side of cell junctions in Pn.p cells in wild-type animals, but in
lin-2, lin-7, and lin-10 mutants LET-23 is broadly localized along the entire cell
membrane (Simske et al., 1996; Whitfield et al., 1999). LIN-2, LIN-7, LIN-10, and
LET-23 form a complex required for proper LET-23 localization (Kaech et al., 1998;
Simske et al., 1996), which is in turn required for LET-23 to receive the LIN-3 inductive
signal from the anchor cell. Unlike other components of EGF signaling, lin-2, lin-7, and
lin-10 are involved only in vulval development, and null mutations do not exhibit other
pleiotropic defects exhibited by most components of EGF signaling (see below)
(Ferguson and Horvitz, 1985; Hoskins et al., 1996).
EGF signaling in vulval development acts through a Ras/MAPK signaling
cascade. Upon binding of LIN-3 to LET-23, LET-23 is likely to dimerize and
autophosphorylate. The phosphorylated LET-23 then binds to SEM-5, an adaptor
protein with one SH2 and two SH3 domains (Clark et al., 1992a). sem-5 was originally
studied in C. elegans for its role in the Ras pathway in vulval development and sex
myoblast migration (Clark et al., 1992a), and the sem-5 homolog Grb2 was later
18
identified as a component of the EGF/Ras pathway in mammals (Lowenstein et al.,
1992). The guanine nucleotide exchange factor SOS-1/LET-341 is recruited, and
subsequently activates LET-60 Ras (Chang et al., 2000). Both sem-5 and sos-1 are
required for normal vulval development, and genetic evidence suggests that sem-5 and
sos-1 act downstream of let-23 EGFR and upstream of let-60 Ras (Chang et al., 2000;
Clark et al., 1992a, b).
Unlike most genes in the C. elegans EGF/Ras pathway that were identified as
Vul mutants, both Vul and Muv alleles of the Ras homolog let-60 were isolated in
screens. Gain-of-function alleles or overexpression of let-60 cause a Muv phenotype,
while loss-of-function alleles cause a Vul phenotype (Beitel et al., 1990; Ferguson and
Horvitz, 1985; Han et al., 1990; Han and Sternberg, 1990). Many of the gain-of-function
mutations in let-60 result in a glycine-to-glutamic acid change at codon 13 (Beitel et al.,
1990). Mutations in that same codon of human Ras genes have been found in cancers
(Bos et al., 1985).
Activation of LET-60 Ras initiates a kinase signaling cascade. The RAF family
serine/threonine kinase LIN-45, the MEK serine/threonine and tyrosine kinase homolog
MEK-2, and the MAP kinase homolog MPK-1/SUR-1 are all required for vulval
development and function downstream of LET-60 (Han et al., 1993; Kornfeld et al.,
1995a; Lackner et al., 1994; Wu and Han, 1994; Wu et al., 1995). Biochemical
evidence shows that MEK-2 functions between LIN-45 and MPK-1/SUR-1 in a kinase
cascade (Wu et al., 1995). The KSR-1 and KSR-2 proteins are similar to Raf and are
thought to function as scaffolds that promote Raf/MEK/MAPK signaling, and animals
lacking both ksr-1 and ksr-2 have a phenotype similar to strong EGF/Ras pathway lossof-function mutants (Kornfeld et al., 1995b; Ohmachi et al., 2002; Sundaram and Han,
1995).
Two transcription factors function directly downstream of the EGF/Ras pathway.
The ETS-domain transcription factor LIN-1 negatively regulates vulval cell fates, and
loss-of-function mutations of lin-1 cause a Muv phenotype (Beitel et al., 1995). The
winged helix transcription factor LIN-31 both positively and negatively regulates vulval
cell fates, and lin-31 loss-of-function leads to a mix of Muv and Vul phenotypes (Miller et
19
al., 1993). LIN-1 and LIN-31 form a complex, and their binding is disrupted by
phosphorylation of LIN-1 and LIN-31 by MPK-1/SUR-1 (Tan et al., 1998). In addition to
the core elements of the Ras pathway described here, many additional positive and
negative regulators promote or inhibit Ras signaling in C. elegans (Sundaram, 2006).
Wnt and Notch signaling are also required for normal vulval development
Besides EGF/Ras signaling, multiple other signaling pathways are required to
form a wild-type vulva. Wnt signaling prevents the Pn.p cells from prematurely fusing
with the hypodermis, which would render them unable to receive and respond to the
inductive signal from the anchor cell (Eisenmann and Kim, 2000; Eisenmann et al.,
1998). Wnt signaling also promotes the adoption of vulval cell fates by Pn.p cells
(Eisenmann and Kim, 2000; Gleason et al., 2002). The role of Wnt signaling in vulval
development is somewhat obscured by the substantial redundancy in the Wnt pathway,
as there are multiple Wnt ligands and receptors involved (Gleason et al., 2006).
In wild-type animals, P6.p adopts a primary vulval cell fate and produces eight
descendants, and P5.p and P7.p adopt secondary vulval cell fates and produce seven
descendants each (Sulston and Horvitz, 1977). Additional differences between the
progeny produced by primary and secondary Pn.p cells can be distinguished by gene
expression reporters (Inoue et al., 2002). The adoption of secondary vulval cell fates
and the precise patterning of primary and secondary fates are regulated by a Notch
pathway that signals between the Pn.p cells (Greenwald et al., 1983; Sternberg, 1988;
Yochem et al., 1988). Patterning the vulva requires crosstalk between the EGF/Ras
and Notch pathways to ensure that the correct cell adopts the primary vulval cell fate
(Chen and Greenwald, 2004; Shaye and Greenwald, 2002; Yoo et al., 2004). In
wild-type animals, primary vulval cell fates are specified by EGF/Ras signaling and
secondary vulval cell fates are induced in P5.p and P7.p by a combination of Notch and
EGF signaling (Katz et al., 1995). P6.p is closest to the anchor cell, so it receives the
strongest inductive signal and adopts the primary fate. P6.p subsequently signals to the
adjacent Pn.p cells to adopt secondary fates. In mosaic animals in which P6.p has wildtype let-23 but P5.p and P7.p have both lost let-23, P5.p and P7.p can still properly
20
adopt secondary vulval cell fates, indicating that Notch signaling alone in the absence of
EGF/Ras signaling can specify secondary fates (Koga and Ohshima, 1995; Simske and
Kim, 1995). In Muv animals resulting from EGF pathway overactivation, Notch signaling
is still present, typically causing an alternating pattern of primary and secondary vulval
cell fates among the six Pn.p cells (Sternberg, 1988).
Additional roles of EGF signaling in C. elegans
In addition to its role in vulval cell-fate specification, EGF signaling also controls
many other aspects of C. elegans development and behavior. Similarly to vulval
development, several cell fates require EGF signaling transduced by a Ras/MAPK
signaling cascade. Null mutations in lin-3 EGF, let-23 EGFR, let-60 Ras, and other
components of the Ras/MAPK pathway cause lethality (Beitel et al., 1990; Ferguson
and Horvitz, 1985; Han et al., 1990). Specifically, animals die during the first larval
stage and exhibit a fluid-filled appearance known as rod-like lethality. Most alleles of
genes in the EGF/Ras pathway identified in screens for abnormal vulval development
are partial loss-of-function alleles that impair vulval development but do not cause
lethality. This rod-like lethality could reflect a general requirement for EGF/Ras
signaling to promote growth of the animal, or it could be caused by a relatively specific
cell-fate defect. Mosaic experiments showed that loss of let-60 in large portions of the
animal does not cause lethality, and that lethality is caused specifically by loss of let-60
in the duct cell (Yochem et al., 1997). The duct cell is required for osmoregulation, and
ablation of the duct cell also causes lethality (Nelson and Riddle, 1984).
EGF signaling also regulates the fate of the P11 and P12 ventral cord precursor
cells (Aroian and Sternberg, 1991; Fixsen et al., 1985; Jiang and Sternberg, 1998). If
either P11 or P12 is ablated in a wild-type animal the other cell adopts a P12 fate,
indicating that P12 is the primary fate (Sulston and White, 1980). Loss of EGF signaling
causes P12 to transform to a P11 fate (Jiang and Sternberg, 1998). EGF signaling in
P11 and P12 acts through let-60 Ras, as a let-60 loss-of-function mutation also causes
a P12-to-P11 transformation (Jiang and Sternberg, 1998). Conversely, expression of a
lin-3 cDNA from a ubiquitous heat-shock promoter causes P11 to transform to a P12
21
fate (Jiang and Sternberg, 1998). Therefore, EGF signaling in P11 and P12 promotes
the adoption of the P12 fate.
EGF signaling also controls development of certain cells in the male tail during
spicule development. Partial loss-of-function mutations in lin-3 EGF, let-23 EGFR,
let-60 Ras, and other components of the EGF/Ras signaling pathway prevent the
adoption of certain anterior cell fates (Chamberlin and Sternberg, 1994). Expression of
lin-3 from a ubiquitous heat-shock promoter causes the ectopic adoption of those fates
(Chamberlin and Sternberg, 1994).
After the vulva is formed a connection must be established between the vulva
and the uterus, and this connection also requires EGF signaling. lin-3 is expressed in
specific descendants of the Pn.p cells in the vulva, and let-23 is expressed in the
ventral-most cells of the uterus (Chang et al., 1999). Partial loss-of-function mutations
in let-23 or let-60 prevent the adoption of the uterine uv1 cell fate necessary to make the
vulval-uterine connection (Chang et al., 1999).
EGF signaling has additional roles outside of cell-fate specification, generally
acting through pathways other than Ras/MAPK signaling. A strong non-null lin-3 allele
and certain let-23 alleles cause hermaphrodite sterility as the result of a defect in
ovulation (Aroian et al., 1990; Ferguson and Horvitz, 1985). The somatic gonad
appears grossly normal and oocytes mature normally, but oocytes do not enter the
spermatheca where they are fertilized in wild-type animals (Clandinin et al., 1998).
let-60 Ras is not involved in this process, and instead let-23 signals through inositol
triphosphate, possibly regulating calcium levels in the spermatheca (Clandinin et al.,
1998).
EGF signaling also regulates behavioral quiescence in C. elegans. Expression of
lin-3 from a ubiquitous heat-shock promoter causes animals to cease pharyngeal
pumping and grow more slowly (Van Buskirk and Sternberg, 2007). The growth and
pumping defects caused by lin-3 overexpression act through phospholipase C-" and
diacylglycerol but not through let-60 Ras (Van Buskirk and Sternberg, 2007). The
phenotype caused by heat-shock lin-3 expression mimics the lethargus period which
precedes each larval molt in C. elegans, and let-23 activation in a specific neuron
22
induces quiescence (Van Buskirk and Sternberg, 2007). In summary, lin-3 has multiple
Ras-independent roles in behavioral regulation, in addition to the multiple
Ras-dependent roles of EGF signaling in cell-fate specification in C. elegans.
A common theme among all of the roles of EGF signaling in C. elegans is that
misexpression of lin-3 causes numerous developmental and behavioral defects.
Consistent with the adverse effects of lin-3 overexpression and ectopic expression, lin-3
expression is normally tightly restricted to specific cells and tissues that correspond to
the different roles of EGF signaling. At the late L2 and early L3 stage when vulval
induction occurs a lin-3::lacZ fusion is expressed specifically in the anchor cell and not
in any other cells near the developing vulva (Hill and Sternberg, 1992). lin-3 is also
expressed in a temporally regulated manner in the male tail, spermatheca, and in
specific vulval cells, likely corresponding to the roles of EGF signaling in male tail
development, ovulation, and uterine development, respectively (Chang et al., 1999;
Hwang and Sternberg, 2004). Additionally, lin-3 expression is seen in the pharynx
throughout development, perhaps to regulate quiescence (Hwang and Sternberg, 2004).
Because lin-3 is expressed in distinct tissues and cells at different times, there
are likely to be multiple cis-regulatory elements in the lin-3 locus controlling the different
aspects of lin-3 expression. Supporting this theory, lin-3::lacZ and lin-3::gfp fusions with
different extents of 5ʼ non-coding regions have differing expression patterns, with more
extensive 5ʼ regions conferring expression in additional tissues (Chang et al., 1999; Hill
and Sternberg, 1992; Hwang and Sternberg, 2004). The only aspect of lin-3 expression
for which the basis of spatial specificity has been studied is the anchor cell. The
lin-3(e1417) mutation causes a vulvaless phenotype, but does not result in any of the
other defects associated with lin-3 loss-of-function (Liu et al., 1999). lin-3(e1417) affects
a conserved 59 bp element located in a lin-3 intron that is both necessary and sufficient
to drive expression in the anchor cell (Hwang and Sternberg, 2004). This element
contains consensus binding sites for the bHLH protein HLH-2 and the nuclear hormone
receptor NHR-25. Both NHR-25 and HLH-2 bind to this element in vitro, and RNAi of
hlh-2 at the appropriate stage eliminates lin-3 expression in the anchor cell (Hwang and
Sternberg, 2004). In summary, lin-3 is expressed in a tightly regulated, spatiotemporally
23
restricted manner. The different sources of lin-3 control a variety of developmental and
behavioral processes, and ectopic expression of lin-3 can disrupt the precise regulation
of these processes.
The synMuv genes antagonize EGF signaling
Because excessive or misplaced EGF signaling can disrupt normal development,
it is important for C. elegans to properly restrict EGF signaling. The synthetic multivulva
(synMuv) genes oppose EGF signaling in vulval development. The synMuv genes are
grouped into two redundant classes, A and B. Animals carrying a mutation in a single
synMuv gene have essentially wild-type vulval development, but animals carrying
mutations in both a class A and a class B synMuv gene exhibit a Muv phenotype
(Ferguson and Horvitz, 1989). Animals with two class A synMuv mutations or two class
B synMuv mutations have mostly normal vulval development. Therefore, the synMuv
genes form two redundant pathways that prevent ectopic vulval development. When
either pathway is mutated, the other pathway is still active and prevents excess vulval
development, but when both pathways are mutated extra Pn.p cells adopt vulval cell
fates. Genes with redundant functions can be difficult to identify by genetic screens.
However, the synMuv genes were found in four distinct ways. The first two synMuv
genes were discovered when a Muv strain was fortuitously isolated with two unlinked
mutations that were both required to cause a Muv phenotype (Horvitz and Sulston,
1980). Second, a number of synMuv mutations were discovered when a strain carrying
a background mutation in a class A synMuv gene was mutagenized, leading to many
Muv strains that carried two synMuv mutations (Ferguson and Horvitz, 1989). Third, the
class A and B synMuv genes lin-15A and lin-15B are located adjacent to each other in
an operon, and mutations that eliminate both lin-15A and lin-15B (called lin-15 or lin15AB mutations) were isolated in screens (Clark et al., 1994; Ferguson and Horvitz,
1989; Huang et al., 1994). Finally, screens have been performed using animals
carrying one synMuv mutation to identify synMuv mutations in the other class. These
approaches, as well as directly testing candidate genes, have led to the identification of
four class A synMuv genes and approximately 25 class B synMuv genes.
24
The class A synMuv genes encode putative transcription factors
Four class A synMuv genes have been discovered: lin-8, lin-15A, lin-38, and
lin-56. lin-8 encodes a novel acidic protein that is the founding member of a family of C.
elegans proteins (Davison et al., 2005). lin-15A and lin-56 encode THAP domain
proteins (Clark et al., 1994; Huang et al., 1994; Davison, 2003). The THAP domain is a
zinc finger-like motif present in many organisms including C. elegans, Drosophila
melanogastar, and humans, and can exhibit sequence-specific DNA-binding activity
(Bessiere et al., 2008; Clouaire et al., 2005; Liew et al., 2007; Roussigne et al., 2003;
Sabogal et al., 2009). THAP domain proteins repress transcription of many genes,
possibly by recruiting corepressor proteins (Cayrol et al., 2007; Dejosez et al., 2008;
Macfarlan et al., 2005). LIN-15A and LIN-56 interact in vitro and are required for each
otherʼs stability in vivo, suggesting that they function in a complex together (Davison,
2003). LIN-8, LIN-15A, and LIN-56 are all expressed broadly throughout the animal and
are primarily localized to the nucleus (Davison, 2003; Davison et al., 2005). Based on
their domains and nuclear localization, the class A synMuv genes might be
transcriptional regulators. However, as none of the class A synMuv proteins has
extensive homology to well-characterized proteins, the specific molecular functions of
the class A synMuv genes are still unclear.
The class B synMuv genes encode transcriptional repressors
Many class B synMuv genes are homologous to genes known to be involved in
chromatin remodeling and transcriptional repression. Nucleosomes, composed of
approximately 146 base pairs of DNA wrapped around an octamer consisting of two
H2A, two H2B, two H3, and two H4 histones, are the core unit of chromatin. These
nucleosomes then form higher order structures that can compact the chromatin,
typically rendering it less accessible to polymerases. Many proteins can affect the state
and structure of chromatin and thereby regulate transcription.
Histone tails extend from the nucleosome and are subjected to many covalent
modifications, including acetylation, methylation, phosphorylation, ubiquitylation, and
sumoylation. Modifications can either promote or repress transcription, depending on
25
both the specific covalent modification and the residue that it is applied to. For example,
methylation of lysine 9 of histone H3 typically leads to a repressive chromatin state that
inhibits transcription, but methylation of lysine 4 of histone H3 typically promotes
transcription (Kouzarides, 2007). The class B synMuv genes include both the histone
deacetlyase HDA-1 and the histone acetylase MYS-1 (Ceol and Horvitz, 2004; Lu and
Horvitz, 1998). It is not known if HDA-1 and MYS-1 affect different residues of histones
at the same target genes, or if they affect two separate target genes that have opposing
effects on vulval development. Two histone lysine methyltransferases have been
identified as class B synMuv genes, MET-1 and MET-2 (Andersen and Horvitz, 2007;
Poulin et al., 2005). MET-1 is homologous to histone H3 lysine 36 histone
methyltransferases, and MET-2 is homologous to histone H3 lysine 9 histone
methyltransferases. Covalent modifications are often “read” by proteins that bind
specific covalently-modified histones to regulate transcription. For example, methylated
lysine 9 of histone H3 serves as a binding site for chromodomain proteins such as HP1,
which represses transcription (Bannister et al., 2001; Lachner et al., 2001; Nakayama et
al., 2001). The C. elegans HP1 homolog hpl-2 is a class B synMuv gene (Couteau et
al., 2002).
ATP-dependent chromatin remodeling enzymes use energy from the hydrolysis
of ATP to remodel chromatin by either sliding nucleosomes along the DNA or by
displacing nucleosomes from the DNA. These actions can alter the accessibility of the
DNA and thereby affect transcription (Saha et al., 2006). One such enzyme is Mi-2, a
component of the nucleosome remodeling and histone deacetlyase (NuRD) complex
(Tong et al., 1998; Wade et al., 1998; Zhang et al., 1998). The C. elegans Mi-2
homolog LET-418 and homologs of several other components of the NuRD complex are
class B synMuv proteins (Lu and Horvitz, 1998; von Zelewsky et al., 2000).
Other class B synMuv genes encode transcription factors that can bind to
specific DNA regulatory sequences and regulate expression of adjacent genes. The
class B synMuv genes lin-35, dpl-1, and efl-1 encode the C. elegans homologs of the
retinoblastoma gene (Rb), DP, and E2F, respectively (Ceol and Horvitz, 2001; Lu and
Horvitz, 1998). Rb is a tumor suppressor, and either Rb or other components of the Rb
26
pathway are inactivated in the majority of human cancers (Hanahan and Weinberg,
2000). E2F and DP proteins form heterodimeric transcription factors that bind DNA in a
sequence-specific manner. Rb can bind to the DP/E2F heterodimer and modulate its
effects on transcription. Rb, DP, and E2F have been extensively studied for their roles
in regulating cell-cycle progression (Dyson, 1998). Additionally, Rb controls the
expression of many genes involved in development and differentiation in Drosophila
(Dimova et al., 2003).
Some class B synMuv genes do not have known functions, and many of these
genes are conserved in humans. For example, LIN-9, LIN-37, LIN-52, and LIN-54 are
conserved proteins, and their specific molecular functions are not well understood
(Beitel et al., 2000; Harrison et al., 2006; Thomas et al., 2003). Other class B synMuv
genes such as lin-13, lin-15B, and lin-36 do not have obvious homologs in mammals,
but do encode proteins with specific domains that are present in many animals,
including humans (Clark et al., 1994; Huang et al., 1994; Melendez and Greenwald,
2000; Reddy and Villeneuve, 2004; Thomas and Horvitz, 1999).
synMuv protein complexes
Based on interaction studies in C. elegans and homology to well-studied yeast
and mammalian complexes, the synMuv genes are thought to form a number of distinct
complexes. The class A synMuv proteins LIN-15A and LIN-56 form a complex
(Davison, 2003). The class B synMuv proteins HDA-1, LIN-53, and LET-418 are the C.
elegans homologs of members of the NuRD complex (Harrison et al., 2006; Lu and
Horvitz, 1998; von Zelewsky et al., 2000). The NuRD complex is a corepressor that
combines histone deacetlyase and ATP-dependent chromatin remodeling activities.
Eight other class B synMuv proteins, including LIN-35 and DPL-1, are present in the
DRM complex, which is distinct from the C. elegans NuRD-like complex (Harrison et al.,
2006). The DRM complex is highly similar to the Drosophila dREAM and Myb-MuvB
complexes that repress the transcription of many E2F target genes (Korenjak et al.,
2004; Lewis et al., 2004). Another group of class B synMuv genes, previously referred
to as the class C synMuv genes (see Chapter Three for explanation), are predicted to
27
form a C. elegans Tip60/NuA4-like histone acetyltranferase complex (Ceol and Horvitz,
2004). The class B synMuv genes hpl-2 and lin-13 both exhibit a Muv phenotype at
high temperatures as single mutants in addition to their class B synMuv phenotype, and
they form a complex in vivo (Coustham et al., 2006; Couteau et al., 2002; Melendez and
Greenwald, 2000). Some synMuv proteins might be members of multiple complexes
that control vulval development and other processes. For example, the RbAp48
homolog LIN-53 is present in both the DRM and NuRD complexes (Harrison et al.,
2006; Lu and Horvitz, 1998). Surprisingly, yeast two-hybrid and in vitro binding assays
indicate that the class A synMuv protein LIN-8 and the class B synMuv protein LIN-35
physically interact (Davison et al., 2005). Given the different roles of lin-8 and lin-35 in
vulval development, this interaction might not be relevant to vulval development but
might instead be important for a non-vulval function of these two genes.
The site of action of the synMuv genes
The site of action of components of the EGF signaling pathway in vulval induction
is clear: lin-3 EGF is expressed in the anchor cell, and let-23 EGFR and all downstream
signaling components including the transcription factor lin-31 function cell-autonomously
in the Pn.p cells (Hill and Sternberg, 1992; Koga and Ohshima, 1995; Lackner et al.,
1994; Miller et al., 1996; Simske and Kim, 1995; Yochem et al., 1997). The site-ofaction of the synMuv genes is less clear. The synMuv phenotype is epistatic to anchor
cell ablation, indicating that the synMuv genes function outside of the anchor cell
(Ferguson et al., 1987; Sternberg and Horvitz, 1989). Mosaic analyses of the synMuv
genes lin-15 and lin-37 clearly showed that they do not act cell autonomously in the
Pn.p cells (Hedgecock and Herman, 1995; Herman and Hedgecock, 1990). However,
mosaic analyses did not clearly implicate any one cell or tissue as the site-of-action of
the synMuv genes (Hedgecock and Herman, 1995; Herman and Hedgecock, 1990;
Thomas and Horvitz, 1999). More recently, Myers and Greenwald (2005) reported that
expression of a lin-35 cDNA driven specifically in the hyp7 hypodermal syncytium using
the dpy-7 promoter was sufficient to rescue the lin-35 synMuv phenotype. However, the
rescue was not complete and I have found that the expression pattern of the dpy-7
28
promoter is broader than was previously thought (Appendix Two), so it is possible that
other cells in addition to hyp7 contribute to the synMuv phenotype.
Relationship between EGF signaling and synMuv genes
The relationship between the EGF/Ras pathway and the synMuv genes in vulval
development has been a longstanding question. Double mutant experiments indicated
that the synMuv phenotype was epistatic to the Vul phenotype caused by a reduction of
lin-3 EGF function, but the Vul phenotype caused by loss-of-function mutations in let-23
EGFR or let-60 Ras was epistatic to the synMuv phenotype (Ceol and Horvitz, 2001,
2004; Huang et al., 1994; Lu and Horvitz, 1998). This observation led to the hypothesis
that LET-23 may have some basal signaling activity in the absence of LIN-3 binding
(Ceol and Horvitz, 2001). However, these epistasis experiments were confounded
because non-null alleles of lin-3 were used, since null alleles of lin-3 cause lethality that
precludes assaying vulval development. The first hint that the synMuv phenotype might
not be epistatic to lin-3 loss-of-function was that a partial loss-of-function mutation of
lin-3 in trans to a lin-3 null mutation partially suppresses a weak synMuv phenotype
(Ferguson et al., 1987). Later, it was found that RNAi directed against lin-3 could
efficiently suppress the synMuv phenotype of many synMuv double mutants (Cui et al.,
2006a). Because the synMuv phenotype requires EGF signaling, EGF signaling must
act either downstream of or in parallel to the synMuv genes. lin-3 mRNA levels are
grossly wild-type in synMuv single mutants, but lin-3 is overexpressed in synMuv double
mutants (Andersen and Horvitz, 2007; Cui et al., 2006a). Thus, the class A and B
synMuv genes redundantly repress lin-3 expression, and lin-3 is likely to be a direct or
indirect target of the synMuv genes in vulval development.
The synMuv genes have numerous functions
synMuv single mutants typically do not have major defects in vulval development,
and most class A synMuv single mutants have a superficially wild-type phenotype.
However, most class B synMuv single mutants exhibit numerous abnormalities.
Microarray analysis of animals with mutations in the class B synMuv genes lin-35, dpl-1,
29
and efl-1 identified a large number of transcripts with altered expression (Chi and
Reinke, 2006; Kirienko and Fay, 2007), and many defects other than those in vulval
development have been identified in class B synMuv single mutants. Some of these
defects are present in many synMuv mutants, while other defects are specific to much
smaller subsets of the synMuv genes. These various processes involve many different
groups of synMuv genes. Most of the defects caused by synMuv mutations do not
resemble the defects that result from either loss-of-function or overexpression of lin-3,
indicating that they result from effects on other target genes.
The initial sign that the synMuv genes might be involved in processes other than
vulval development came from studies of lin-9, the first class B synMuv gene identified.
The first allele of lin-9 was a partial loss-of-function allele, and a non-complementation
screen isolated strong lin-9 loss-of-function mutations that cause sterility (Ferguson and
Horvitz, 1989). Subsequently, many class B synMuv mutations were found to cause
sterility (Ceol and Horvitz, 2001; Ceol et al., 2006; von Zelewsky et al., 2000). The
sterility of these mutants might not indicate a specific role in fertility for these genes.
Rather, these genes could be absolutely essential for all aspects of growth and viability,
and defects are seen in vulval development and fertility because the maternal
contribution of the gene has been exhausted by the time those processes occur. The
divisions of the Pn.p cells are some of the latest divisions in the animal (Sulston and
Horvitz, 1977), which may be why defects are seen in vulval development and not in
other aspects of development.
The Rb gene has an important role in mammals controlling progression through
the cell cycle. Likewise, the C. elegans Rb homolog lin-35 is a negative regulator of the
G1/S progression (Boxem and van den Heuvel, 2001). Many other synMuv genes,
including efl-1, dpl-1, lin-9, lin-15B, and lin-36 also function in cell cycle progression
(Boxem and van den Heuvel, 2002). Screens for mutants synthetically lethal with lin-35
loss-of-function implicated lin-35 Rb and other class B synMuv genes in pharyngeal
development, gonadal development, and general growth and viability (Bender et al.,
2007; Bender et al., 2004; Cui et al., 2004; Fay et al., 2003; Fay et al., 2004).
30
Therefore, lin-35 Rb is required for multiple development processes where its role is
masked by redundancy, much like the function of lin-35 in vulval development.
Many class B synMuv mutants exhibit a set of abnormalities that can be
attributed to an underlying defect in maintaining the distinction between germline and
soma. In these mutants, somatic cells adopt a more germline-like appearance and
inappropriately express PGL-1, a component of P granules that is normally expressed
only in the germline (Kawasaki et al., 1998; Unhavaithaya et al., 2002; Wang et al.,
2005). Transgenes in C. elegans created by injecting DNA into the gonad are large,
multicopy, and repetitive arrays (Mello et al., 1991; Stinchcomb et al., 1985). These
transgenes are expressed in somatic cells, but are typically silenced in the germline
(Kelly et al., 1997). In some class B synMuv mutant backgrounds, repetitive transgenes
are also silenced in somatic cells, perhaps reflecting the adoption of germline
characteristics that include transgene silencing (Hsieh et al., 1999). Many of these
class B synMuv mutants are also hypersensitive to RNAi, possibly as a result of
misexpression of the germline-specific RNA-dependent RNA polymerase EGO-1 (Wang
et al., 2005). Some class B synMuv genes, such as the histone methyltransferases
met-1 and met-2, do not affect the distinction between germline and soma (Andersen
and Horvitz, 2007). Other synMuv mutants exhibit only some aspects of the soma to
germline transformation. For example, mutations in the class B synMuv genes tam-1
and lex-1 cause silencing of repetitive transgenes but do not affect expression of the
germline marker PGL-1 (Hsieh et al., 1999; Tseng et al., 2007). It is also possible that
tam-1 and lex-1 do not play any role in distinguishing germline and somatic fates, and
that their effects on transgene silencing are caused by other mechanisms. Furthermore,
it is not known if the class B synMuv genes impart somatic identity by directly repressing
many or all germline-specific genes, or if the class B synMuv genes control a small
number of master regulators that specify germline and somatic fates and thereby
indirectly repress the expression of germline genes.
31
Conclusion
The C. elegans vulva has been an informative system for the study of EGF/Ras
signaling. The synMuv genes, which oppose EGF signaling in vulval development,
function in chromatin remodeling and transcriptional repression to inhibit the ectopic
adoption of vulval cell fates. My thesis work has focused on identifying synMuv genes,
determining how the synMuv genes interact with each other, and elucidating the
relationship between the synMuv genes and the EGF/Ras pathway. I have specifically
focused on the class A synMuv genes, as they have been studied much less extensively
than the class B synMuv genes. In Chapter Two I show that the class A synMuv genes
antagonize vulval development primarily by repressing lin-3, and that the class A and B
synMuv genes function globally to prevent ectopic lin-3 expression. In Chapter Three I
show that the class A synMuv genes function in multiple parallel pathways to repress
lin-3 expression. In Chapter Four I report the identification of class A synMuv alleles of
mcd-1 and the cloning and characterization of lin-38. Finally, I discuss what future
approaches might be taken to study further the synMuv genes and their role in
repressing lin-3 EGF expression.
Acknowledgments
I thank Shunji Nakano and Christoph Engert for helpful comments about this chapter.
32
Chapter Two
The C. elegans synthetic multivulva genes prevent Ras pathway activation by
tightly repressing ectopic expression of lin-3 EGF
Adam M. Saffer, Dong hyun Kim, Alexander van Oudenaarden, and H. Robert
Horvitz
Chapter Two: The C. elegans synthetic multivulva genes prevent Ras pathway
activation by tightly repressing ectopic expression of lin-3 EGF
I fixed animals for in situ hybridizations, Dong hyun Kim performed the hybridizations
and collected the images, and I analyzed the images. I performed all other experiments
in this chapter and wrote the manuscript.
This manuscript is being prepared for submission.
33
Summary
The C. elegans class A and B synthetic multivulva (synMuv) genes redundantly
antagonize an EGF/Ras pathway to prevent ectopic vulval induction. We identify a
class A synMuv mutation in the promoter of the lin-3 EGF gene, establishing that lin-3 is
the key biological target of the class A synMuv genes in vulval development. Using
FISH with single mRNA molecule resolution, we find that lin-3 EGF expression is tightly
restricted to only a few tissues in wild-type animals, including the germline. In synMuv
double mutants lin-3 EGF is also ectopically expressed at low levels throughout the
animal. Our findings reveal that the ectopic expression of extremely low levels of a
growth factor mRNA – an average of one to two molecules per cell – can abnormally
activate the Ras pathway and alter cell fates. Our results suggest a mechanistic basis
for the functional redundancy between the tumor-suppressor-like class A and B synMuv
genes: the class A synMuv genes function to specifically repress ectopic lin-3
expression, while the class B synMuv genes either directly or indirectly repress lin-3
EGF as a consequence of their role in preventing cells from adopting a germline-like
fate. Analogous genes in mammals might function as tumor suppressors by preventing
broad ectopic expression of EGF-like ligands.
34
Introduction
Signaling by epidermal growth factor (EGF) family ligands and EGF receptor
(EGFR) family tyrosine kinases controls many aspects of mammalian development and
can drive cancers: EGFRs are commonly overexpressed or constitutively activated by
mutations in tumor cells (Normanno et al., 2006), and EGF-family ligands can be
misregulated in cancer. For example, the EGF-family ligands heparin-binding EGF-like
growth factor, amphiregulin, and TGF-! are upregulated in cancer cells from many
different cancer types (Massague, 1990; Yotsumoto et al., 2008), and TGF-!
overexpression causes widespread epithelial hyperplasia in mice (Jhappan et al., 1990;
Sandgren et al., 1990). Growth factors often signal through a Ras pathway, and
approximately 20% of tumors carry a constitutively active Ras mutations (Downward,
2003).
In the nematode Caenorhabditis elegans the EGF-family ligand LIN-3 acts
through the EGFR LET-23 and the Ras protein LET-60 to control many aspects of
development, including the induction of the hermaphrodite vulva (Aroian et al., 1990;
Beitel et al., 1990; Han and Sternberg, 1990; Hill and Sternberg, 1992). In wild-type
animals, of a set of six equipotent cells, three (P5.p, P6.p and P7.p) adopt vulval cell
fates, while the other three (P3.4, P4.p, and P8.p) adopt non-vulval fates (Sternberg and
Horvitz, 1986). The expression of vulval cell fates requires EGF/Ras signaling, and
mutations that reduce EGF/Ras signaling cause a vulvaless (Vul) phenotype in which
none of the six cells adopts vulval cell fates (Aroian et al., 1990; Beitel et al., 1990; Han
and Sternberg, 1990; Hill and Sternberg, 1992). The anchor cell, located closest to
P6.p, is the only cell that both expresses LIN-3 EGF and is located near the six Pn.p
cells (Hill and Sternberg, 1992), and laser ablation of the anchor cell results in a Vul
phenotype (Kimble, 1981) like that seen in mutants defective in lin-3 EGF or let-23
EGFR. Overactivation of the EGF/Ras pathway, by overexpression of lin-3 EGF or by an
activating mutation in either let-23 EGFR or let-60 Ras, causes a multivulva (Muv)
phenotype in which all six Pn.p cells adopt vulval cell fates (Beitel et al., 1990; Han and
Sternberg, 1990; Hill and Sternberg, 1992; Katz et al., 1996).
35
In vulval development, EGF signaling and Ras pathway activation are
antagonized by the synthetic multivulva (synMuv) genes. The synMuv genes define two
classes, A and B (Andersen et al., 2008; Ferguson and Horvitz, 1989). In synMuv single
mutants or in class A double mutants or class B double mutants, vulval development is
mostly normal. By contrast, animals mutant in both a class A synMuv gene and a class
B synMuv gene exhibit a strong Muv phenotype. Many class B synMuv genes have
homologs that function in histone modification, chromatin remodeling, and
transcriptional repression. For example, the class B synMuv genes encode a
DP/E2F/Rb complex (Ceol and Horvitz, 2001; Lu and Horvitz, 1998), a nucleosome
remodeling and deacteylase (NuRD) complex (Solari and Ahringer, 2000; Unhavaithaya
et al., 2002), two histone methyltransferases (Andersen and Horvitz, 2007), a
heterochromatin protein 1 homolog (Couteau et al., 2002), and a Tip60/NuA4 histone
acetyltransferase complex (Ceol and Horvitz, 2004). Of the four identified class A
synMuv genes, three encode proteins with either a zinc-finger domain or a zinc-fingerlike THAP domain (Clark et al., 1994; Davison et al., 2003; Huang et al., 1994; Chapter
Four). The expression patterns of three class A synMuv proteins have been studied,
and all three are localized to the nucleus, suggesting that class A synMuv proteins also
regulate transcription (Clark et al., 1994; Davison et al., 2003; Davison et al., 2005;
Huang et al., 1994).
The synMuv genes function at least in part by repressing expression of lin-3
EGF. Loss-of-function mutations in either let-23 EGFR or lin-3 EGF can suppress the
synMuv phenotype (Cui et al., 2006; Ferguson et al., 1987; Lu and Horvitz, 1998),
indicating that the synMuv genes act upstream of or in parallel to lin-3. Furthermore, lin3 mRNA levels are increased in synMuv double mutants but not in synMuv single
mutants (Cui et al., 2006), and overexpression of lin-3 EGF causes a Muv phenotype
(Hill and Sternberg, 1992). Laser ablation of the anchor cell, the source of LIN-3 in wildtype vulval development, does not fully suppress the Muv phenotype of synMuv double
mutants (Ferguson et al., 1987), indicating that synMuv genes cannot simply prevent
overexpression of lin-3 from the anchor cell. Heterologous expression experiments
have indicated that the class B synMuv gene lin-35 functions primarily in the
36
hypodermal syncytium to prevent adoption of vulval cell fates (Myers and Greenwald,
2005). It is not known where lin-3 is overexpressed in synMuv mutants, how the
synMuv genes control lin-3 expression, or if the synMuv genes control targets other than
lin-3 important for vulval development.
Here we report the identification of a lin-3 EGF promoter mutation that causes a
dominant class A synMuv phenotype. The effect of this mutation reveals that the only
major role of the class A synMuv genes in vulval development is to repress lin-3. We
find that lin-3 mRNA is ectopically expressed throughout the animal in synMuv mutants.
Our results show that low levels of ectopic lin-3 expression outside the cells that
normally produce and respond to lin-3 can adversely alter the development of C.
elegans. We propose that the class A and class B synMuv genes prevent ectopic lin-3
expression by two distinct mechanisms: the class A synMuv genes act through an
element in the lin-3 promoter to tightly repress ectopic lin-3 expression, while the class
B synMuv genes either directly or indirectly repress lin-3 as a consequence of their role
in preventing somatic cells from adopting a germline-like fate. When both the class A
and B synMuv genes are inactivated, somatic cells adopt a germline-like fate that
includes lin-3 expression and because there is no mechanism to tightly repress lin-3,
widespread ectopic expression of lin-3 and a Muv phenotype results.
37
Materials and Methods
Strains and genetics
C. elegans strains were cultured by standard methods on OP50 bacteria
(Brenner, 1974). All animals were grown at 20˚C, except where otherwise noted. The
wild-type strain was N2, except in SNP mapping experiments in which the polymorphic
CB4856 strain was also used (Wicks et al., 2001). The following mutations were used
in this study and were described by Riddle (1997) unless otherwise noted:
LGI: dpy-5(e61), lin-61(n3447) (Harrison et al., 2007)
LGII: lin-8(n2731) (Thomas et al., 2003), lin-56(n2728) (Thomas et al., 2003), lin38(n751), syIs12 (Katz et al., 1995)
LGIII: dpy-17(e164), unc-32(e189), lin-52(n771) (Thomas et al., 2003)
LGIV: lin-3(n4441), lin-3(n4929), lin-3(n4951) (this study), lin-3(n1059)
LGX: lin-15A(n767), lin-15B(n744), lin-15(e1763)
The balancer strain nT1[qIs51] IV:V (Siegfried et al., 2004) was used; qIs51 is a GFPexpressing transgene integrated onto the nT1 translocation.
Quantitative PCR assay
Synchronized animals were harvested at or near the L2-to-L3 larval transition,
when vulval induction occurs. N2 animals were harvested 33 hours after starved L1
larvae were placed on plates with food. Some mutants grew more slowly and were
harvested after 39 hours. Total RNA was extracted using Trizol (Invitrogen). Firststrand cDNA was prepared from 1 µg total RNA using the SuperScript III First-Strand
Synthesis Supermix for qRT-PCR (Invitrogen). Each real-time reverse transcriptase
(RT) PCR reaction contained 10 ng of RT products, 1x SyBR Green PCR Master Mix
(Applied Biosystems) and 0.4 µM of each primer. The real-time PCR was performed in
triplicate using an Eppendorf Mastercycler realplex2. Two independent biological
replicates were tested for each genotype. The #CT values for lin-3 were determined
using rpl-26 as the internal reference, and the ##CT values were calculated for each
genotype relative to that of the wild type. The error shown is the standard deviation of
38
relative lin-3/rpl-26 ratios for the biological replicates. lin-3 was amplified using the
primers CGCATTTCTCATTGTCATGC and CTGGTGGGCACATATGACTC.
Fluorescence in situ hybridization
Animals were grown to the L2-to-L3 transition as in the quantitative RT-PCR
experiments. Fixation and hybridization were performed as described previously (Raj et
al., 2008), except that worms were fixed for one hour instead of 45 minutes. The lin-3
probes (Biosearch Technologies, Inc) were conjugated to the fluorophore Cy5 using the
Amersham Cy5 Mono-reactive Dye pack (GE Healthcare). The probe sequences used
are shown in Supplementary Table 1. Figures 2 and 3 are maximum intensity
projections of a Z-stack of images processed with the find edges and smooth operations
in ImageJ. Approximately five animals were imaged for each genotype.
39
Results
n4441 causes a dominant class A synMuv phenotype
During a screen for new class A synMuv mutations, we identified a Muv animal in
the F1 generation after ethyl methanesulfonate (EMS) mutagenesis of the class B
synMuv mutant lin-52(n771). We named the mutation that caused this defect n4441. To
seek additional n4441-like mutations, we screened approximately 492,000 F1 progeny
of lin-52(n771) animals mutagenized by EMS and approximately 89,000 progeny of
animals mutagenized by N-ethyl-N-nitrosourea (ENU), but we did not identify any
additional n4441-like mutants. As a single mutant, n4441 animals are wild-type at 20˚C
and exhibit a low penetrance Muv defect at 25˚C (Table 1), comparable to that of most
class A synMuv mutants (Andersen et al., 2008). Double mutants between n4441 and
the class B synMuv mutations lin-15B(n744), lin-52(n771), or lin-61(n3447) exhibit a
strong synMuv phenotype. n4441 causes a fully penetrant Muv defect as a heterozygote
in the class B synMuv mutant background lin-15B(n744), indicating that n4441
dominantly causes a class A synMuv phenotype. n4441 causes a 97% penetrant
synMuv defect in the weak class B synMuv mutant background lin-61(n3447) at 22.5˚C,
comparable to the previously reported phenotype of double mutants between lin61(n3447) and the strong class A synMuv mutations lin-15A(n767) or lin-38(n751)
(Andersen et al., 2008).
To determine how n4441 interacts with other class A synMuv mutations, we built
double mutants between n4441 and an allele of each known class A synMuv gene. We
used the putative null alleles lin-8(n2731), lin-15A(n767), and lin-56(n2728) and the nonnull allele lin-38(n751), since a null allele of lin-38 causes lethality (Chapter Four). At
20˚C and 25˚C, the double mutants n4441; lin-15A(n767), lin-38(n751); n4441, and lin56(n2728); n4441 were enhanced for the Muv phenotype when compared to their
respective single mutants (Table 1). The lin-8(n2731); n4441 double mutant was
roughly comparable to n4441 alone when scored at 25˚C and also exhibited a low
penetrance Muv defect at 20˚C, which neither n4441 or lin-8(n2731) did on their own
(Table 1). Thus, mutations in all known class A synMuv genes can enhance the Muv
40
phenotype of n4441, but do so very weakly compared to the enhancement caused by
class B synMuv mutations.
n4441 is an allele of lin-3
By performing SNP mapping experiments using the CB4856 polymorphic strain
of C. elegans, we mapped the n4441 mutation to a 661 kb region containing
approximately 170 genes between SNPs dbP6 and uCE4-1148 (Figure 1A). n4441
dominantly causes a synMuv phenotype and thus might well be a gain-of-function
mutation, so we sought loss-of-function mutations in the gene affected by n4441.
n4441/nT1[qIs51]; lin-15B(n744) animals, which display a fully penetrant Muv defect,
were mutagenized with EMS. The nT1[qIs51] translocation causes inviability when
homozygous and suppresses recombination across an interval that includes lin-3
(Siegfried et al., 2004). Approximately 6,800 F1 progeny were screened, and two
animals were identified that were non-Muv and produced only non-Muv progeny,
indicating that they contained a suppressor mutation tightly linked to n4441. We named
these mutations n4929 and n4951. n4441 n4929; lin-15B(n744) animals were sterile
and exhibited a very low penetrance Muv defect (Figure 1D). n4441 n4951/nT1[qIs51]
animals were superficially wild-type with no Muv defect, and n4441 n4951 homozygotes
died as L1 larvae with a rod-like appearance (Figure 1D). The rod-like lethal phenotype
is characteristic of loss-of-function mutations in genes in the EGF/Ras pathway required
for vulval induction (Han et al., 1990). The only known gene in the EGF/Ras pathway in
the genetic interval containing n4441 is lin-3, which encodes the EGF ligand. Strong
loss-of-function alleles of lin-3 cause a rod-like lethal phenotype, and lin-3 mutations can
also cause sterility (Ferguson and Horvitz, 1985; Liu et al., 1999). The n4929 mutant
carries a G-to-A transition in the first nucleotide of exon 8 of lin-3 and is predicted to
mutate an arginine to a lysine at amino acid 347 of LIN-3 (Figure 1B). The n4951
mutant carries a G-to-A transition that results in a nonsense mutation predicted to
truncate LIN-3 after only 26 amino acids, before the EGF domain (Figure 1B). The lin3(n1059) nonsense mutation failed to complement the sterility caused by n4929 and the
lethality caused by n4951, proving that n4929 and n4951 are alleles of lin-3. Since the
41
lin-3(n4951) nonsense mutation suppressed the n4441 synMuv defect in cis, but the lin3(n1059) nonsense mutation did not suppress the n4441 synMuv defect in trans (Figure
1D), a lin-3 loss-of-function mutation is a cis dominant suppressor of n4441, indicating
that n4441 is a gain-of-function allele of lin-3.
We determined the sequences of all exons and introns of lin-3 and of
approximately 11 kb of upstream DNA in lin-3(n4441) mutants. The only mutation was
a G-to-A transition at nucleotide 30904 of cosmid F36H1, approximately 200 bp
upstream of the start of lin-3 transcription (Figure 1C). To prove that the F36H1(30904)
mutation is responsible for the class A synMuv phenotype caused by lin-3(n4441), we
sought recombinants between lin-3(n4441) and the lin-3(n4951) nonsense mutation,
which is 5.3 kb downstream of F36H1(30904). We screened approximately 90,000
progeny from lin-3(n4441 n4951)/+; lin-15B(n744) animals, identified five independent
Muv animals and established homozygous lines. None of the five lines contained the
lin-3(n4951) mutation, and all five carried the F36H1(30904) G-to-A mutation. Thus, the
lin-3(n4441) mutation that causes the class A synMuv phenotype must be to the left of
lin-3(n4951), because if it were to the right then the recombinants would not carry the
F36H1(30904) mutation. The 5.3 kb between F36H1(30904) and lin-3(n4951), as well
as 10.8 kb of DNA upstream of F36H1(30904), carried no additional mutations in lin3(n4441) animals. If the mutation that causes the lin-3(n4441) synMuv phenotype is not
the F36H1(30904) mutation, then the lin-3(n4441) mutation must be at least 10.8 kb to
the left of the F36H1(30904) mutation. However, in that case, assuming a constant
recombination rate throughout the lin-3 interval, the likelihood that all five recombination
events would have occurred between F36H1(30904) and lin-3(n4951) is
((5.3)/(5.3+10.8))5, or <0.004. We conclude that the G-to-A mutation at nucleotide
30904 of cosmid F36F1 is responsible for the class A synMuv phenotype caused by lin3(n4441).
lin-3(n4441) might be a class A synMuv specific allele of lin-3. Alternatively, lin3(n4441) might cause weak overexpression of lin-3 if weak overexpression of lin-3
behaves like a class A synMuv mutation. To differentiate between these alternatives,
we overexpressed lin-3 weakly using the syIs12 integrated transgene. syIs12 expresses
42
the EGF domain of lin-3 under the control of a heat-shock promoter (Katz et al., 1995).
At 20˚C in the absence of heat-shock, syIs12 did not cause a Muv phenotype (Table 2).
syIs12; lin-15B(n744) animals were mostly wild-type, with only a 1% penetrant Muv
defect, whereas syIs12; lin-15A(n767) animals exhibited a Muv defect with 40%
penetrance (Table 2). Thus, weak overexpression of lin-3 from the syIs12 transgene
was enhanced by a class A synMuv mutation but not by a class B synMuv mutation. By
contrast, lin-3(n4441) was enhanced much more strongly by class B synMuv mutations
than by class A synMuv mutations (Table 1). We conclude that lin-3(n4441) is a class A
synMuv specific allele of lin-3 and does not simply cause weak overexpression of lin-3.
lin-3(n4441) specifically prevents repression of lin-3
The class A and B synMuv genes redundantly repress expression of lin-3 mRNA
(Cui et al., 2006). To test if the lin-3(n4441) mutation affects lin-3 mRNA levels similarly
to other class A synMuv mutations, we assayed lin-3 mRNA levels using real-time RTPCR. As previously reported, the class B synMuv mutant lin-15B(n744) has wild-type
lin-3 levels (Figure 2B). The class A synMuv mutants lin-15A(n767) and lin-3(n4441)
both had slightly increased levels of lin-3 mRNA. The synMuv double mutants lin15AB(e1763) and lin-3(n4441); lin-15B(n744) had substantially increased lin-3 mRNA
levels (Figure 2B). Therefore, the lin-3(n4441) mutation behaves as a class A synMuv
mutation with respect to lin-3 mRNA repression.
The lin-3(n4441) mutation is located 211 bp upstream of the lin-3 transcriptional
start and is also 465 bp upstream of the predicted transcriptional start of the gene
F36H1.12, which is upstream of lin-3 in the opposite orientation (Figure 2A). To
determine if lin-3(n4441) or other synMuv mutations also affect expression of F36H1.12,
we assayed F36H1.12 mRNA levels by real-time RT-PCR. F36H1.12 mRNA levels
were roughly equivalent to those of the wild type in all possible single and double
mutant combinations involving lin-15A(n767), lin-3(n4441), and lin-15B(n744) (Figure
2C). Therefore, the synMuv proteins specifically repress lin-3 and do not establish a
broad domain of repression.
43
Expression pattern of lin-3 in wild-type animals and synMuv mutants
Although lin-3 is overexpressed in synMuv double mutants (Cui et al., 2006; also,
Figure 2), it is not known where this overexpression occurs. GFP- and LacZ-tagged lin-3
repetitive transgene arrays have been used as reporters for lin-3 expression (Chang et
al., 1999; Hill and Sternberg, 1992; Hwang and Sternberg, 2004), but these reporters
might not be appropriate for determining lin-3 expression in synMuv mutants: first, the
level of ectopic lin-3 expression might be too low to visualize using a GFP reporter;
second, many synMuv mutations affect the expression of repetitive transgene arrays,
potentially confounding interpretation of the expression pattern of such reporters (Hsieh
et al., 1999). Instead, we assayed lin-3 expression using a fluorescence in situ
hybridization (FISH) technique that has sufficient sensitivity to detect single mRNA
molecules (Raj et al., 2008).
We first determined the expression pattern of lin-3 in wild-type animals at the late
L2 to early L3 stage when vulval induction occurs. Previous studies found that at the
early L3 stage lin-3 is expressed in the anchor cell and in the pharynx (Hill and
Sternberg, 1992; Hwang and Sternberg, 2004). We indeed observed robust expression
of lin-3 in the anchor cell and throughout the pharynx. In addition, we saw expression of
lin-3 in the germline (Figure 3A). In some wild-type animals we also observed a few
copies of lin-3 mRNA in one or more cells in the tail, on the ventral side slightly anterior
to the anus. In addition, a few copies of lin-3 mRNA were seen on the ventral side of
the animal, slightly behind the posterior gonad arm. We imaged a single animal that was
slightly older, in the late L3 stage, and observed expression of several copies of lin-3
mRNA in the region where P6.p and its descendants are located (data not shown),
consistent with previous reports of expression of lin-3 in the descendants of P6.p by the
L4 stage (Chang et al., 1999). Other tissues typically expressed zero copies of lin-3
mRNA, although a few animals expressed a single lin-3 mRNA molecule elsewhere,
including one molecule in an intestinal cell (Figure 3A) and one molecule in either a
hypodermal or body wall muscle cell (data not shown). The restricted expression pattern
of lin-3 was not a result of some tissues being inaccessible to FISH probes, as probes
directed against ama-1 and eft-2 robustly detected mRNA in all cells (data not shown).
44
Overall, outside those tissues that highly expressed lin-3 there was very tight repression
of lin-3.
lin-15AB(e1763) animals expressed lin-3 in the pharynx, germline, and anchor
cell at levels grossly similar to those of wild-type animals. In addition there was
widespread ectopic expression of lin-3, with approximately 500 to 1000 ectopic copies
of lin-3 mRNA observed per animal (Figure 3D). This ectopic expression was much
weaker than the normal expression in the anchor cell; whereas approximately 30-40
copies of lin-3 mRNA were seen in the anchor cell in wild-type animals (Figure 3A and
data not shown), only one or a few copies of lin-3 mRNA were observed in most cells in
lin-15AB(e1763) mutants. Because we could not see cell boundaries, we could not
determine if every cell ectopically expressed lin-3, but there were no tissues that
appeared to lack ectopic lin-3 mRNA (data not shown). Cells around the perimeter of
the animal expressed lin-3 in the lin-15AB(e1763) mutant, consistent with ectopic
expression in the hypodermis (Figure 3D and data not shown). There were also many
ectopic lin-3 mRNA copies that clearly were not in the hypodermis (data not shown).
There was no region of the worm that did not exhibit at least some ectopic lin-3
expression.
We also determined lin-3 expression in lin-15A(n767) and lin-15B(n744) single
mutants. lin-15B(n744) animals had a lin-3 expression pattern similar to that of wildtype animals (Figure 3C). In lin-15B(n744) mutants there was a low level of ectopic lin-3
expression, with up to a total of approximately 10 ectopic lin-3 mRNA molecules per
animal, but lin-3 was still tightly repressed outside of the germline, anchor cell, and
pharynx. lin-15A(n767) animals exhibited broad ectopic expression of lin-3, but at a
much lower level than that of lin-15AB(e1763) animals (Figure 3B). An average of
approximately 50 copies of lin-3 mRNA were seen outside of the pharynx, germline, and
anchor cell in lin-15A(n767) animals. Unlike in lin-15AB(e1763) animals, in any given
lin-15A(n767) animal most cells did not have ectopic lin-3 expression. However, we
observed no obvious cell or tissue specificity to the ectopic expression among several
lin-15A(n767) animals. Rather, it appeared that in lin-15A(n767) animals lin-3 is globally
derepressed, but the level of derepression is much less than one copy per cell, as there
45
were hundreds of cells that normally did not express lin-3 and only about 50 ectopic lin3 mRNA molecules per lin-15A(n767) animal.
The lin-3(n4441) mutation could cause global derepression of lin-3 similarly to lin15A(n767), or it could affect lin-3 expression in a subset of tissues. We examined the
expression of lin-3 mRNA in lin-3(n4441) and lin-3(n4441); lin-15B(n744) animals. lin3(n4441) animals had broad but weak ectopic expression of lin-3, similar to lin15A(n767) animals (Figure 4A). lin-3(n4441); lin-15B(n744) animals exhibited ectopic
lin-3 expression in most if not all cells and were indistinguishable from lin-15AB(e1763)
animals (Figure 4B).
46
Discussion
lin-3 is the major target of the class A synMuv genes in vulval development
Identifying the biologically relevant targets of transcriptional regulators that
control development is a challenging problem. The synMuv genes encode putative
transcriptional repressors that prevent ectopic vulval development. Mutating a synMuv
binding site in a target gene might relieve repression of that target, and if that repression
were essential to prevent ectopic vulval development could cause a dominant synMuv
phenotype. We isolated a mutation in the lin-3 EGF gene that derepresses lin-3
transcription and causes a dominant class A synMuv phenotype. This finding
establishes that lin-3 is a functionally important target of the class A synMuv genes,
consistent with a previous report that lin-3 expression is repressed by the synMuv
genes and that double-stranded RNA directed against lin-3 can suppress the synMuv
phenotype (Cui et al., 2006). Importantly, the lin-3(n4441) mutation fully recapitulates
the class A synMuv phenotype with regard to vulval development and lin-3 expression
and causes a class A synMuv phenotype equivalent to that caused by alleles of the
strong class A synMuv genes lin-15A and lin-38. If the class A synMuv genes
repressed multiple targets to prevent ectopic vulval development, then a mutation that
abolished class A synMuv-mediated repression of lin-3 would recapitulate only partially
the class A synMuv phenotype. We conclude that lin-3 is likely to be the only key
biologically relevant target of the class A synMuv genes in vulval development.
The simplest interpretation of the effect of the lin-3(n4441) mutation is that this
mutation abolishes a binding site for a transcriptional repressor consisting of or
controlled by synMuv proteins. However, the effect of the lin-3(n4441) mutation is
slightly enhanced by mutations in all other class A synMuv genes. If the lin-3(n4441)
mutation completely inactivated a binding site that responds to only one of the known
class A synMuv proteins, then mutation of that class A synMuv gene should not
enhance the synMuv phenotype caused by lin-3(n4441). One possibility is that a
complex consisting of multiple class A synMuv proteins binds to the lin-3 promoter, the
lin-3(n4441) mutation strongly reduces but does not completely eliminate that binding,
47
and removing any one class A synMuv protein does not fully abrogate the ability of the
complex to bind to the lin-3 locus and repress transcription. This model is consistent
with the observation that most class A synMuv mutations, including lin-3(n4441), are
enhanced by class A synMuv mutations in other genes (Andersen et al., 2008). Many
of the class A synMuv genes encode proteins with domains, such as zinc fingers, that
are consistent with a role in transcriptional regulation (Clark et al., 1994; Davison et al.,
2003; Huang et al., 1994; Chapter Four). Alternatively, the class A synMuv genes might
regulate the expression or activity of another protein that binds to the lin-3 promoter to
prevent ectopic transcription.
The class A and B synMuv genes repress lin-3 by two distinct mechanisms
lin-3 expression in the germline had not been previously observed, likely because
the reporters used to assay lin-3 expression were either silenced in the germline (Kelly
et al., 1997) or lacked distant regulatory regions necessary to drive germline expression.
Mutations in the FOG and FBF translational inhibitor RNA-binding proteins cause a
germline-dependent Muv phenotype, and the FBF proteins can bind to the 3ʼ UTR of lin3 in vitro, suggesting that germline lin-3 mRNA is translationally repressed during the
larval stage when vulval induction occurs (Thompson et al., 2006). In many class B
synMuv mutants, somatic cells express normally germline-specific genes (Unhavaithaya
et al., 2002; Wang et al., 2005). Given our finding that lin-3 is normally expressed in the
germline, we propose that the class B synMuv genes repress ectopic lin-3 expression in
somatic cells as a consequence of their role in ensuring that somatic cells do not
inappropriately adopt germline-like fates. The class B synMuv genes might repress a
few master regulators of germline fates, which then promote the expression of germline
genes including lin-3. Alternatively, some or all class B synMuv genes might impart
somatic identities by directly repressing the expression of germline genes such as lin-3.
In class B synMuv single mutants, the somatic cells adopt a more germline-like fate that
would include lin-3 expression except that the class A synMuv proteins still tightly
repress lin-3, mostly preventing ectopic lin-3 expression. In class A synMuv single
mutants, lin-3 is not tightly repressed, but most somatic cells are not fated to express
48
lin-3, so there is only a low level of leaky ectopic lin-3 expression. However, in class AB
synMuv double mutants, the somatic cells adopt a germline-like fate that includes lin-3
expression, and there is no class A synMuv mechanism to tightly repress lin-3, resulting
in widespread and substantial ectopic lin-3 expression. In short, we propose that the
synthetic Muv phenotype caused by mutations in the synMuv genes is a consequence
of two distinct functions of the class A and class B synMuv genes: the class A synMuv
genes tightly repress ectopic lin-3 transcription, and the class B synMuv genes prevent
somatic expression of germline-expressed genes, which include lin-3; only if both
functions are lost will somatic cells ectopically express sufficient lin-3 mRNA to cause
ectopic vulval induction. These findings raise the possibility that the development of
some human tumors might require the loss of one tumor suppressor gene that prevents
cells from adopting a fate that is permissive for the expression of a growth factor and the
loss of a second tumor suppressor gene that specifically represses the expression of
that growth factor.
The synMuv genes repress lin-3 throughout the animal
In lin-15AB mutants, lin-3 is ectopically expressed throughout the animal in a
broad range of cells and tissues. Site-of-action experiments have shown that the
synMuv genes function at least in large part in the hyp7 hypodermal syncytium to
prevent ectopic vulval development (Myers and Greenwald, 2005). The expression
pattern of lin-3 in synMuv mutants does not directly address the site-of-action of synMuv
genes in regulating vulval development but does show that the synMuv genes function
throughout the animals to keep lin-3 very tightly repressed in numerous cells and
tissues. lin-3 EGF regulates other cell fates in C. elegans development, and at least
some of these fates, such as the P11/P12 fate, are also regulated by the synMuv genes
in a manner analogous to that of vulval development (Jiang and Sternberg, 1998). Thus,
the synMuv genes act throughout the animal to prevent ectopic lin-3 expression, which
can cause a variety of developmental abnormalities. Mutants with a displaced anchor
cell show that lin-3 can act at a significant distance (Thomas et al., 1990), so a cell
ectopically expressing lin-3 could affect fates in both nearby and distant cells. We
49
suggest that for any given cell-fate decision, the site of action of the synMuv genes is
likely to be spread across multiple cells and determined by the size and proximity of
those cells to the cell being regulated by lin-3. For vulval development, hyp7 plays the
major role, given its large size and close proximity to the Pn.p cells, with likely lesser
contributions from many other cells. The site at which the synMuv genes repress lin-3
to ensure proper vulval development is therefore probably a combination of the Pn.p
cells themselves and neighboring cells that do not normally either express or respond to
lin-3. This situation is similar to that in which both tumor cells and the microenvironment
surrounding the tumor provide factors that drive tumor development (Hu and Polyak,
2008). We suggest that analogously to the synMuv genes some tumor suppressor
genes function by repressing growth factor expression in both tumor cells and the
surrounding microenvironment.
Conclusion
In synMuv double mutants, lin-3 was ectopically expressed but at a much lower
level than at its major normal site of function, the anchor cell. Class A synMuv single
mutants exhibit low penetrance defects in vulval development, despite causing fewer
than one ectopic copy of lin-3 mRNA per cell. Furthermore, synMuv double mutants
that displayed a fully penetrant Muv defect had an average of only one to two ectopic
copies of lin-3 mRNA per cell. Thus, normal C. elegans development requires lin-3 to
be exceedingly tightly repressed outside of a few cells, and only slight expression of lin3 throughout the animal can cause abnormal cell-fate transformations. Such very low
levels of ectopic expression would likely be missed by most techniques used to assay
gene expression. We suggest it could be important to examine the expression of EGFfamily ligands in tumors using highly sensitive techniques with single-molecule
resolution to determine if broad low-level misexpression of EGF-family ligands plays a
role in oncogenic growth. In C. elegans, the tight repression of lin-3 EGF requires both
the class A synMuv gene pathway and the class B gene synMuv pathway, which
includes homologs of known tumor suppressor genes, such as lin-35 Rb. Therefore,
some tumor suppressor genes in mammals might function by tightly repressing low-level
50
ectopic expression of EGF-family ligands in many cells, possibly in both the tumor and
the microenvironment surrounding the tumor.
Acknowledgments
We thank Dave Harris for helpful comments concerning this manuscript, and
Beth Castor, Elissa Murphy and Rita Droste for technical assistance. Some nematode
strains used in this work were provided by the Caenorhabditis Genetics Center, which is
funded by the NIH National Center for Research Resources (NCRR). H.R.H. is the
David H. Koch Professor of Biology at MIT and an Investigator of the Howard Hughes
Medical Institute. This work was supported by NIH grant GM24663. A.v.O. was
supported by an NIH Pioneer Award (1DP1OD003936).
51
Table 1: lin-3(n4441) causes a dominant class A synMuv phenotype
genotype
wild-type
lin-3(n4441)
lin-3(n4441)/+b
lin-15B(n744)
lin-3(n4441); lin-15B(n744)
lin-3(n4441)/+; lin-15B(n744)c
lin-52(n771)
lin-3(n4441); lin-52(n771)
lin-61(n3447)
lin-61(n3447); lin-3(n4441)
lin-8(n2731)
lin-8(n2731); lin-3(n4441)
lin-15A(n767)
lin-3(n4441); lin-15A(n767)
lin-38(n751)
lin-38(n751); lin-3(n4441)
lin-56(n2728)
lin-56(n2728); lin-3(n4441)
a
% multivulva ± s.d. (n)a
20ºC
25˚ C
0±0
(1422)
0 ± 0 (1368)
0±0
(954)
1 ± 0.4 (1105)
0±0
(621)
1 ± 2 (830)
0±0
(1058)
0 ± 0 (642)
100 ± 0 (729)
100 ± 0 (248)
100 ± 0 (795)
100 ± 0 (152)
0±0
(1029)
0 ± 0 (1233)
98 ± 0.1 (974)
100 ± 0 (1244)
0±0
(1039)
0 ± 0 (986)
17 ± 13 (778)
100 ± 0 (1201)
0±0
(902)
0 ± 0 (910)
0.4 ± 0.5 (1093)
1 ± 2 (788)
0.1 ± 0.2 (1022)
2 ± 1 (1009)
5±3
(831)
60 ± 17 (609)
0±0
(947)
1 ± 1 (1307)
1±2
(1028)
12 ± 3 (889)
0 ±0
(932)
1 ± 1 (733)
0.3 ± 0.3 (930)
6 ± 4 (843)
Animals were scored as Muv if any ventral ectopic protrusions were observed. For
each strain, animals were scored on three separate days, and % multivulva shown is
the average for those three days. SD, standard deviation. n, total number of animals
scored.
b
These animals were also heterozygous for dpy-17(e164) and unc-32(e189) and
descended from lin-3(n4441) homozygous parents.
c
These animals were also heterozygous for dpy-5(e61) and descended from
dpy-5(e61); lin-3(n4441); lin-15B(n744) parents.
52
Table 2: lin-3 overexpression is enhanced more
strongly by a class A synMuv mutation than by a
class B synMuv mutation
genotype
% multivulva (n)a
syIs12b
0 (92)
syIs12; lin-15A(n767)
38 (105)
syIs12; lin-15B(n744)
1 (100)
a
Animals were scored as Muv if any ventral ectopic protrusions were observed. n, total
number of animals scored.
b
syIs12 is an integrated transgene expressing the lin-3 EGF domain under the control of
a heat-shock promoter (Katz et al., 1995). Animals were assayed in the absence of
heat-shock.
53
Figure 1: n4441 is an allele of lin-3.
(A) Genetic map showing n4441 on LGIV. n4441 was localized between dpy-13 and
unc-30 by three-factor mapping. SNP mapping using polymorphisms present in the
CB4856 strain further localized n4441 to a 661 kb region between the SNPs dbP6 at
10909553 and uCE4-1148 at 11570158 of LGIV (data not shown).
(B) The lin-3 locus. The lin-3a isoform (Wormbase web site, http://www.wormbase.org,
release WS200, Mar 20 2009) is shown. Solid boxes, exons; open boxes, UTRs. The
predicted start of transcription is indicated by an arrow. Arrowheads indicate the
locations of mutations. n4951 is a nonsense mutation that truncates LIN-3 after 26
amino acids, and n4929 is a missense mutation that converts an arginine to a lysine at
amino acid 347.
(C) n4441 is a G-to-A mutation at nucleotide 30904 of cosmid F36H1, approximately
200 bp upstream of the start of the lin-3 transcript. No other mutations were present in
n4441 mutants in the region shown in (B).
(D) A lin-3 loss-of-function mutation suppresses the n4441 synMuv phenotype in cis but
not in trans. lin-3(n1059) is a nonsense mutation in lin-3 (Liu et al., 1999). lin-3(n4441
n4951) and lin-3(n4441 n4929) heterozygotes also carried the nT1[qIs51] translocation.
All animals were grown at 20˚C. Animals were scored as Muv if any ventral ectopic
protrusions were observed. n, total number of animals scored.
54
Figure 1
A
n4441 interval (661 kb)
dpy-13
dbP6
unc-30
lin-3 uCE4-1148
LG IV
B
W27amber
n4951
R347K
n4929
1 kb
n4441
C
GAATTTTGAA
GATGTTGCGAC
n4441
G A
D
Genotype
% Muv (n)
lin-3(n4441 n4951)/+; lin-15B(n744)
0 % (461)
lin-3(n4441 n4929); lin-15B(n744)
3 % (76)
lin-3(n4441 n4929)/+; lin-15B(n744)
1 % (163)
lin-3(n4441)/lin-3(n1059); lin-15B(n744)
99 % (472)
Figure 2: The lin-3(n4441) mutation specifically prevents repression of lin-3
(A) The lin-3(n4441) mutation is located 211 bp upstream of the start of lin-3
transcription. The gene F36H1.12 is upstream of lin-3 in the opposite orientation, and
lin-3(n4441) is located 465 bp from the predicted start of F36H1.12 transcription. Solid
boxes, exons; open boxes, UTRs.
(B) lin-3 mRNA levels in lin-3(n4441) single and double mutants. As reported previously
(Cui et al., 2006), lin-3 mRNA levels are substantially increased in lin-15AB double
mutants but not in lin-15A or lin-15B single mutants. Like other class A synMuv
mutations, lin-3(n4441) caused a substantial increase in lin-3 mRNA levels only in a
class B synMuv mutant background. Realtime RT-PCR experiments were performed
using RNA harvested at the late L2 or early L3 stage from each strain shown. Relative
lin-3 mRNA levels were normalized to the levels of mRNA encoding the ribosomal
protein subunit rpl-26 using the ∆∆Ct method (Schmittgen and Livak, 2008). The means
and standard deviations of relative lin-3 mRNA levels from two independent trials are
shown. The lin-15A(n767), lin-15B(n744) and lin-15AB(e1763) alleles were used in this
experiment.
(C) F36H1.12 mRNA levels in synMuv single and double mutants. No combination of
lin-3(n4441), lin-15A, and lin-15B mutations affected F36H1.12 mRNA levels. Realtime
RT-PCR experiments were performed using RNA harvested at the late L2 or early L3
stage from each strain shown. Relative F36H1.12 mRNA levels were normalized to the
levels of mRNA encoding the ribosomal protein subunit rpl-26 using the ∆∆Ct method.
The means and standard deviations of relative F36H1.12 mRNA levels from two
independent trials are shown.
56
Figure 2
A
F36H1.12
lin-3
lin-3(n4441)
relative lin-3 expression
B
10
9
8
7
6
5
4
3
2
1
0
relative F36H1.12 expression
C
10
9
8
7
6
5
4
3
2
1
0
500 bp
Figure 3: The synMuv genes prevent widespread ectopic expression of lin-3
mRNA.
FISH of lin-3 mRNA in late L2 to early L3 animals. Each dot represents a single mRNA
molecule (Raj et al., 2008). lin-3 mRNAs are shown in red, and 4′,6-diamidino-2phenylindole (DAPI) staining of nuclei is shown in blue. The images shown are
maximum intensity projections of a z-stack of images. The anchor cell (AC) is indicated
by an arrowhead in each panel.
(A) Wild type. lin-3 mRNA is expressed in the pharynx, anchor cell, and germline and is
tightly repressed elsewhere.
(B) lin-15A(n767). There is a low level of ectopic lin-3 expression, with approximately
60 ectopic copies of lin-3 mRNA seen outside the pharynx, anchor cell, and germline.
(C) lin-15B(n744). There is a very low level of ectopic lin-3 expression, with
approximately 10 ectopic copies of lin-3 mRNA seen outside the pharynx, anchor cell,
and germline.
(D) lin-15AB(e1763). lin-3 is ectopically expressed throughout the animal, with
approximately 900 ectopic copies of lin-3 mRNA seen outside the pharynx, anchor cell,
and germline.
58
Figure 3
A
germline
pharynx
D
A
P
V
wild type
B
lin-15A(n767)
C
lin-15B(n744)
D
lin-15AB(e1763)
Figure 4: The lin-3(n4441) mutation causes widespread ectopic expression of lin3 mRNA.
FISH of lin-3 mRNA in late L2 to early L3 animals. Each dot represents a single mRNA
molecule (Raj et al., 2008). lin-3 mRNAs are shown in red, and 4′,6-diamidino-2phenylindole (DAPI) staining of nuclei is shown in blue. The images shown are
maximum intensity projections of a z-stack of images.
(A) lin-3(n4441). there is a low level of ectopic lin-3 expression, with approximately 45
ectopic copies of lin-3 mRNA seen outside the pharynx, anchor cell, and germline.
(B) lin-3(n4441); lin-15B(n744). lin-3 is ectopically expressed throughout the animal,
with approximately 1000 ectopic copies of lin-3 mRNA seen outside the pharynx, anchor
cell, and germline.
60
Figure 4
A
lin-3(n4441)
B
D
A
P
V
lin-3(n4441); lin-15B(n744)
Supplemental Table 1: Oligonucleotides in the lin-3 in situ hybridization probe
CAAGCCGACGATCCGTTATT
AATTGATGCATAAGGGGTGG
GCGACGATATCTTATGAAAC
CCGCATTTTGGAAACTTGTG
GAGGCATAAAGAGTAGAAGG
GGGAGACACGATTCTGTAAA
TACGTTCTTGACGAAACCAC
TCTGCAGATTGAAGCTGTTC
GCCACTATTTTCAGCTGCAT
ATTTCGAGAAGTATCGGGTG
GGTGCATCACCTATTTCGTT
TAGGTGTTTCAGGTGTCGAA
GCTTCGGAAATCGTAGTTTC
GTTCGTTTTTCATCGTCTCC
CACCCTCATATTCTGCTTCT
CTTCTACTTCTTCATCAACC
CATCTTGAGTGGCATCTTCA
TTCTCGATTTCCTTCCGAAC
GTGATGACAGTAGTCTTTGC
ATATCACTTCCACGTGGCAT
CTGAAACTCTATCTTCACGG
CAATGGCAAGAAGGAACAAC
ACGATCACAACGAGTGCCTT
ATAGAACGCCTGAACGTAGT
TATATCTGCCGTTGATTGGG
TCATCGTGCTCAAACGTACA
TTTCCTTGAACGAGGAGTTG
GGCGTTGTAGAAATCGTTTG
CTCTCATAAAGCCCACTGAA
GCTGATGTTGAAGATTGCAC
AGAAATGCGAACGCAGGAAT
GGGCACATATGACTCATTGT
AGGACATTGAATGCTTCTGG
GGAATATGTCGTCCATTTGG
CGGTGTTGGGATAGTATAAG
CTTGATCCAGGAGTTGATGA
AATAGCTTGTTGACGAGTGG
GTTGTTCCGTGCTTGTTCAT
CTTGACTTCTGAGAATGCTG
TACTCCTGGATGGAATGGTA
ACTGACTTGTAGTGCTTCGG
TGCTGAAACTTCAACACGTG
CTTCTGATTCAGTCGACTGA
GTGGACATGTTACTGATGCT
ATTAATTACAGTGTGCGCCG
ATCTGCAGAATCCAACTCGA
TGTTCTCCAGAACTTCGAGA
TTCACATGTTGCTGGTGATC
62
Chapter Three
Multiple levels of redundant processes inhibit C. elegans vulval cell fates
Erik C. Andersen, Adam M. Saffer and H. Robert Horvitz
Chapter Three: Multiple levels of redundant processes inhibit C. elegans vulval
cell fates
I constructed and scored all class A synMuv single mutants, class AA double mutants
and class AAB triple mutants. Erik Andersen constructed and scored all class B
synMuv single mutants, class BB double mutants, and class BBA triple mutants. I
performed all real-time RT-PCR experiments. Erik wrote the manuscript and I helped
edit it.
This manuscript was published as Andersen et al. (2008) Genetics 179: 2001-2012
63
Summary
Many mutations cause obvious abnormalities only when combined with other
mutations. Such synthetic interactions can be the result of redundant gene functions. In
Caenorhabditis elegans, the synthetic multivulva (synMuv) genes have been grouped
into multiple classes that redundantly inhibit vulval cell fates. Animals with one or more
mutations of the same class undergo wild-type vulval development, whereas animals
with mutations of any two classes have a multivulva phenotype. By varying temperature
and genetic background, we determined that mutations in most synMuv genes within a
single synMuv class enhance each other. However, in a few cases no enhancement
was observed. For example, mutations that affect an Mi2 homolog and a histone
methyltransferase are of the same class and do not show enhancement. We suggest
that such sets of genes function together in vivo and in at least some cases encode
proteins that interact physically. The approach of genetic enhancement can be applied
more broadly to identify potential protein complexes as well as redundant processes or
pathways. Many synMuv genes are evolutionarily conserved, and the genetic
relationships we have identified might define the functions not only of synMuv genes in
C. elegans but also of their homologs in other organisms.
64
Introduction
Global analyses of loss-of-function mutants have revealed that many null
mutations do not cause obvious phenotypic abnormalities (Fraser et al., 2000; Kamath
et al., 2003; Park and Horvitz, 1986; Winzeler et al., 1999). In some cases, the wild-type
phenotypes of such mutants result from genetic redundancy, which has been suggested
to confer phenotypic robustness to developmental and behavioral processes (Harrison
et al., 2007b; Sieber et al., 2007). Genetic redundancy can be studied using synthetic
interactions, in which an animal mutant in two genes displays a phenotype that is not
seen in either single mutant. Genes that display such a synthetic interaction are typically
thought to act in separate processes or pathways to mediate the same broad biological
function. Numerous synthetic-lethal interactions have been identified in S. cerevisiae
(Ooi et al., 2003; Tong et al., 2001; Tong et al., 2004). In the nematode
Caenorhabditis elegans, mutations in the synthetic multivulva (synMuv) genes cause
synthetic defects in the development of the hermaphrodite vulva (Ferguson and Horvitz,
1989).
In C. elegans, three of six equipotent progenitor cells are specified to adopt vulval
cell fates by three cell-signaling cascades involving receptor tyrosine kinase (RTK)/Ras,
Notch and Wnt pathways (Sternberg, 2006; Sundaram, 2005). The three cells that adopt
vulval fates divide several times and give rise to the vulva. The remaining three cells
divide once, and their progeny adopt non-vulval fates and fuse with the underlying
hypodermis (Sulston and Horvitz, 1977). The synMuv genes prevent the adoption of
ectopic vulval cell fates (Ferguson and Horvitz, 1989).
The synMuv genes originally were grouped into two classes, A and B; animals
mutant for one or more genes within the same synMuv class do not have vulval
abnormalities, but a multivulva (Muv) phenotype is observed in class A-B double
mutants (Ferguson and Horvitz, 1989). This synthetic interaction indicates that the class
A and class B synMuv genes act in two redundant pathways or processes to inhibit the
adoption of vulval cell fates. The class A synMuv genes encode nuclear proteins
proposed to regulate transcription (Clark et al., 1994; Davison et al., 2005; Huang et al.,
1994). Many class B synMuv genes encode homologs of regulators of transcription and
65
chromatin, including a Nucleosome Remodeling and Deacetylase (NuRD) complex
(Solari and Ahringer, 2000; Unhavaithaya et al., 2002), an Rb/E2F4/DP complex (Ceol
and Horvitz, 2001; Lu and Horvitz, 1998), two histone methyltransferases (Andersen
and Horvitz, 2007), heterochromatin protein 1 (HP1) (Couteau et al., 2002) and the
DP/Rb/MuvB (DRM) complex (Harrison et al., 2006). Ceol and Horvitz (2004) proposed
the existence of a third class of synMuv gene, called C. These genes encode proteins
similar to members of the Tip60/NuA4 histone acetyltransferase complex. In this
manuscript, we show that these putative class C synMuv genes are not distinct from
and should be considered class B synMuv genes. The gene lin-3, which encodes the
EGF-like ligand of the RTK/Ras pathway required for the specification of vulval cell fates
(Hill and Sternberg, 1992), is an apparent direct or indirect transcriptional target of at
least some synMuv genes (Cui et al., 2006a). Given this observation and the molecular
nature of many synMuv proteins, the synMuv proteins likely act as transcriptional
repressors to prevent the ectopic expression of the RTK/Ras pathway ligand LIN-3 and
in this way prevent ectopic vulval induction.
To date, there are at least four class A synMuv genes and 25 class B synMuv
genes (Andersen and Horvitz, 2007; Ceol et al., 2006; Couteau et al., 2002; Dufourcq et
al., 2002; Ferguson and Horvitz, 1989; Hsieh et al., 1999; Lu and Horvitz, 1998; Poulin
et al., 2005; Thomas et al., 2003; Tseng et al., 2007; Unhavaithaya et al., 2002; von
Zelewsky et al., 2000). Two class B synMuv genes, met-1 and met-2, act redundantly
with each other to inhibit vulval cell fates (Andersen and Horvitz, 2007), and the Muv
phenotypes caused by mutations in the class B synMuv genes let-418 and hpl-2 are
enhanced by mutations in other class B synMuv genes (Andersen and Horvitz, 2007;
Couteau et al., 2002; von Zelewsky et al., 2000). These genetic interactions along with
the molecular identities of the class B synMuv proteins led us to hypothesize that the
class B genes do not act in a single pathway or process to inhibit vulval cell-fate
specification.
By scoring the vulval phenotypes of strains sensitized using temperature and
genetic background, we sought to determine whether genes within each of the two
synMuv classes act together in the same pathway or process. In these assays, we
66
compared the Muv phenotypes caused by single mutants with the Muv phenotypes
caused by double mutants within the same synMuv class. We found that most but not all
synMuv genes within a single class act in separate pathways or processes, as indicated
by the more penetrant Muv phenotypes of double mutants within a class than those of
the respective single mutants. The class C synMuv genes were originally postulated to
define a separate class from the class B synMuv genes, because the putative class C
mutations were weakly redundant with class B synMuv mutations. However, as our
results show that nearly all class B synMuv genes are redundant with each other, the
putative class C synMuv genes are not different from the class B synMuv genes. For the
rest of this paper, we consider there to be only two synMuv classes, A and B.
In a few cases, we found that two genes within the same class did not show
enhancement and hence might act in the same pathway or process. In several of these
cases, the proteins encoded by these genes have been identified biochemically to be in
a complex. We believe that such lack of genetic redundancy can identify sets of genes
that mediate the same molecular function. Additionally, we showed that many synMuv
proteins repress lin-3 transcription, and that the redundancy observed in the vulval
phenotype between class B synMuv genes likely results from an increase in lin-3
expression.
67
Materials and Methods
Strains and genetics
C. elegans was cultured on the bacterial strain OP50 as described (Brenner,
1974). N2 was the wild-type strain. Mutant alleles used are listed below and in Table 1.
Many alleles were described previously (Riddle, 1997) unless otherwise noted:
LGI: lin-35(n745) (Lu and Horvitz, 1998), lin-53(n833) (Lu and Horvitz, 1998), lin61(n3447, n3809) (Harrison et al., 2007a), met-1(n4337) (Andersen and Horvitz, 2007).
LGII: lin-8(n2731) (Thomas et al., 2003), dpl-1(n3316) (Ceol and Horvitz, 2001),
trr-1(n3712) (Ceol and Horvitz, 2004), lin-56(n2728) (Thomas et al., 2003), lin-38(n751).
LGIII: lin-37(n4903) (Andersen et al., 2006), met-2(n4256) (Andersen and Horvitz,
2007), lin-9(n112) (Beitel et al., 2000), lin-52(n771) (Thomas et al., 2003), lin-36(n766)
(Thomas and Horvitz, 1999).
LGIV: ark-1(sy247) (Hopper et al., 2000).
LGV: let-418(n3536) (Ceol et al., 2006), hda-1(e1795) (Dufourcq et al., 2002),
mys-1(n3681) (Ceol and Horvitz, 2004).
LGX: lin-15B(n744), lin-15A(n433, n767), lin-15AB(e1763) (Ferguson and Horvitz,
1985).
The following balancer chromosomes were used: mIn1 [mIs14 dpy-10(e128)] (Edgley
and Riddle, 2001) hT2 [qIs48], nT1 [qIs51] (Mathies et al., 2003) and qC1 [nIs189]
(Andersen et al., 2006).
RNAi analyses
RNAi of efl-1 was performed by injection using clone yk617e4 to synthesize
dsRNA, as previously described (Andersen et al., 2006).
Scoring the vulval cell fate
Using a dissecting microscope, we scored the vulval phenotypes of each strain
from at least three and in many cases nine independently grown cultures. At least 100
68
animals were scored for each genotype. Animals were scored as Muv if one or more
ectopic ventral protrusions were observed. We scored vulval induction of a few strains
using Nomarski optics and found the frequencies of ectopic inductions of vulval cell
fates to be equivalent to the frequencies of the Muv phenotypes observed using a
dissecting microscope. The penetrance of the Muv phenotype was calculated as the
fraction of total animals that were Muv multiplied by 100. The average percent Muv and
standard deviation of the replicates were calculated. A strain was scored as enhanced
when the Muv phenotype penetrance of the double mutant was more than one standard
deviation greater than the Muv phenotype penetrances of the respective single mutants.
In some cases, enhancement was observed only when comparing the Muv phenotypes
of double mutants to the respective single mutants at one of a number of temperatures
tested.
Quantitative PCR assay
Synchronized wild-type and mutant animals were grown, and larvae were
harvested at or near the L2-to-L3 larval transition, when vulval induction occurs. Total
RNA was extracted using Trizol (Invitrogen). First-strand cDNA was prepared from 1 µg
total RNA using the SuperScript III First-Strand Synthesis Supermix for qRT-PCR
(Invitrogen). Each real-time reverse transcriptase (RT) PCR mix contained 10 ng of RT
products, SyBR Green PCR Master Mix (Applied Biosystems) and 0.4 µM of each
primer. The real-time PCR was performed in triplicate on an Eppendorf Mastercycler ep
realplex2. Two or three independent samples of each genotype were prepared, and
levels of lin-3 and rpl-26 were quantified from each biological replicate. The #CT values
for lin-3 were determined using rpl-26 as the internal reference, and the ##CT values
were calculated for each genotype by comparison with the wild type. All changes were
normalized to the wild type. The error shown is the standard deviation of relative
lin-3/rpl-26 ratios for the biological replicates.
69
Results
To assess whether different synMuv genes function in the same process, we
combined null or strong loss-of-function mutations (Table 1) and quantified the
penetrance of the phenotype as compared to the phenotype of either single mutant. For
simplicity below, we will use the word “process” instead of the phrase “pathway or
process” to describe how groups of gene act together, but in each case such genes
could act simultaneously (e.g., by encoding members of a protein complex) or
sequentially (e.g., in a linear pathway). If a double synMuv mutant had a Muv phenotype
equivalent in penetrance to that of either of the single mutants, we concluded that the
two genes could act in the same process. By contrast, if a double synMuv mutant had a
Muv phenotype that was higher in penetrance than that of either single mutant, we
concluded that the two genes function in separate processes. The only way to score an
increase in penetrance was to score strains in which the penetrance of the Muv
phenotype is incomplete. We used both temperature and the addition of partial loss-offunction alleles of synMuv genes to sensitize the vulval phenotype to incomplete
penetrances, increasing our ability to detect enhancement.
Temperature sensitizes the vulval phenotype
As observed previously (Andersen and Horvitz, 2007; Beitel et al., 2000; Ceol
and Horvitz, 2001; Ceol et al., 2006; Clark et al., 1994; Ferguson and Horvitz, 1989;
Harrison et al., 2007a; Hsieh et al., 1999; Lu and Horvitz, 1998; Thomas et al., 2003;
Unhavaithaya et al., 2002; von Zelewsky et al., 2000), single null mutations of synMuv
genes did not cause a Muv phenotype at 20ºC or 25ºC, but class A mutations combined
with class B mutations caused Muv phenotypes. The Muv phenotype caused by synMuv
mutations is temperature-sensitive: at higher temperatures the penetrance of the Muv
phenotype increases for all tested synMuv double mutants (Ferguson and Horvitz,
1989) and see below. For example, the penetrance of the lin-61(n3447); lin-56(n2728)
Muv phenotype increased from 5% at 20ºC to 81% at 22.5ºC, and the penetrance of the
lin-35(n745); lin-15A(n433) Muv phenotype increased from 11% at 15ºC to 100% at
20ºC. Both lin-56(n2728) and lin-35(n745) are presumptive null alleles, and some
70
synMuv class A-B null double mutants have been shown to have temperature-sensitive
Muv phenotypes (Andersen and Horvitz, 2007; Ceol and Horvitz, 2004; Ceol et al.,
2006). Therefore, it is likely that it is the synMuv process and not the synMuv gene
products that is temperature-sensitive. Most synMuv double mutants have more
penetrant Muv phenotypes at high temperatures and incompletely penetrant Muv
phenotypes at low temperatures.
Partial loss-of-function mutations in class A or class B synMuv genes can
sensitize the vulval phenotype
In addition to temperature, genetic background can sensitize the vulval
phenotype of synMuv mutant strains. To seek redundancy between class A genes, we
partially inhibited a class B synMuv gene using the missense mutation lin-61(n3447)
(Harrison et al. 2007a), which causes an incompletely penetrant synMuv defect in
combination with strong or null class A mutations. This incompletely penetrant Muv
phenotype might be enhanced by other null or strong class A mutations if the genes act
in separate processes to inhibit vulval fates. To seek redundancy between class B
genes, we partially inhibited a class A synMuv gene using the missense mutation
lin-15A(n433), which causes a weak class A synMuv defect (Clark et al., 1994). The
Muv phenotype caused by a partial loss of lin-15A function in combination with a null
class B synMuv mutation might be enhanced by other null or strong class B mutations if
these two class B synMuv genes act in separate process to inhibit vulval fates.
Most class A-A double mutants have Muv phenotypes
To look for enhancement, we scored strains at temperatures in which the Muv
phenotypes were less than 100% penetrant and phenotypic enhancement could be
quantified easily. We observed the vulval phenotypes of animals with null mutations in
lin-8, lin-15A and lin-56 (Table 1). We used a partial loss-of-function allele of lin-38,
because a null mutation caused larval lethality, which precluded scoring of the vulval
phenotype (A.M.S. and H.R.H., unpublished results). Whereas class A single mutants
do not have Muv phenotypes at 20ºC (Davison et al., 2005; Ferguson and Horvitz, 1989;
71
Thomas et al., 2003), some class A synMuv mutations caused very weak Muv
phenotypes at 25ºC, with less than 1% penetrance (Table 2). We also observed the
vulval phenotypes of class A-A double mutants (Table 2). Most strains were not Muv at
20ºC, but many had Muv phenotypes at 25ºC, ranging from 1% to 33% penetrance.
Only the class A-A double mutants lin-8 lin-56 and lin-56; lin-15A did not have a Muv
phenotype greater than that of either single mutant at 25ºC.
Therefore, not only do the class A and B synMuv genes act in separate
processes, but, given these results, most class A synMuv genes can act in processes
separate from those of other class A synMuv genes. Given that lin-8 lin-56 and
lin-56; lin-15A double mutants were not enhanced when compared to their respective
single mutants, the two genes in each of these gene pairs might act in the same
process. Alternatively, they might act in different processes, with the decrease in
synMuv gene function in these experiments being insufficient to reach the threshold
necessary to cause a Muv phenotype, just as certain synMuv gene double mutants
were Muv only if strong loss-of-function alleles were used, e.g., lin-8(n2741);
lin-15B(n2245) was 1% Muv at 15ºC, while lin-8(n2376); lin-15B(n2245) was 98% Muv
at 15ºC (Davison et al., 2005). Because the lin-8; lin-15A double mutant had an
enhanced vulval phenotype, indicating that lin-8 and lin-15A act in separate processes,
one or both of the class A-A synMuv gene pairs lin-8 lin-56 and lin-56; lin-15A must act
in parallel.
lin-15A and lin-56 act in the same process to inhibit the specification of vulval cell
fates
For class A-A double mutants that contained at least one null mutation and for
which no enhancement was observed, either the two synMuv genes act in the same
process, or they act in different processes and loss of function of both genes did not
cause sufficient loss of inhibition of vulval cell fates to result in a Muv phenotype.
Addition of a partial loss-of-function mutation in a synMuv gene from a different synMuv
class could allow the detection of subtle redundancy by increasing the loss of inhibition
72
of vulval fates to result in a Muv phenotype. We combined class A single or class A-A
double mutations with the class B synMuv mutation lin-61(n3447).
We found that single class A synMuv mutations in combination with lin-61(n3447)
caused Muv phenotypes at 20ºC and 22.5ºC (Table 3). In a lin-61(n3447) background,
the Muv phenotypes caused by lin-15A or lin-38 mutations were more penetrant than
those caused by lin-8 or lin-56, indicating that lin-15A and lin-38 cause a stronger
inhibition of vulval cell fates. Because lin-8 and lin-56 mutations each cause a relatively
weak inhibition of vulval cell fates, the lack of enhancement observed in lin-8 lin-56 and
lin-56; lin-15A double mutants at 25ºC could be because neither class A-A double
mutant had sufficient loss of vulval cell-fate inhibition to cause a Muv phenotype.
To address possible redundancy between the class A synMuv genes more
sensitively, we constructed triple mutants with two class A synMuv genes and the class
B synMuv gene lin-61 (Table 3). The group of class A-A mutants that with an added
lin-61 mutation had Muv phenotypes greater than that of each respective class A; lin-61
double mutant included the group of class A-A double mutants that had Muv
phenotypes at 25ºC; thus, the two class A synMuv genes in each of these double
mutant combinations were shown to act in separate processes by two different
observations. The Muv phenotype of the lin-61; lin-8 lin-56 triple mutant was stronger
than the Muv phenotypes of either lin-61; lin-8 or lin-61; lin-56 animals at 20ºC and
22.5ºC. Therefore, lin-8 and lin-56 act in different processes. By contrast, the Muv
phenotype of the lin-61; lin-56; lin-15A triple mutant was not stronger than lin-61; lin-56
or lin-61; lin-15A. Because all other class A; lin-15A; lin-61 and class A; lin-56; lin-61
triple mutants had Muv phenotypes enhanced as compared to the respective class A;
lin-61 double mutants, the lack of enhancement observed in the lin-61; lin-56; lin-15A
triple mutant suggests that lin-56 and lin-15A act in the same process.
Most class B-B double mutants do not have Muv phenotypes
We also assayed redundancy among the class B synMuv genes using
temperature to sensitize the vulval cell-fate decision. We used null mutations of the
class B synMuv genes dpl-1, lin-35, lin-61, lin-37, met-1, met-2 and trr-1 (Table 1). We
73
used partial loss-of-function alleles of let-418, lin-52 and mys-1, because null alleles
cause larval lethality or sterility. We studied the pairwise interactions among this set of
10 class B synMuv genes comprehensively. We did not test all 378 pairwise interactions
involving all 28 class B genes. Unlike the class A genes, most class B single and class
B-B double synMuv mutants did not have Muv phenotypes at 20ºC or 25ºC (Table 4).
The exceptions were let-418(n3536) and trr-1(n3712), each of which caused very low
penetrance Muv defects as single mutants. Many class B-B double mutants had larvallethal phenotypes at 25ºC, precluding scoring vulval phenotypes at high temperature.
These larval-lethal phenotypes indicate that in the control of viability the class B synMuv
genes act redundantly, but this redundancy might not be indicative of functions in vulval
development. Also, the class B-B double null mutants lin-61(n3809) lin-35(n745) and
lin-61(n3809); lin-37(n4903) had very low penetrance Muv phenotypes. Because most
class B-B double mutants were not appreciably Muv at high temperature, the class B
synMuv genes might all act in the same process. Given that there are a number of
examples of redundancy within the class B synMuv genes (Andersen and Horvitz, 2007;
Couteau et al., 2002; von Zelewsky et al., 2000), it is more likely that many of these
genes act in separate processes, and the loss of inhibition of vulval cell fates caused by
the class B synMuv mutations was insufficient to reach the threshold necessary to
cause a Muv phenotype.
Most class B synMuv genes act redundantly with genes of the same class to
inhibit vulval cell fates
To observe subtle redundancies among the class B synMuv genes and to assess
the relative input of each of the class B genes into vulval cell-fate inhibition, we
combined class B single and class B-B double mutants with the weak class A mutation
lin-15A(n433) (Tables 5 and 6). We tested many class B genes with viable null
phenotypes. dpl-1, lin-35, lin-37 and met-2 caused the strongest inhibition of vulval cell
fates, as mutants in each of these genes had highly penetrant Muv phenotypes at
17.5ºC and 20ºC in combination with lin-15A(n433). Mutants in lin-61, lin-52, let-418,
met-1, mys-1 and trr-1 did not have appreciably Muv phenotypes at 17.5ºC or 20ºC in
74
combination with lin-15A(n433), suggesting that these genes caused a weaker inhibition
of vulval cell fates.
We found using a sensitized genetic background that most class B genes act in
separate processes to inhibit vulval cell fates (Tables 5 and 6). In combination with the
weak class A mutation lin-15A(n433), most combinations of class B-B double mutants
had more highly penetrant Muv phenotypes than those found in the respective single
mutants. This difference was observed readily in strong double mutant combinations at
15ºC, intermediate combinations at 17.5ºC and weak combinations at 20ºC. For
example three weak synMuv genes, let-418, lin-52 and met-1, show insignificant
enhancement at 17.5ºC, but at 20ºC enhancement is observed between let-418 and
lin-52 but not between let-418 and met-1.
Some synMuv genes within the same class function non-redundantly to inhibit
vulval cell fates
Several class B-B double mutants had Muv phenotypes that were not enhanced
as compared to the single class B mutants in a lin-15A(n433) background, indicating
that these gene pairs likely act in the same process to inhibit vulval cell fates (Tables 5
and 6). Specifically, three pairs of genes — lin-35 and dpl-1; let-418 and met-1; and
mys-1 and trr-1 — did not show synthetic enhancement in a lin-15A(n433) background.
This lack of genetic enhancement was detected even though these combinations
involve alleles that in other combinations are sufficiently strong to cause Muv
phenotypes. We suggest that our findings identify sets of genes that act nonredundantly, i.e. in the same processes.
lin-35 and dpl-1 encode the C. elegans homologs of human Rb and DP,
respectively. Rb mediates transcriptional repression of the genes bound by the
heterodimeric transcription factor E2F, which is composed of the proteins DP and E2F
(Krek et al., 1993). In C. elegans, DPL-1 DP and EFL-1 E2F interact in vitro (Ceol and
Horvitz, 2001), and their homologs interact both in vitro and in vivo in other organisms
(Bandara et al., 1993; Helin et al., 1993; Krek et al., 1993). We tested whether RNAi of
the C. elegans homolog of E2F, efl-1 (Ceol and Horvitz, 2001), would enhance the Muv
75
phenotypes of lin-35; lin-15A and dpl-1; lin-15A mutants. RNAi of efl-1 in a lin-15A(n433)
background caused a Muv phenotype that was 8 ± 12% penetrant at 17.5ºC (average ±
standard deviation, see Materials and Methods). At 17.5ºC, dpl-1(n3316); lin-15A(n433)
and lin-35(n745); lin-15A(n433) animals had 2 ± 2% and 65 ± 10% penetrant Muv
phenotypes, respectively. RNAi of efl-1 in dpl-1(n3316); lin-15A(n433) or lin-35(n745);
lin-15A(n433) mutants did not enhance the Muv phenotypes at 17.5ºC (7 ± 10% Muv
and 72 ± 11% Muv, respectively). These data indicate that efl-1 E2F and dpl-1 DP,
which together encode a heterodimeric transcription factor, act to inhibit vulval fates in
the same molecular process both with each other and with lin-35 Rb.
We note that because null mutations of dpl-1 and trr-1 cause sterility, the dpl-1
and trr-1 homozygous animals we scored were descended from heterozygous mothers.
Therefore, these animals could have maternal rescue of dpl-1 or trr-1. Additionally,
RNAi of efl-1 and mys-1(n3681) likely caused partial loss of gene function, as null
alleles of each gene cause sterility. We believe that the loss of gene function of each of
these four genes was sufficient to allow us to detect enhancement of the Muv
phenotype by our assays, because each allele or RNAi enhanced the Muv phenotypes
of other synMuv combinations. For example, RNAi of efl-1 enhanced the Muv
phenotype of lin-37(n4903); lin-15A(n433) mutants from 59% to 100% at 17.5ºC, and
mys-1(n3681) enhanced other Muv phenotypes (Tables 5 and 6).
We chose several other class B synMuv genes to test in a limited number of
class B-B double mutant combinations in a lin-15A(n433) background. Null or strong
mutations in ark-1, lin-9 or lin-36 enhanced the Muv phenotypes caused by other class
B genes (Table 6). The mutation hda-1(e1795) causes a recessive Muv phenotype
(Dufourcq et al., 2002), and the Muv phenotype is enhanced by both class A and class
B synMuv mutations (E.C.A. and H.R.H., unpublished results). Heterozygous hda1(e1795) in three different mutant class B synMuv backgrounds caused enhancement of
those Muv phenotypes, indicating that lin-35, lin-37 and lin-61 function in separate
processes from hda-1. Using a strong partial loss-of-function allele of the class B
synMuv gene lin-53 RbAp48 (Lu and Horvitz, 1998), we found that in a lin-15A(n433)
background lin-53(n833) failed to enhance the Muv phenotypes caused by lin-9(n112),
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lin-37(n4903), lin-52(n771) and let-418(n3536) at 17.5ºC or 20ºC but did enhance the
Muv phenotype caused by mys-1(n3681). Because four class B; lin-53; lin-15A triple
mutants were weakly Muv at 17.5ºC and completely Muv at 20ºC, we scored each of
these strains at the intermediate temperature 18.5ºC. At this temperature, weak to
moderate enhancement of the Muv phenotype was observed (Table 6), indicating that
these genes act in separate processes.
In short, we identified four sets of synMuv genes — lin-15A and lin-56; lin-35,
dpl-1 and efl-1; let-418 and met-1; and mys-1 and trr-1 — for which the genes within
each set likely act together to inhibit vulval fates.
The penetrances of synMuv vulval phenotypes correlate with the level of lin-3
mRNA expression
Cui et al. (2006a) reported that the lin-3 gene, which encodes the EGF ligand of
the Ras pathway is a transcriptional target of some synMuv proteins. Three double
mutants carrying a class A and a class B synMuv mutation had increased lin-3 EGF
mRNA levels. We determined whether each of the class A genes and the class B genes
we studied repress lin-3 transcription and whether the redundancy we found to control
the vulval phenotype also controls lin-3 transcription.
We found that all class A-B double mutant strains between mutations in each of
the four class A genes and lin-15B(n744) had increased lin-3 mRNA levels as compared
to the wild type (Figure 1A). These results suggest that all four class A synMuv protein
repress the transcription of lin-3 EGF. All class A-B double mutants strains between
mutations in each of the 13 class B genes used in this study and lin-15A(n767) also had
increased lin-3 mRNA levels as compared to the wild type (Figure 1B). This analysis
greatly expands the number of genes implicated in the transcriptional repression of lin-3.
The level of increased lin-3 transcription in the class A-B synMuv double mutants
roughly correlated with the phenotypic strength of the respective Muv phenotypes
(Figure S1). For example, double mutants between lin-9 or lin-35 and a class A gene
had very strong Muv phenotypes and high levels of lin-3 transcription. By contrast,
77
mys-1; lin-15A animals had a weak Muv phenotype and a lower increase in lin-3
transcription.
Notably, ark-1(sy247); lin-15A(n767) had an increased level of lin-3 transcription,
even though ark-1 encodes a protein proposed to directly down-regulate Ras pathway
activity through SEM-5 (Hopper et al., 2000). One possibility is that there is positive
feedback through the Ras pathway during vulval induction, so that Ras pathway activity
elevates levels of lin-3 mRNA. We measured the level of lin-3 transcription in mutants
carrying the gain-of-function let-60 Ras allele n1046 and found lin-3 transcription was
slightly increased (2.8 fold ± 0.9 fold) as compared to the wild type. Therefore, it is
possible that at least some of the increase in lin-3 mRNA levels in the ark-1(sy247);
lin-15A(n767) mutant is caused by positive feedback.
We found that the Muv phenotypes of many class A-B-B synMuv triple mutants
were enhanced as compared to the respective class A-B double mutants. We wondered
if this redundancy extends to the control of lin-3 transcriptional repression. We
compared the lin-3 transcription levels of lin-61(n3809); lin-15A(n433), lin-37(n4903);
lin-15A(n433) and met-2(n4256) double mutants to the lin-3 expression of lin-61(n3809);
lin-37(n4903); lin-15A(n433) and lin-61(n3809); met-2(n4256); lin-15A(n433) triple
mutants. We found that in each case there was an increase in lin-3 transcription in the
triple mutants as compared to the respective double mutants (Figure 1C), indicating that
for these synMuv genes there is redundant control of lin-3 transcriptional repression.
78
Discussion
Genetic enhancement tests can distinguish processes that act in series or in
parallel
Genetic enhancement experiments are most simply interpreted when one or both
of the mutations used in the study are null. If a null mutation is combined with another
mutation, the phenotype can be more severe than the phenotypes of the respective
single mutants if the genes act in parallel (redundant) processes but cannot be more
severe if the genes act in the same process. At least one of the two mutations must be
null for such a test, because enhancement of the mutant phenotype of a double mutant
between two partial loss-of-function mutations could result even if the genes act in the
same process. Additionally, the penetrance of the mutant phenotype must be
incomplete, so that differences can be observed.
This type of genetic pathway analysis of biological systems defines logical
relationships equivalent to “and” versus “or” statements in mathematics and to “series”
versus “parallel” circuits in electrical engineering. If a double mutant has a phenotype
more severe than that of either single mutant, the two genes must provide separate
functions, e.g., act in parallel as a logical “or”; if either gene is active, some function is
provided. By contrast, if a double mutant has a phenotype equivalent to that observed in
either single mutant, the two genes might provide the same function, e.g., act together
in series as a logical “and”; if either gene is inactive, no function is provided.
By these criteria, the class A and B synMuv genes act in parallel processes, as
class A-B double mutants have almost completely penetrant Muv phenotypes at 20ºC
(Ferguson and Horvitz, 1989). It has been previously thought that within a synMuv gene
class there is no enhancement in double mutants, in which case all of the synMuv
genes within each synMuv class could act in a single process. However, we found that
there are multiple levels of redundant processes involving the functions of both the class
A and class B synMuv gene classes in inhibiting vulval cell fates (Figure 2). Most class
A-A or class B-B double mutants either had Muv phenotypes or showed enhancement
in a sensitized genetic background or at higher temperatures. By contrast, a few sets of
79
genes within a single synMuv class failed to show enhancement and hence might act
within the same process.
The synMuv genes define two distinct classes
The class B synMuv genes met-1, met-2, hda-1, hpl-2, and let-418 cause a Muv
phenotype in combination with either class A or class B synMuv mutations (Andersen
and Horvitz, 2007; Ceol and Horvitz, 2004; Couteau et al., 2002; Dufourcq et al., 2002;
von Zelewsky et al., 2000). Mutations in each of these genes cause stronger Muv
phenotypes when combined with class A synMuv mutations than with class B synMuv
mutations. The class C genes were considered a separate class because of their
redundancy with both class A and class B genes. However, our findings indicate that the
class C synMuv genes are not distinctive in this respect, as most class B synMuv genes
are similarly weakly redundant with each other. Furthermore, many class A-A double
mutants had weak Muv defects comparable in strength to or stronger than those of
class B-C double mutants.
We propose that the synMuv genes should be classified based on the strength of
the Muv phenotype observed in a double mutant with a class A synMuv mutation as
compared to a double mutant with a class B synMuv mutation. The double mutant
combination with the more penetrant Muv phenotype distinguishes the class. For
example, if a synMuv mutation when combined with a class A synMuv mutation caused
a 80% penetrant Muv phenotype and when combined with a class B synMuv mutations
caused a 15% penetrant Muv phenotype, this synMuv gene would be assigned to class
B, because it is more Muv in combination with class A mutations than with class B
mutations. Because mutations in met-1, met-2, hda-1, hpl-2, lin-13, let-418 or any of the
class C genes all exhibit strong Muv phenotypes when combined with mutations in class
A genes and weak Muv phenotypes when combined with mutations in class B genes,
we suggest that each of these genes should be assigned as class B synMuv genes.
80
Lack of genetic enhancement can identify proteins that function in a complex or
a process
A few sets of synMuv genes in the same class did not display synthetic
interactions. While all synMuv genes act redundantly with some other synMuv genes to
inhibit vulval cell fates, a set of synMuv genes that acts non-redundantly might act
together in a more specific molecular process, as discussed below.
Specifically, some sets of synMuv genes that did not show synthetic
enhancement encode proteins expected or known to function together in vivo. For
example, we found that dpl-1 DP, efl-1 E2F, and lin-35 Rb define a single process in the
inhibition of vulval cell fates. The corresponding C. elegans proteins likely form a
complex (Ceol and Horvitz, 2001), and their homologs function together in a complex
(Bandara et al., 1993; Helin et al., 1993; Krek et al., 1993). Similarly as shown
previously (Ceol and Horvitz, 2004), mys-1 HAT and trr-1 TRRAP define a single
process, and their homologs have been identified together in a histone acetyltransferase
complex (Ikura et al., 2000). Our data also indicate that the class A genes lin-15A and
lin-56 act together in the same process. This observation is consistent with recent
molecular data that indicate LIN-56 and LIN-15A are each required for the stability of the
other in vivo and bind each other in vitro, supporting the hypothesis that LIN-15A and
LIN-56 function in a protein complex (E.M. Davison and H.R.H., unpublished results).
We suggest that our genetic enhancement tests have identified groups of genes that
function together in protein complexes or that act sequentially in a pathway.
From our genetic enhancement tests of class B synMuv genes, we identified one
pair of genes that had not been found previously to act together, met-1 and let-418. The
two histone methyltransferases met-1 and met-2 act redundantly to control vulval cellfate inhibition; met-1 is similar to the yeast histone H3 lysine 36 methyltransferase Set2p
(Andersen and Horvitz, 2007). let-418 encodes the C. elegans homolog of human Mi2,
an ATP-dependent chromatin-remodeling enzyme found in the NuRD complex (von
Zelewsky et al., 2000). To date, neither MET-1 nor its homologs have been found in
NuRD-like complexes. Perhaps the NuRD-like complex requires MET-1 to function in
the inhibition of vulval fates. For example, MET-1 might methylate histone H3 lysine 36
81
at genes that normally promote vulval cell fates, leading to recruitment of the NuRD-like
complex and transcriptional repression.
It is possible that members of a protein complex could display synthetic
enhancement. However, genes that display synthetic enhancement cannot encode
proteins that work as essential components of the same complex (i.e., if either
component is absent, the complex has no function) or as members of a linear pathway
in which each step requires the prior step for activity (i.e., if any component is absent,
the pathway has no output). Many class B synMuv proteins, including LIN-9, LIN-35,
LIN-37, LIN-52, LIN-53, LIN-54, DPL-1 and EFL-1, are components of the C. elegans
DRM complex (Harrison et al., 2006). Nonetheless, most double mutants among lin-9,
lin-35, lin-37, lin-52 and lin-53 showed weak redundancy, suggesting that most proteins
in the DRM complex have redundant functions during vulval development. These results
suggest either the DRM complex is composed of members that can functionally
substitute for each other or the complex (which was isolated from embryos) does not
function in the inhibition of vulval cell fates. The synthetic interactions between many
DRM complex members were much weaker than interactions between non-DRM
complex members, suggesting that complex components might have subtle redundant
functions.
Genetic enhancement tests can identify functionally related groups of
evolutionarily conserved but uncharacterized genes
The gene lin-3, which encodes the EGF ligand for the receptor tyrosine kinase
RTK/Ras signal transduction cascade that drives vulval development, is a transcriptional
target of some synMuv proteins (Cui et al., 2006a). We found that all of the synMuv
genes we examined repress lin-3 transcription. Each set of synMuv genes we
hypothesize to act together in a single process to inhibit vulval cell fates might mediate a
specific mechanism of lin-3 transcriptional repression. For example, two sets of class B
synMuv genes, dpl-1 - efl-1 - lin-35 and mys-1 - trr-1, probably act redundantly to control
two different processes of transcriptional repression, namely the DNA binding of a
transcriptional repressor and histone acetylation, respectively. We suggest that a third
82
set of class B synMuv genes, let-418 and met-1, mediates a movement and methylation
of histones important for transcriptional repression in a process distinct from those of
the previous two examples.
An understanding of the distinct mechanisms used by different sets of synMuv
genes in lin-3 repression could define how the homologs of these genes control
repression of target genes in other organisms. We suggest that genetic enhancement
studies of additional synMuv gene interactions might implicate the homologs of
conserved but uncharacterized synMuv genes in well characterized processes and in
this way facilitate the understanding of how these genes act and of how they interact
with other gene processes in both development and disease.
Acknowledgments
We thank Na An for strain management and Hillel Schwartz and David Harris for critical
reading of this manuscript. Strains were provided by the Caenorhabditis Genetics
Center, which is supported by the National Institutes of Heath National Center for
Research Resources. Yuji Kohara of the National Institute of Genetics in Japan kindly
provided the efl-1 cDNA clone. E.C.A. is an Anna Fuller Cancer Research Fellow, and
H.R.H. is the David H. Koch Professor of Biology at the Massachusetts Institute of
Technology and an Investigator of the Howard Hughes Medical Institute. This work was
supported by National Institutes of Health grant GM24663.
83
Table 1: synMuv alleles used in this study.
class A gene
lin-8
lin-15A
lin-15A
lin-38
lin-56
class B gene
ark-1
dpl-1
efl-1
hda-1
let-418
lin-9
lin-15B
lin-35
lin-36
lin-37
lin-52
lin-53
lin-61
lin-61
met-1
met-2
mys-1
trr-1
allele
n2731
n767
n433
n751
n2728
mutation
Q113ochre
deletion
A250Va
R517Cb
deletion
null?
Yes
Yes
No
Noc
Yes
sy247
n3316
RNAie
e1795
n3536
n112
n744
n745
n766
n4903
n771
n833
n3809
n3447
n4337
n4256
n3681
n3712
Q528ochre
deletion
NA
G182E
P675L
G341E
W485opal
W151opal
Y796ochre
deletion
E36K
L292F
Q159ochre
S354N
deletion
deletion
G341R
W2593amber
No
Yesd
No
Noc
Noc
Noc
Yes
Yes
No
Yes
Noc
Noc
Yes
No
Yes
Yes
Noc
Yesd
complex
DP/E2F/Rb, DRM
DP/E2F/Rb, DRM
NuRD
NuRD
DRM
DP/E2F/Rb, DRM
DRM
DRM
DRM, NuRD
NuA4
NuA4
a
E. Davison and H.R.H., unpublished results.
b
A.M.S., E. Davison, and H.R.H., unpublished results.
c
This mutation is non-null. Null alleles cause sterility or larval arrest.
d
This mutation causes a recessive sterile phenotype. Homozygotes descended from
heterozygous mothers could possess some maternally provided wild-type gene
activity.
e
For efl-1, we used RNAi to create a partial loss of gene function.
84
Table 2: Most class A synMuv double
mutants have multivulva phenotypes.
genotypea
wild-type
lin-8
lin-56
lin-38
lin-15Ac
genotypea
lin-8 lin-56
lin-8 lin-38
lin-8; lin-15Ac
lin-56 lin-38
lin-56; lin-15Ac
lin-38; lin-15Ac
% multivulva ± s.d.b
20ºC
25ºC
0 ±0
0 ±0
0 ±0
0 ±0
0 ±0
1 ±0
0 ±0
1 ±1
0 ±0
0 ±0
0
1
0
0
0
0
±0
±1
±0
±0
±0
±0
0
7
13
6
1
33
±0
±2
±6
±4
±0
± 10
a
The alleles used are described in Table 1.
b
% multivulva was determined as described in
Materials and Methods. s.d., standard deviation.
c
The null allele lin-15A(n767) was used for these
analyses.
85
Table 3: Mutations in most class A synMuv genes enhance
the Muv phenotypes caused by mutations in other class A
synMuv genes in a class B synMuv mutant background
20ºC
lin-8
lin-15A
lin-8
8±6
60 ± 7
69 ± 8
21 ± 11
21 ± 8
78 ± 10
14 ± 7
32 ± 18
49 ± 13
lin-15A
lin-38
lin-38
lin-56
22.5ºC
lin-8
lin-15A
lin-38
lin-56
lin-56
5±1
lin-8
69 ± 3
lin-15A
lin-38
lin-56
100 ± 1
100 ± 0
95 ± 2
97 ± 2
100 ± 0
97 ± 2
99 ± 1
100 ± 0
81 ± 8
Each strain was grown at the temperature shown in the top left corner of each table.
All strains were homozygous for the class B mutation lin-61(n3447). The alleles used
are described in Table 1. lin-15A(n767) was used in these analyses. Class A-A-B
triple mutants with Muv phenotypes not enhanced when compared to the respective
class A-B double mutants are shaded. The % Muv was determined as described in
Materials and Methods, and the average % Muv and standard deviation are shown.
Table 4: Class B synMuv single and class
B-B double mutants do not have
appreciable Muv phenotypes.
% multivulva ± s.d.b
genotypea
20ºC
25ºC
wild-type
0 ±0
0 ±0
lin-35
0 ±0
0 ±0
c
lin-61
0 ±0
0 ±0
dpl-1d
0 ±0
0 ±0
d
trr-1
2 ±1
3 ±2
lin-37
0 ±0
0 ±0
lin-52
0 ±0
0 ±0
let-418
0 ±0
1 ±0
mys-1
0 ±0
0 ±0
efl-1
0 ±0
0 ±0
a
genotype
lin-35 lin-61c
0 ±0
1 ±0
lin-35; lin-37
0 ±0
0 ±0
lin-35; lin-52
0 ±0
0 ±1
lin-35; let-418
0 ±0
Lvl
lin-35; mys-1
0 ±0
Lvl
lin-35; efl-1
0 ±0
0 ±0
c
lin-61 ; lin-37
0 ±0
1 ±1
lin-61c; lin-52
0 ±0
0 ±0
c
lin-61 ; let-418
2 ±2
Lvl
lin-61c; mys-1
0 ±0
Lvl
d
dpl-1; efl-1
0 ±0
0 ±0
d
trr-1; mys-1
4 ±3
Lvl
lin-37; let-418
0 ±0
0 ±0
lin-37; mys-1
0 ±0
Lvl
lin-37; efl-1
0 ±0
0 ±0
lin-52; let-418
0 ±0
0 ±0
lin-52; mys-1
0 ±0
Lvl
let-418 mys-1
0 ±0
Lvl
a
The alleles used are described in Table 1.
b
% multivulva was determined as described in
Materials and Methods. s.d., standard deviation.
c
The null allele lin-61(n3809) was used for these
analyses.
87
d
These animals were descended from synMuv
mutant heterozygotes, because the mutations cause
recessive sterility, as described in Table 1.
Lvl, Larval lethality.
88
Table 5: Mutations of most class B and C genes enhance the Muv phenotypes caused by mutations
in other class B genes in a class A synMuv mutant background
15ºC
lin-35
lin-35
11 ± 3
met-1
met-1
dpl-1a
93 ± 5
13 ± 3
0±0
dpl-1
trr-1a
lin-37
met-2
lin-52
lin-61
Lvl
72 ± 6
98 ± 3
23 ± 3
97 ± 3
50 ± 5
67 ± 9
33 ± 15
65 ± 6
100 ± 0
62 ± 10
2±1
4±3
1±1
1±1
0±0
55 ± 13
69 ± 5
90 ± 11
0±0
46 ± 21
0±0
0±0
1±1
Lvl
61 ± 6
11 ± 12
18 ± 10
6±9
1±2
4±3
100 ± 0
22 ± 16b
95 ± 3
33 ± 5
29 ± 5
79 ± 7
67 ± 20
39 ± 4
20 ± 10
0±1
17 ± 7
0±0
1±1
0±0
1±1
1±1
0±0
1±1
trr-1
lin-37
met-2
7±7
lin-52
lin-61
let-418
mys-1
All strains were homozygous for the class A mutation lin-15A(n433)
17.5ºC
lin-35
met-1
dpl-1a
lin-35
65 ± 10
96 ± 4
60 ± 3
0±0
met-1
dpl-1
trr-1
lin-37
met-2
lin-52
trr-1a
0±0
met-2
Lvl
100 ± 0
100 ± 0
93 ± 10
100 ± 1
100 ± 0
81 ± 15
90 ± 8
100 ± 0
95 ± 3
26 ± 18
16 ± 3
2±2
15 ± 16
2±2
90 ± 2
100 ± 0
99 ± 2
13 ± 3
91 ± 2
3±1
0±1
Lvl
79 ± 4
64 ± 2
88 ± 3
3±4
5±7
59 ± 11
100 ± 1
92 ± 5b
100 ± 0
96 ± 6
92 ± 7
86 ±14
98 ± 1
89 ± 7
95 ± 6
0±0
58 ± 18
1±2
6±6
1±1
9±2
9±6
0±0
9±2
4±5
59 ± 19
lin-52
mys-1
lin-37
lin-61
let-418
mys-1
let-418
All strains were homozygous for the class A mutation lin-15A(n433)
lin-61
let-418
mys-1
99 ± 1
0±0
20ºC
lin-35
met-1
dpl-1a
100 ± 0
100 ± 0
8±4
99 ± 1
70 ± 7
lin-35
100 ± 0
met-1
dpl-1
trr-1
trr-1a
lin-37
met-2
lin-52
lin-61
100 ± 0
100 ± 0
100 ± 0
100 ± 0
100 ± 0
100 ± 0
100 ± 1
100 ± 0
100 ± 1
86 ± 10
96 ± 6
6±1
88 ± 6
100 ± 0
100 ± 0
100 ± 0
87 ± 2
99 ± 1
97 ± 1
84 ± 5
100 ± 0
98 ± 2
100 ± 0
53 ± 11
6±6
100 ± 0
100 ± 0b
100 ± 0
100 ± 0
100 ± 0
99 ± 1
100 ± 0
100 ± 0
100 ± 0
100 ± 0
15 ± 6
100 ± 0
Lvl
12 ± 5
lin-37
Lvl
100 ± 0
met-2
lin-52
lin-61
19 ± 17
let-418
mys-1
let-418
mys-1
89 ± 10
97 ± 1
94 ± 5
93 ± 6
0±0
31 ± 7
All strains were homozygous for the class A mutation lin-15A(n433)
2±1
Each strain was grown at the temperature shown in the top left corner of each table. The alleles used are described
in Table 1. lin-61(n3809) was used in these analyses. Class A-B-B triple mutants with Muv phenotypes not
enhanced when compared to the respective class A-B double mutants are shaded. The % Muv was determined
as described in Materials and Methods, and the average % Muv and standard deviation are shown.
a
These animals were descended from synMuv mutant heterozygotes, because the mutations cause recessive sterility,
as described in Table 1.
b
RNAi of lin-52 was used in the lin-37(n4903); lin-15A(n433) strain for this experiment.
Table 6: Mutations in most class B synMuv genes enhance the
Muv phenotypes caused by mutations of other class B synMuv
genes in a class A synMuv mutant background.
% multivulva ± s.d.b
genotypea
15ºC
17.5ºC
18.5ºC
20ºC
wild-type
0 ±0
0 ±0
0 ±0
0 ±0
ark-1
0 ±0
0 ±1
4 ±2
c
dpl-1
0 ±0
2 ±2
70 ± 7
efl-1
0 ±0
8 ± 12
92 ± 7
hda-1/+
0 ±0
0 ±0
0 ±0
let-418
0 ±0
0 ±0
0 ±0
0 ±0
lin-9
4 ±3
63 ± 11
81 ± 6
100 ± 0
lin-35
10 ± 3
65 ± 10
86 ± 5
100 ± 0
lin-36
0 ±0
0 ±0
5 ±2
lin-37
4 ±3
59 ± 11
32 ± 9
100 ± 0
lin-52
0 ±1
0 ±0
0 ±0
15 ± 6
lin-53
0 ±0
1 ±1
7 ±3
100 ± 1
lin-61d
0 ±0
1 ±1
19 ± 17
mys-1
0 ±0
0 ±0
2 ±1
trr-1c
1 ±1
4 ±5
12 ± 5
% multivulva ± s.d.b
genotypea
15ºC
17.5ºC
18.5ºC
20ºC
lin-35; ark-1
37 ± 23
92 ± 3
100 ± 0
lin-35; hda-1/+
36 ± 2
97 ± 1
100 ± 0
lin-36; let-418
1 ±2
1 ±0
99 ± 1
lin-36; mys-1
1 ±1
15 ± 15
99 ± 1
lin-53; let-418
0 ±1
4 ±2
46 ± 13
99 ± 1
lin-53; lin-9
13 ± 4
67 ± 9
93 ± 5
100 ± 0
lin-53; lin-37
13 ± 1
42 ± 14 100 ± 0
100 ± 0
lin-53; lin-52
0 ±0
3 ±2
16 ± 5
99 ± 1
lin-53; mys-1
8 ± 13
47 ± 11
100 ± 1
lin-61d; ark-1
7 ±8
34 ± 16
84 ± 26
lin-61d; hda-1/+
1 ±1
7 ±5
100 ± 0
lin-61d; lin-9
86 ± 12 100 ± 0
100 ± 0
d
lin-61 ; lin-36
1 ±1
21 ± 16
99 ± 1
a
These strains were homozygous for the class A synMuv mutation
lin-15A(n433). The alleles used are described in Table 1.
91
b
% multivulva was determined as described in Materials and Methods.
s.d., standard deviation.
c
These animals were descended from synMuv mutant heterozygotes,
because the mutations cause recessive sterility, as described in Table 1.
d
lin-61(n3809) was used in these analyses.
92
Figure 1: Regulation of lin-3 transcription by the synMuv genes.
Real-time RT-PCR experiments were performed on the strains shown. Mean ##CT
values were used to calculate relative changes in lin-3 expression normalized to levels
of rpl-26. Mean values and standard deviations of relative lin-3/rpl-26 ratios for the trials
are shown.
A. Mutation of each class A synMuv gene in a class B synMuv mutant background has
increased lin-3 transcription.
B. Mutation of each class B synMuv gene in a class A synMuv mutant background has
increased lin-3 transcription. lin-15A(n767) and lin-61(n3809) were used for this
experiment.
C. Two class B-B-A synMuv triple mutants have increased levels of lin-3 transcription
compared to their respective class B-A double mutants. lin-15A(n433) and lin-61(n3809)
were used for this experiment.
93
lin
et
-2
;l
in
-1
5A
5A
5A
5A
-1
-1
lin
in
2
5A
-1
-1
lin
lin
;l
7;
-2
-3
et
lin
m
1;
1;
-6
-6
lin
m
7;
1;
-3
-6
lin
lin
N
Relative lin-3 expression
N
2
k - lin - 1
1;
5
d p lin A
lle 1 ; 1 5 A
t- 4 lin
18 15
;
lin lin A
-9 -1 5
lin ; lin A
-3
5 -1 5
lin ; lin A
-3 -1
6
5
lin ; lin A
-3
-1
7
5
lin ; lin A
-5
2 1
lin ; lin 5 A
-5 -1
3
lin ; lin 5 A
-6
1 -1
m ; li 5 A
et n-1 1
m ; li 5 A
et n1
m 2; l 5A
y s in -1 1 5
;l
in A
-1
5A
ar
Relative lin-3 expression
;l
-5
6;
8;
-1
5B
5B
B
5B
-1
lin
lin
2
5B
5A
-1
-1
-1
in
lin
-8
-3
lin
lin
lin
lin
N
Relative lin-3 expression
Figure 1
A
B
C
Figure 2: Model: The synMuv classes function in two redundant pathways or
processes, each composed of separate molecular pathways or processes, to
mediate the inhibition of vulval cell fates.
The class A and B synMuv genes act in two distinct pathways to inhibit the expression
of vulval cell fates. Additionally, each class is composed of many separate pathways as
defined by genetic redundancy tests. A few synMuv genes act non-redundantly with
each other: lin-15A and lin-56 act together within the class A genes, while within the
class B genes dpl-1 DP, efl-1 E2F and lin-35 Rb; mys-1 HAT and trr-1 TRRAP; and
let-418 Mi2 and met-1 Set2 act together to inhibit vulval fates.
95
Figure 2:
class A
class B
lin - 1 5
A , l in
-5 6
lin - 3 8
lin - 8
b
-3 5 R
n
i
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Relative lin-3 expression
Supplemental Figure 1
The class B synMuv genes were ranked based on the penetrance of
the Muv defect they caused when mutated in a lin-15A(n433)
background (Tables 5 and 6). The rank order of phenotypic strength for
each strain graphed against the relative lin-3 levels (Figure 1) of that
strain is shown. The correlation of phenotypic strength to lin-3 mRNA
levels is significant (! =0.60, p=.001, Spearman rank correlation).
Chapter Four
lin-38 and mcd-1 antagonize the class A synMuv pathway to promote C. elegans
vulval development
Adam M. Saffer, Erik C. Andersen, Peter Reddien, Ewa M. Davison, and H. Robert
Horvitz
Chapter Four: lin-38 and mcd-1 antagonize the class A synMuv pathway to
promote C. elegans vulval development
Ewa Davison performed mapping experiments with lin-38 that guided the rescue
experiments shown in Figure 1. I performed all other lin-38 experiments. Peter
Reddien and Erik Andersen discovered that mcd-1(n4005) causes a class A synMuv
phenotype. Erik Andersen isolated mcd-1(n4140) and built all of the mcd-1(n4005)containing strains used in Table 3. Erik performed the RNAi experiment with mcd-1S. I
performed all other mcd-1 experiments and wrote the manuscript.
98
Summary
The class A and B synthetic multivulva (synMuv) genes of Caenorhabditis
elegans redundantly repress ectopic expression of lin-3 EGF to inhibit Ras-mediated
vulval development. The class B synMuv genes encode transcriptional repressors,
including C. elegans homologs of the NuRD and Myb-MuvB/dREAM complexes. Much
less is known about how the class A synMuv genes repress lin-3, although the nuclear
localization of many class A synMuv proteins is consistent with a role in transcription.
To investigate the function of the class A synMuv genes, we molecularly characterized
lin-38 and mcd-1, which can both be mutated to cause a class A synMuv phenotype.
lin-38 had not been previously cloned, and class A synMuv alleles of mcd-1 had not
been previously studied. We find that lin-38 and mcd-1 encode paralogous zinc-finger
proteins. We also find that mcd-1 has two distinct opposing functions: it can both
promote and inhibit the adoption of vulval cell fates. Finally, we show that both lin-38
and mcd-1 control multiple aspects of C. elegans development. Our results indicate that
lin-38 and mcd-1 antagonize the class A synMuv pathway to promote EGF/Rasmediated vulval development and provide insight into the mechanisms that regulate
expression of EGF-like ligands.
99
Introduction
Development of the C. elegans vulva requires multiple signaling pathways,
making it an excellent system to study cell-cell interactions, signaling cascades, and
cell-fate determination. An epidermal growth factor (EGF) and Ras signaling pathway
plays a central role in regulating vulval development, and EGF/Ras signaling has been
extensively studied in that context. In mammals, increased EGF signaling is a common
occurrence in cancers (Normanno et al., 2001; Normanno et al., 2006), and genes that
negatively regulate EGF signaling might therefore function as tumor suppressor genes.
In C. elegans, the six cells P(3-8).p are each capable of adopting either a vulval
or a non-vulval cell fate (Sternberg and Horvitz, 1986). In wild-type animals, the three
cells P(5-7).p are induced to adopt vulval cell fates by a signal secreted from the nearby
anchor cell, part of the somatic gonad (Kimble, 1981). P(5-7).p divide several times, and
their 22 progeny form the adult vulva (Sulston and Horvitz, 1977). P3.p, P4.p, and P8.p
do not receive the anchor cell signal, and they adopt a non-vulval cell fate, divide once,
and their progeny fuse with the hypodermis (Sulston and Horvitz, 1977).
The EGF-like ligand LIN-3 is expressed in the anchor cell and constitutes the
inductive signal that specifies vulval cell fates (Hill and Sternberg, 1992). LIN-3 binds to
the EGF receptor (EGFR) LET-23, which then activates a Ras/MAP kinase signaling
cascade that transduces this signal to transcription factors in the nucleus (Aroian et al.,
1990; Beitel et al., 1990; Beitel et al., 1995; Han and Sternberg, 1990; Kornfeld et al.,
1995a; Miller et al., 1993; Wu and Han, 1994; Wu et al., 1995). Loss-of-function
mutations in genes in the EGF/Ras pathway cause a vulvaless phenotype in which none
of the P(3-8).p cells adopt vulval fates (Aroian et al., 1990; Beitel et al., 1990; Han and
Sternberg, 1990; Hill and Sternberg, 1992). Overactivation of the EGF/Ras pathway, by
overexpression of lin-3 EGF or by activating mutations in let-60 Ras or let-23 EGFR,
leads to a multivulva (Muv) phenotype in which more than three P(3-8).p cells adopt
vulval cell fates (Beitel et al., 1990; Han and Sternberg, 1990; Hill and Sternberg, 1992;
Katz et al., 1996).
In opposition to EGF signaling, the synthetic multivulva (synMuv) genes prevent
the adoption of vulval cell fates. The synMuv genes are grouped into two redundant
100
classes, A and B (Ferguson and Horvitz, 1989). synMuv single mutants or double
mutants carrying two mutations within the same class have mostly wild-type vulval
development, but animals mutant for both a class A synMuv gene and a class B synMuv
gene exhibit a strong Muv phenotype (Andersen et al., 2008; Ferguson and Horvitz,
1989). Four class A synMuv genes have been identified: lin-8 encodes a novel acidic
protein, lin-15A and lin-56 both encode THAP domain proteins, and lin-38 has not been
cloned (Davison et al., 2005; Ferguson and Horvitz, 1989; Chapter Five). The THAP
domain is a C2CH motif that can exhibit zinc-dependent DNA binding activity (Bessiere
et al., 2008; Clouaire et al., 2005; Liew et al., 2007; Roussigne et al., 2003; Sabogal et
al., 2009). All molecularly characterized class A synMuv proteins are localized to the
nucleus, consistent with a role in transcription (Davison et al., 2005; Chapter Five). A
large number of class B synMuv genes have been identified, and their molecular
identities suggest that they function in chromatin remodeling and transcriptional
repression. Some class B synMuv genes encode proteins predicted to covalently
modify histones, including the mys-1 histone acetyltransferase and the met-1 and met-2
histone lysine methyltransferases (Andersen and Horvitz, 2007; Ceol and Horvitz,
2004). Other class B synMuv genes encode putative ATP-dependent chromatin
remodeling activites, such as the Mi-2 homolog let-418, which is part of a C. elegans
NuRD nucleosome remodeling complex (von Zelewsky et al., 2000). The class B
synMuv genes also encode proteins that bind to specific DNA sites and control
transcription: lin-35, dpl-1, and efl-1 encode the C. elegans homologs of the Rb, DP,
and E2F transcription factors (Ceol and Horvitz, 2001; Lu and Horvitz, 1998).
The synMuv genes ensure proper vulval development by repressing ectopic
expression of lin-3. In synMuv double mutants global lin-3 mRNA levels are
substantially increased compared to those of wild-type (Cui et al., 2006a). RNAi of lin-3
can suppress the synMuv phenotype, suggesting that lin-3 acts downstream of the
synMuv genes (Cui et al., 2006a). A mutation in the promoter of lin-3 causes a dominant
class A synMuv phenotype, proving that lin-3 is the major target of the class A synMuv
genes in vulval development (Chapter Two). In wild-type animals, lin-3 is expressed in
the anchor cell to induce vulval development (Hill and Sternberg, 1992). The class A
101
and B synMuv genes redundantly repress lin-3 expression in the rest of the animal, and
in synMuv double mutants lin-3 is ectopically expressed throughout the animal, leading
to excess vulval cell fates (Chapter Two).
In addition to their role in vulval development, many synMuv genes are required
for other developmental and non-developmental processes. A subset of synMuv genes,
including the class A synMuv gene lin-8 and several class B synMuv genes, are
required to prevent inappropriate expression from a cryptic pharyngeal promoter (Hillel
Schwartz, personal communication). No other defects besides those resulting from lin-3
overexpression have been reported for class A synMuv mutants. By contrast, mutations
in different class B synMuv genes can cause numerous pleiotropies, including sterility,
altered sensitivity to RNAi, silencing of expression from repetitive DNA, cell-cycle
defects, and other abnormalities (Boxem and van den Heuvel, 2001, 2002; Ceol and
Horvitz, 2001; Ceol et al., 2006; Ferguson and Horvitz, 1989; Hsieh et al., 1999; von
Zelewsky et al., 2000; Wang et al., 2005). A subset of class B synMuv genes, including
dpl-1 DP, efl-1 E2F, lin-35 Rb, and lin-37, promote programmed cell death (Reddien et
al., 2007).
While the class B synMuv genes have been extensively studied, much less is
known about the class A synMuv genes. To learn more about how the class A synMuv
genes repress ectopic expression of lin-3 EGF, we molecularly characterized class A
synMuv alleles of lin-38 and mcd-1. lin-38 had not been cloned, and mcd-1 was initially
studied for a role in programmed cell death but had not been previously implicated in
vulval development. We report that lin-38 and mcd-1 encode paralogous zinc-finger
proteins. Both mcd-1 and lin-38 control multiple aspects of development in C. elegans.
We also find that mcd-1 has two opposing functions in vulval development: one function
that promotes vulval cell fates and a separable function that inhibits vulval cell fates as
part of the class A synMuv pathway.
102
Materials and Methods
Strains and genetics
C. elegans strains were cultured using standard methods on OP50 bacteria
(Brenner, 1974). All animals were grown at 20˚C except where noted otherwise. The
wild-type strain used was N2, except in SNP mapping experiments with the polymorphic
CB4856 strain (Wicks et al., 2001). The following mutations were used in this study and
were described previously (Riddle, 1997) unless noted otherwise:
LGI: met-1(n4337) (Andersen and Horvitz, 2007), lin-35(n745) (Ferguson and Horvitz,
1989), nIs133 (Schwartz and Horvitz, 2007).
LGII: lin-8(n2731) (Thomas et al., 2003), dpy-10(e128), rol-6(e187), lin-56(n2728)
(Thomas et al., 2003), rol-1(e91), lin-38(n751) (Ferguson and Horvitz, 1989),
lin-38(n2727) (Thomas et al., 2003), lin-38(tm736) (this study), mcd-1(n3376) (Reddien
et al., 2007), mcd-1(n4005) (Reddien et al., 2007), mcd-1(n4140) (this study),
mcd-1(n4418) (this study), mcd-1(n761) (Ferguson and Horvitz, 1989), mcd-1(n2402)
(Thomas et al., 2003), unc-52(e444).
LGIII: lin-37(n758) (Ferguson and Horvitz, 1989), lin-36(n766) (Ferguson and Horvitz,
1989), lin-52(n771) (Ferguson and Horvitz, 1989).
LGIV: ced-3(n2427) (Shaham et al., 1999).
LGV: rde-1(ne219) (Tabara et al., 1999).
LGX: lin-15AB(n765) (Ferguson and Horvitz, 1989), lin-15A(n767) (Ferguson and
Horvitz, 1989), lin-15B(n744) (Ferguson and Horvitz, 1989), lin-15B(n2245) (Thomas et
al., 2003), nIs106 (Reddien et al., 2001).
The balancer strains hT2[qIs48] I:III (Mathies et al., 2003) and mnC1 (Herman, 1978)
were used. qIs48 is a GFP-expressing transgene integrated onto the hT2 translocation
(Mathies et al., 2003).
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SNP mapping
Previous studies had placed lin-38 between rol-1 and unc-52 on linkage group II
(Ferguson and Horvitz, 1989). To map lin-38 within this interval relative to DNA
polymorphisms between the wild isolate strains N2 and CB4856, we generated a strain
carrying the lin-15B(n744) mutation and CB4856 sequences throughout the entire
region covered by the mnC1 balancer, which includes at least dpy-10 to unc-52
(Herman, 1978). The rest of the genome was a mix of N2 and CB4856 sequences.
CB4856; lin-15B(n744) males were mated with rol-1(e91) lin-38(n751) unc-52(e444);
lin-15B(n744) hermaphrodites to generate rol-1(e91) lin-38(n751) unc-52(e444) /
CB4856; lin-15B(n744) animals. The progeny of those animals were then screened for
Rol non-Muv non-Unc and Rol Muv non-Unc phenotypes. Rol Unc animals cannot be
easily distinguished from non-Rol Unc animals, so we screened for neither class.
Homozygous lines were then established from each Rol non-Muv non-Unc and Rol Muv
non-Unc recombinant animal. Polymorphisms were assayed by PCR, followed by either
restriction digestion or DNA sequence determination. To identify previously
unannotated polymorphisms between N2 and CB4856 we determined the DNA
sequences of regions near lin-38 in both N2 and CB4856. Comparing N2 to CB4856, we
found an A-to-C difference at nucleotide 52914 of YAC Y48E1B and an A-to-G
difference at nucleotide 61805 of Y48E1B. Four Rol non-Muv non-Unc recombinant
animals placed lin-38(n751) to the right of the SNP at nucleotide 52914 of Y48E1B, and
one Rol Muv non-Unc recombinant animal placed lin-38(n751) to the left of the SNP at
nucleotide 61805 of Y48E1B.
RNAi
RNAi by injection was performed as previously described (Andersen et al., 2006).
For zygotic RNAi (Herman, 2001), dsRNA was injected into the gonads of dpy-10(e128);
rde-1(ne219); lin-15B(n744) hermaphrodites, which are insensitive to RNAi because
they lack rde-1 function. rde-1(+); lin-15B(n744) males were then mated with the
injected dpy-10(e128); rde-1(ne219); lin-15B(n744) hermaphrodites and non-Dpy cross
progeny were assayed. In this assay, the maternal contribution of lin-38 is unaffected
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because the mother is insensitive to RNAi, but the fertilized embryo carries a wild-type
rde-1 allele allowing RNAi to affect the zygotic contribution of lin-38. Feeding RNAi was
performed essentially as previously described (Timmons et al., 2001).
Characterization of the lin-38 cDNA
The 5ʼ end of the lin-38 transcript was determined by 5ʼ rapid amplification of
cDNA ends (RACE). The 5´ RACE System, Version 2.0 (Invitrogen) was used with the
primers 5ʼ-CCCACGCAAATTAAGACGATGG-3ʼ and 5ʼ-CAACGGTTCCCATGCACACA3ʼ. An SL1-spliced transcript was identified with a six nucleotide 5ʼ UTR starting at
nucleotide 51547 of Y48E1B. No additional transcripts were found.
The 3ʼ end of the lin-38 transcript was isolated from a poly(dT)-primed C. elegans
cDNA library in the Uni-ZAP XR vector (Stratagene). To amplify the 3ʼ end of the lin-38
transcript, a primary PCR was performed using the library as template with the primers
M13F(-20) and 5ʼ-GTTGGGTATCATCAGGAGG-3ʼ, and a secondary PCR was
performed using the primers T7 and 5ʼ-CTGGACGAGTTGTGAAGAAG-3ʼ. The PCR
product that was obtained extended to nucleotide 60,496 of Y48E1B and terminated in a
poly-A region.
First-strand synthesis was performed using RNA isolated from mixed-stage wildtype animals, random hexamers, and SuperScript III reverse transcriptase (Invitrogen).
The resulting cDNA was used as template for PCR to characterize the lin-38 transcript.
To determine if Y48E1B.6 and Y48E1B.7 were part of a single transcript, PCR was
performed with the primers 5ʼ-TGTAGATGACTCTGGAGACCAC-3ʼ and 5ʼCAATCGAAGTCCGCTTCTGTGA-3ʼ. To assemble the full-length transcript, one PCR
was performed with primers 5ʼ-GAAAAGATGTCGCTTCAAACTATCG-3ʼ and 5ʼCTCAGGATATTCGACAAGTGC-3ʼ, and another PCR was performed with primers 5ʼGCAATCGTCTCCAAACACTTTCC-3ʼ and 5ʼ-GGGTTTTTTTTGGGCTTA-3ʼ. The
resulting PCR products were cloned into the pGEM-T Easy vector. Both plasmids were
digested with PstI and HindIII and ligated together to create a lin-38 cDNA containing all
exons and part of the 3ʼ UTR.
105
Characterization of the mcd-1 cDNA
To determine if Y51H1A.7 was transcribed separately from mcd-1, 5ʼ RACE was
used with the 5´ RACE System, Version 2.0 (Invitrogen) and the primers 5ʼTTCAATGTCGTGCTCGTAGGCG-3ʼ and 5ʼ-TTCCTCCGAACCTTCACCTC-3ʼ. No
transcripts were detected that did not include mcd-1.
The 3ʼ end of the Y51H1A.7-containing transcript was isolated from a poly(dT)primed C. elegans cDNA library in the Uni-ZAP XR vector (Stratagene). To amplify the
3ʼ end of the transcript, a primary PCR was performed using the library as template with
the primers M13F(-20) and 5ʼ-ACGAGCACGACATTGAAATCG-3ʼ, and a secondary
PCR was performed using the primers T7 and 5ʼ-GACGCCCCGATGCTTATGAT-3ʼ.
The PCR product that was obtained extended to nucleotide 41397 of Y51H1A and
terminated in a poly-A region.
To determine the full-length mcd-1 transcript that includes Y51H1A.7, RT-PCR
was performed using the primers 5ʼ-ATGGAGACCGCGCCAGACGAAGCGC-3ʼ and 5ʼTTTCGGAGCATCGTTTCCC-3ʼ; this reaction amplified a transcript of 2795 nucleotides
that includes mcd-1, Y51H1A.7, and part of the Y51H1A.7 3ʼ UTR.
Isolation of the mcd-1(n4140) deletion mutant
mcd-1(n4140) was isolated by using PCR to screen libraries of mutagenized
worms as previously described (Ceol and Horvitz, 2001). mcd-1(n4140) removes
nucleotides 32661 to 33823 of Y51H1A.
Plasmids
To make the pAS1 plasmid, which carries a 17.7 kb fragment from Y48E1B near
lin-38, a PCR fragment was amplified from genomic DNA using the Expand Long
Template PCR System (Roche) with primers 5ʼ-CGCCGCTAAAAACCAACCATTTCC-3ʼ
and 5ʼ-GGATGACTATTGGTATCACAGGGGCTT-3ʼ, and the resulting PCR product was
cloned into the pCR-XL-TOPO vector (Invitrogen). To make pAS2, which contains a
subclone of the pAS1 Y48E1B fragment, pAS1 was digested with BstEII and an 11.2 kb
fragment was purified and religated. To make pAS3, which contains a subclone of the
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pAS1 Y48E1B fragment, pAS1 was digested with PstI and a 14.7 kb fragment was
purified and religated. To make pAS13, which is identical to pAS3 except for a
frameshift introduced into Y48E1B.7, pAS3 was digested with SalI, the overhang was
filled with T4 DNA polymerase, and the plasmid was religated. DNA sequence
determination showed that pAS13 has a four base pair insertion in the SalI site and
causes a predicted frameshift.
To make the lin-38 feeding RNAi construct, a fragment of the lin-38 transcript
was amplified by RT-PCR using the primers 5ʼ-TGTAGATGACTCTGGAGACCAC-3ʼ
and 5ʼ-TCACAGAAGCGGACTTCGATTG-3ʼ and cloned into the pGEM-T Easy vector
(Promega). A SacII/PstI fragment was then excised from this vector and cloned into the
L4440 vector.
107
Results
lin-38 encodes a zinc-finger protein
lin-38 previously had been mapped to linkage group II between rol-1 and unc-52
(Ferguson and Horvitz, 1989). Using polymorphisms between the N2 and CB4856
strains, we mapped the lin-38(n751) mutation to a region between nucleotides 52914
and 61805 of the YAC Y48E1B (Figure 1). The pAS1 plasmid, containing a 17.7 kb
fragment of this region with the predicted genes Y48E1B.6, Y48E1B.7, Y48E1B.8,
Y48E1B.16, and part of Y48E1B.9, efficiently rescued the Muv phenotype of a lin38(n751); lin-15B(n2245) strain (Figure 1). The pAS3 plasmid, containing a 10.2 kb
fragment of pAS1 with the predicted genes Y48E1B.6 and Y48E1B.7, also efficiently
rescued the Muv phenotype of a lin-38(n751); lin-15B(n2245) strain (Figure 1). The
pAS13 plasmid, which is identical to the pAS3 plasmid except for a frameshift mutation
introduced into Y48E1B.7, was unable to rescue the synMuv phenotype of lin-38(n751);
lin-15B(n2245) animals (Figure 1), indicating that Y48E1B.7 is lin-38. Homology of the
predicted C. briggsae gene CBG20757 to both Y48E1B.6 and Y48E1B.7 led us to
suspect that they might in fact be a single gene. We performed RT-PCR experiments
showing that there is a single transcript containing both Y48E1B.6 and Y48E1B.7,
hereafter referred to as Y48E1B.7. 5ʼ RACE experiments were unable to detect any
additional Y48E1B.7 transcripts that did not contain the exons previously predicted to
define Y48E1B.6. By a combination of RT-PCR and 5ʼ RACE experiments, we identified
an SL1-spliced full-length lin-38 cDNA of 2493 bp, including a 6 bp 5ʼ UTR and a 384 bp
3ʼ UTR (Figure 2A). Expression of this cDNA driven by a heat-shock promoter was able
to rescue the Muv phenotype of a lin-38(n751); lin-15B(n744) strain, indicating that it is
functional (data not shown).
Based on this cDNA, lin-38 has nine exons and encodes a 700-amino acid
protein (Figure 2B). The only known domain identified by SMART (http://smart.emblheidelberg.de/) and PFAM (http://pfam.sanger.ac.uk/) searches is a C2H2 zinc-finger
spanning amino acids 213-236. There is also a repeated VEEE motif in the C-terminus
of the protein (Figure 2B). LIN-38 is conserved in the related nematodes
108
Caenorhabditis remanei, Caenorhabditis brenneri, and Caenorhabditis briggsae but has
no obvious homology to any non-nematode proteins outside of the zinc finger domain.
AC8.5 has substantial homology to lin-38, including two nearly identical regions of 70
and 160 amino acids. However, AC8.5 is predicted to be a pseudogene
(http://ws190.wormbase.org/), and we found that a deletion that removes the first two
exons and the predicted translational start site of AC8.5 did not cause a synMuv
phenotype and was grossly wild-type (data not shown).
We determined the sequence of all lin-38 exons in strains carrying the lin38(n751) and lin-38(n2727) mutations. lin-38(n751) carries a C-to-T mutation at
nucleotide 59558 of Y48E1B that is predicted to mutate a methionine to an isoleucine at
amino acid 509 of LIN-38 (Figure 2). lin-38(n2727) contains a G-to-A mutation at
nucleotide 59536 of Y48E1B that is predicted to mutate an arginine to a cysteine at
amino acid 517 of LIN-38 (Figure 2).
Characterization of lin-38 class A synMuv alleles
Four lin-38 alleles that cause a class A synMuv phenotype were previously
identified (Ferguson and Horvitz, 1989; Thomas et al., 2003). However, as we show
below, two of these alleles, n761 and n2402, are not alleles of lin-38. We determined
the relative strength of the lin-38(n751) and lin-38(n2727) alleles by varying the
temperature and genetic background. The synMuv phenotype is temperature-sensitive,
and all synMuv mutants, including null mutants, exhibit temperature-sensitivity
(Ferguson and Horvitz, 1989). Both lin-38(n751) and lin-38(n2727) single mutants were
essentially wild-type, although lin-38(n751) animals exhibited a very low penetrance
Muv defect at 25˚C (Table 1). When combined with the class B synMuv null mutation
lin-15B(n744), both lin-38 alleles caused a completely penetrant Muv phenotype at all
temperatures. lin-38(n751); lin-15B(n744) double mutants were inviable at 25˚C. lin15B(n744) single mutants are very sick and slow-growing at 25˚C (A.M.S and H.R.H,
unpublished observations). The lethality of the double mutant might indicate a specific
interaction between lin-38 and lin-15B in promoting viability, or it could be a result of
enhancing the sickness of lin-15(n744) mutants by a slight sickness caused by either
109
lin-38(751) or background mutations. We combined both lin-38 alleles with the weak
class B synMuv mutant lin-15B(n2245) (Thomas et al., 2003). In a lin-15B(n2245)
background, lin-38(n751) caused a higher penetrance Muv defect than lin-38(n2727) at
15˚C, indicating that lin-38(n751) causes a stronger class A synMuv phenotype than lin38(n2727). In a lin-15B(n744) background, both lin-38(n751) and lin-38(n2727) were
mostly recessive (Table 1).
Loss of lin-38 causes larval lethality and suppresses the class A synMuv
phenotype of lin-38(n751)
Because the existing class A synMuv lin-38 alleles were missense, we wanted to
determine the null phenotype of lin-38. The lin-38(tm736∆) deletion removes 632
nucleotides, including the last four nucleotides of exon five of lin-38 and most of intron
five. If the shortened exon five splices to exon six, then the resulting transcript will be
out of frame, and the last 352 amino acids of LIN-38 will be missing. lin-38(tm736∆)
failed to complement the class A synMuv phenotype of lin-38(n751) in a lin-15B(n744)
background. lin-38(tm736∆) homozygous animals descended from lin38(tm736∆)/mnC1 mothers arrested as young larvae, mostly at the L1 stage. The pAS3
plasmid, which contains lin-38, was able to efficiently rescue the lin-38(tm736∆)
lethality. lin-38(tm736∆) homozygous animals that lost the lin-38 rescuing transgene
from rescued mothers arrested at various larval stages, ranging from L1 to L3 based on
size. Injection of dsRNA corresponding to exon three of lin-38 into the gonads of
lin-15B(n744) hermaphrodites caused a highly penetrant larval lethal phenotype among
the progeny (Table 2). RNAi by injection of dsRNA corresponding to exon four of lin-38
in either a wild-type or lin-15B(n744) background caused a highly penetrant larval lethal
phenotype (Table 2). Of the few animals that survived to adulthood, none exhibited a
Muv phenotype. The phenotypes caused by lin-38(tm736) and lin-38 RNAi indicate that
lin-38 is an essential gene and that loss of lin-38 causes larval arrest. However, the
stage at which the larvae arrest is variable, suggesting that lin-38 is required for general
growth and viability and not for a specific essential cell fate.
110
We also attempted to inactivate lin-38 using zygotic RNAi (Herman, 2001), which
affects the zygotic but not maternal contribution of lin-38 (see Materials and Methods).
We injected dsRNA corresponding to exon three or four of lin-38 into RNAi-deficient
dpy-10(e128); rde-1(ne219); lin-15B(n744) hermaphrodites and then mated them with
lin-15B(n744) males. The resulting animals were all wild-type (Table 2). We also
inactivated lin-38 by feeding animals bacteria expressing dsRNA corresponding to
exons two through six of lin-38. Feeding RNAi of lin-38 to wild-type animals or lin52(n771) class B synMuv mutants caused a low penetrance larval-lethal phenotype;
and to lin-15B(n744) class B synMuv mutants caused a highly penetrant larval-lethal
phenotype (Table 2). In addition to having a class B synMuv phenotype, lin-15B(n744)
mutants are also hypersensitive to RNAi, which could explain the more severe
phenotype caused by lin-38 feeding RNAi to lin-15B(n744) mutants (Wang et al., 2005).
Animals that survived to adulthood after lin-38 feeding RNAi to wild-type, lin-52(n771),
or lin-15B(n744) animals were superficially wild-type.
Because RNAi of lin-38 did not cause a synMuv phenotype, we tested if RNAi of
lin-38 might suppress the synMuv phenotype of lin-38 class A synMuv mutants. The
strain lin-38(n751); lin-15B(n2245) exhibits a fully penetrant Muv phenotype at 20˚C.
Feeding RNAi of lin-38 in lin-38(n751); lin-15B(n2245) animals caused a moderate
penetrance larval lethal phenotype. Escaper animals that survived to adulthood were a
mix of Muv and non-Muv animals, indicating that RNAi of lin-38 can partially suppress
the synMuv phenotype of lin-38(n751); lin-15B(n2245) mutants. Therefore the synMuv
alleles of lin-38 are not loss-of-function, but rather are gain-of-function mutations.
Because lin-38 synMuv alleles are mostly recessive and are rescued by an
extrachromosomal array carrying wild-type lin-38, the gain-of-function lin-38 synMuv
alleles must cause altered function of lin-38 that is antagonized by wild-type gene
activity. The observation that lin-38 RNAi only partially suppresses the synMuv
phenotype likely reflects the fact that only animals that are weakly affected by the RNAi
will survive to adulthood to allow for the assaying of vulval development. RNAi by
feeding of lin-38 in a lin-15AB(n765) background caused a fully penetrant larval lethal
phenotype that precluded the assaying of vulval development.
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Some mcd-1 mutations cause a class A synMuv phenotype
MCD-1 shares homology with LIN-38, and BLAST searches indicate these two
proteins are each otherʼs closest paralogs (Figure 3). LIN-38 and MCD-1L (see below)
are 18% identical and 39% similar. mcd-1 promotes programmed cell death, and the
mcd-1(n3376) mutant was isolated in a screen seeking mutations that enhanced the
cell-death defect of a partial loss-of-function allele of the proapoptotic caspase gene
ced-3 (Reddien et al., 2007). The deletion allele mcd-1(n4005) causes a cell-death
defect and is also synthetically lethal with mutations in some class B synMuv genes
(Reddien et al., 2007).
In the course of constructing strains with mcd-1 mutants, we found that
mcd-1(n4005) caused a class A synMuv phenotype. mcd-1(n4005) alone did not cause
an appreciable Muv phenotype (Table 3). Double mutants between mcd-1(n4005) and
the class B synMuv mutations lin-52(n771) and met-1(n4337) were viable and displayed
a Muv phenotype (Table 3). mcd-1(n4005) was previously shown to be synthetically
lethal with the class B synMuv mutants lin-35(n745) and lin-37(n758), causing a larvalarrest phenotype (Reddien et al., 2007). We found that maternally rescued lin-35(n745);
mcd-1(n4005) or mcd-1(n4005); lin-37(n758) animals derived from lin-35(n745)/+;
mcd-1(n4005) or mcd-1(n4005); lin-37(n758)/+ parents, respectively, were viable and
displayed a Muv phenotype (Table 3). mcd-1(n4005) combined with other class A
synMuv mutations caused a weak Muv defect (Table 3), comparable to that of many
other class A; class A double mutant strains (Andersen et al., 2008). Therefore,
mcd-1(n4005) causes a class A synMuv phenotype.
We isolated a second independent deletion in mcd-1, mcd-1(n4140). mcd1(n4140) removes part of exon three and is similar in extent to mcd-1(n4005) (Figure 4).
mcd-1(n4140) caused a slightly weaker synMuv phenotype than mcd-1(n4005) in a lin36(n766) class B synMuv mutant background (Table 3). The missense allele mcd1(n3376) did not cause a Muv phenotype either on its own or in combination with the
class B synMuv mutation lin-52(n771) (Table 3).
Additionally, we isolated the mutation n4418 in a screen for new class A synMuv
mutations by mutagenizing lin-52(n771) class B synMuv mutants. n4418 caused a Muv
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phenotype in a lin-52(n771) background but not as a single mutant, indicating that it is a
class A synMuv mutation (Table 3). We mapped n4418 to linkage group II,
approximately 21 map units from rol-6, by two-factor mapping. n4418 failed to
complement the class A synMuv phenotype of mcd-1(n4005) in a lin-52(n771)
background, indicating that n4418 is an allele of mcd-1. In a lin-52(n771) background,
mcd-1(n4418) caused a fully penetrant Muv phenotype at 20˚C, 22.5˚C, and 25˚C
(Table 3). By contrast, in a lin-52(n771) background, mcd-1(4005) caused only a very
low penetrance Muv defect at 20˚C and 22.5˚C and a 74% penetrant Muv defect at 25˚C
(Table 3). Like most class A synMuv mutations, mcd-1(n4418) caused a low
penetrance synMuv defect in combination with some other class A synMuv mutations at
high temperatures (Table 3).
n2402 and n761 had previously been classified as lin-38 mutations. However, as
we show below, both of these are actually alleles of mcd-1. mcd-1(n2402) caused a
fully penetrant Muv phenotype in a lin-52(n771) background at 20˚C, 22.5˚C, and 25˚C,
indicating that mcd-1(n2402) causes a relatively strong class A synMuv phenotype
(Table 3). In a lin-52(n771) background, mcd-1(n761) caused a Muv phenotype only at
25˚C, indicating that mcd-1(n761) causes a very weak class A synMuv phenotype
(Table 3). Taken together, these data show that mcd-1(n4005), mcd-1(n4140), and
mcd-1(n761) each cause a relatively weak class A synMuv phenotype, while mcd1(n4418) and mcd-1(n2402) both cause a relatively strong class A synMuv phenotype.
In the background of the moderate strength class B synMuv mutation lin52(n771), the class A synMuv phenotype of mcd-1(n4418) was completely recessive.
However, in the background of the strong class B synMuv mutation lin-15B(n744), mcd1(n2402) and mcd-1(n4418) both semidominantly caused a class A synMuv phenotype
(Table 3).
mcd-1 and lin-38 class A synMuv alleles exhibit intergenic non-complementation
We found that animals heterozygous for both the mcd-1(n4418) and lin-38(n751)
mutations in a lin-15B(n744) background displayed a Muv phenotype (Table 4),
suggesting that class A synMuv alleles of mcd-1 and lin-38 display intergenic non-
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complementation. Because no mutations had been identified in lin-38 in n761 or n2402
mutants we thought that n761 and n2402 might in fact be alleles of mcd-1. We then
performed complementation tests between different lin-38 and mcd-1 alleles and found
that each pair of lin-38 and mcd-1 alleles tested failed to complement each other for the
class A synMuv phenotype (Table 4). lin-38 and mcd-1 are both located on the right arm
of chromosome II, approximately 3 m.u. apart. To confirm which mutations are alleles
of lin-38 and which are alleles of mcd-1 we performed recombination experiments. We
generated animals doubly heterozygous for two alleles of lin-38 and/or mcd-1 in a lin15B(n744) class B synMuv mutant background and assayed the vulval phenotype of
their progeny to determine the frequency of recombination between the two mutations.
We did not observe any recombination between lin-38(n751) and lin-38(n2727),
confirming that n2727 is an allele of lin-38 (Table 5). By contrast, we did observe a low
but appreciable rate of recombination between lin-38(n751) and n2402 consistent with
the genetic distance between mcd-1 and lin-38, making it highly unlikely that n2402 is
an allele of lin-38 (Table 5). We did not observe any recombination between mcd1(n4418) and either n761 or n2402, strongly supporting the hypothesis that n761 and
n2402 are alleles of mcd-1 (Table 5). Finally, we found that strains carrying both the
n761 and n2402 alleles had missense mutations in the mcd-1 gene. Therefore we
conclude that n761 and n2402 are alleles of mcd-1 and that class A synMuv alleles of
mcd-1 and lin-38 exhibit intergenic non-complementation. The intergenic noncomplementation between lin-38 and mcd-1 alleles is weaker than the noncomplementation between two lin-38 or two mcd-1 alleles, as in the moderate strength
class B synMuv mutant background lin-52(n771) the mcd-1(n4418) and lin-38(n751)
mutations complemented each other for the class A synMuv phenotype (data not
shown). Also, animals doubly heterozygous for lin-38(n751) and the weak mcd-1 allele
mcd-1(n761) had a lower penetrance Muv defect than animals doubly heterozygous for
mcd-1(n761) and mcd-1(n4418) in a lin-15B(n744) background (Table 4).
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mcd-1 encodes two distinct transcripts
We determined the DNA sequences of all five previously reported mcd-1 exons
(Reddien et al., 2007) in strains carrying mcd-1(n4418) but did not identify any
mutations. We did identify a single mutation in the predicted downstream gene
Y51H1A.7, a C-to-T transition at nucleotide 39208 of YAC Y51H1A. RT-PCR
experiments showed that there is a single transcript that includes both mcd-1 and
Y51H1A.7. 5ʼ RACE experiments did not detect any transcript containing Y51H1A.7
without the other previously identified mcd-1 exons, suggesting that Y51H1A.7 exists
only as exons of an mcd-1 transcript and not as a separate gene. RT-PCR experiments
identified a transcript in which the first four exons are identical to the previously
described mcd-1 transcript, and the fifth exon is 47 bases shorter than in the previously
described transcript and spliced to the two exons of Y51H1A.7. We refer to the
previously identified shorter mcd-1 transcript as mcd-1S and to the longer mcd-1
transcript as mcd-1L. The mcd-1L transcript encodes a predicted protein of 901 amino
acids. The mcd-1(n761), mcd-1(n2402), and mcd-1(n4418) mutations specifically affect
mcd-1L but not mcd-1S (Figure 4). mcd-1(n761) is predicted to mutate an alanine to a
glutamic acid at position 680, mcd-1(n2402) is predicted to mutate a valine to an alanine
at position 685, and mcd-1(n4418) is predicted to mutate a cysteine to an arginine at
position 641. The other known mcd-1 mutations, mcd-1(n4005), mcd-1(n3376), and
mcd-1(n4140), affect both transcripts (Figure 4).
To test the activities of the two mcd-1 transcripts, we attempted to rescue the
class A synMuv phenotype of mcd-1 mutations with mcd-1 cDNAs driven by the dpy-7
promoter. Expression of the corresponding cDNA driven by the dpy-7 promoter is
sufficient to rescue mutations in many synMuv genes, including the class A synMuv
genes lin-15A and lin-56 (Appendix Two). Neither dpy-7p::mcd-1S nor dpy-7p::mcd-1L
were able to rescue the synMuv phenotype of mcd-1(n4418); lin-52(n771) animals, and
dpy-7p::mcd-1S was also unable to rescue the synMuv phenotype of mcd-1(n4005); lin52(n771) animals. We hypothesized that mcd-1(n4418) might be a gain-of-function
mutation, in which case expressing the mcd-1L cDNA with the n4418 mutation might
cause a class A synMuv phenotype. The expression of an mcd-1L(n4418) cDNA driven
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by dpy-7p did not cause a Muv phenotype in a lin-52(n771) background. Therefore,
either the two cDNAs we have identified are not functional for the role of mcd-1 in vulval
development or dpy-7p cannot drive mcd-1 expression at the correct level or in the
correct cells to rescue or phenocopy.
A subset of mcd-1 mutations cause cell-death and synthetic lethality defects
The mcd-1(n3376) and mcd-1(n4005) mutations both impair the ability of mcd-1
to promote cell death. This defect is readily apparent in the background of the partial
loss-of-function allele ced-3(n2427), which causes a weak cell death defect on its own
(Reddien et al., 2007). In wild-type animals, the six ventral cord (VC) motor neurons
P3-8.aap survive and express GFP driven by the lin-11 promoter, while the seven
lineally similar cells W.ap, P1.aap, P2.aap, and P9-12.aap undergo programmed cell
death. If programmed cell death is completely blocked, then the seven neurons that are
supposed to die instead survive, and five of them reliably express lin-11::gfp (Reddien et
al., 2001). In animals with defects in programmed cell-death, up to five extra GFPexpressing VC neuron-like cells can therefore be assayed. We used the integrated lin11::gfp transgene nIs106 to assay cell death in different mutant backgrounds. Wild-type
animals consistently had zero extra GFP-expressing cells (Figure 5A). The
ced-3(n2427) mutation caused an average of 1.7 extra cells to survive and express GFP
(Figure 5B). The mcd-1(n3376) mutation caused a very weak cell-death defect on its
own in this assay, with an average of 0.2 extra GFP-expressing cells per animal, and
strongly enhanced the cell-death defect caused by ced-3(n2427) (Figure 5C and D),
comparable to previous findings (Reddien et al., 2007). By contrast, the mcd-1(n4418)
mutation did not cause a cell-death defect as a single mutant in this assay (Figure 5E),
nor did mcd-1(n4418) enhance the cell-death defect caused by ced-3(n2427) (Figure
5F).
The mcd-1(n4005) mutation is synthetically lethal with a subset of class B
synMuv mutations, including lin-35(n745) and lin-37(n758) (Reddien et al., 2007). We
did not observe synthetic lethality between mcd-1(n4418) and class B synMuv
mutations. Specifically, the double mutants lin-35(n745); mcd-1(n4418) and
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mcd-1(n4418); lin-37(n758) were viable and did not appear to develop more slowly or to
be sicker than the lin-35(n745) and lin-37(n758) single mutants. Therefore, despite
causing a stronger class A synMuv phenotype than mcd-1(n4005), mcd-1(n4418) did
not cause cell-death or synthetic lethality defects, indicating that the role of mcd-1 in
vulval development is separable from its role in cell death and synthetic lethality.
mcd-1 null phenotype
The mcd-1(n4005) deletion caused more severe cell-death and synthetic lethality
defects than mcd-1(n4418), but mcd-1(n4418) caused a stronger synMuv phenotype,
suggesting that neither mutation is a null. mcd-1(n4005) removes part of the third exon,
but leaves most of the coding sequences of mcd-1 intact. Using RT-PCR, we found that
in mcd-1(n4005) mutants exon two splices to exon three, just after the end of the
mcd-1(n4005) deletion. This event is predicted to result in a frameshift. However, there
are several in-frame methionines after this frameshift at which translation could
potentially be initiated, resulting in up to a 729 amino acid in-frame protein. Furthermore,
there could be additional mcd-1 splice variants in mcd-1(n4005) mutants that we did not
detect by RT-PCR.
Previously, RNAi of mcd-1 was performed by injecting double-stranded RNA
corresponding to a region from exon three of mcd-1. This RNAi caused a cell-death
defect and synthetic lethality with some class B synMuv mutations but no noticeable
defects in viability in a wild-type background (Reddien et al., 2007). To better determine
the phenotype of strong mcd-1 loss-of-function, we performed RNAi by injection using
dsRNA corresponding to the entire mcd-1L transcript. RNAi of mcd-1 in a wild-type
background caused severe abnormalities. Animals were very slow-growing, most had
protruding vulvas and were egg-laying defective, most were sterile, and some dead
eggs were observed. RNAi of mcd-1 in a lin-52(n771) background caused similar
defects as did RNAi in a wild-type background, and no Muv animals were observed.
RNAi of mcd-1 in a lin-15B(n744) background caused early larval arrest. Because RNAi
of mcd-1 did not cause a class A synMuv phenotype, we tested if RNAi of mcd-1 might
suppress the synMuv phenotype of class A synMuv alleles of mcd-1. RNAi of mcd-1 in
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a Muv mcd-1(n4418); lin-52(n771) strain resulted in complete suppression of the
synMuv phenotype (Figure 6). RNAi of mcd-1 also suppressed the synMuv phenotype
of lin-52(n771); lin-15A(n767) mutants (data not shown). We also performed RNAi of
mcd-1 by injecting dsRNA corresponding to the entire mcd-1S transcript. This RNAi
treatment suppressed the Muv phenotype of a mcd-1(n4005); lin-52(n771) strain from a
penetrance of 89% (n=101) to 46% (n=46) when assayed at 25˚C. Because our RNAi
experiments indicated that loss of mcd-1 might suppress the synMuv phenotype, and
because the mcd-1(n3376) mutation is a missense mutation that affects the zinc-finger
in both MCD-1 isoforms, we tested if mcd-1(n3376) might be a synMuv suppressor
mutation. mcd-1(n3376) weakly suppressed the synMuv phenotype caused by lin15AB(n765) (Figure 6A), indicating that mcd-1(n3376) is a synMuv suppressor
mutation.
mcd-1 is in an operon with several upstream genes (Blumenthal et al., 2002), so
it is possible that RNAi of mcd-1 affects the mRNA of other genes in the operon and that
some or all of the effects seen with mcd-1 RNAi are caused by loss-of-function of one of
those genes. However, for two out of three operons tested, RNAi of one gene in the
operon does not affect upstream genes (Bosher et al., 1999), so RNAi of mcd-1 might
not affect its upstream neighbors. Furthermore, because the mcd-1(n3376) point
mutation also suppresses the synMuv phenotype, it is highly unlikely that the synMuv
suppression seen with mcd-1 RNAi results from off-target effects. The phenotype of
animals injected with mcd-1 dsRNA suggests that mcd-1 has a greater role in
development and viability than had previously been revealed by mutations and that the
mcd-1 locus normally functions antagonistically to the class A synMuv genes as a
synMuv suppressor gene.
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Discussion
LIN-38 and MCD-1 are putative transcription factors
LIN-38 and MCD-1 each contain a single C2H2 zinc-finger domain. Zinc-finger
domains are best known for binding to DNA in a sequence-specific manner. Many zincfinger proteins have multiple zinc-fingers that together bind to specific DNA sequences
(Wolfe et al., 2000), while proteins with only one or a few zinc-fingers sometimes have
other domains or structures that also mediate DNA-binding (Bowers et al., 1999; Dutnall
et al., 1996). The class A synMuv proteins LIN-15A and LIN-56 both contain a single
atypical THAP domain (Chapter Five), a C2CH zinc-finger-like motif that has been
implicated in DNA-binding (Bessiere et al., 2008; Clouaire et al., 2005; Liew et al.,
2007; Roussigne et al., 2003; Sabogal et al., 2009). LIN-15A and LIN-56 function
together as a complex (Chapters Three and Five). Perhaps LIN-15A, LIN-56, LIN-38,
and MCD-1, which together have a total of four zinc-finger and zinc-finger-like domains,
interact as a complex to form a sequence-specific DNA-binding activity that interacts
with the class A synMuv element in the lin-3 promoter and regulates lin-3 expression.
This model is consistent with the observation that mutations in different class A synMuv
genes cause very similar phenotypes but typically slightly enhance each other (Chapter
Three).
A role for lin-38 and mcd-1 in transcription is also supported by the set of genes
with which they interact genetically. lin-38 and mcd-1 act redundantly with the class B
synMuv genes, which include transcription factors, chromatin modifying enzymes, ATPdependent chromatin remodeling enzymes, and other transcriptional repressors. Loss
of mcd-1 function can suppress the synMuv phenotype, and many other synMuv
suppressor genes are involved in chromatin remodeling (Andersen et al., 2006; Cui et
al., 2006b). Finally, mcd-1 promotes programmed cell death together with some class B
synMuv genes, including the transcription factors lin-35 Rb, dpl-1 DP, and efl-1 E2F
(Reddien et al., 2007).
119
lin-38 is required for viability and likely functions as a synMuv suppressor
lin-38 was originally identified as a class A synMuv gene (Ferguson and Horvitz,
1989). Additionally, we have found that lin-38 is required for viability: both a deletion
allele of lin-38 and RNAi of lin-38 caused a larval-arrest phenotype. The developmental
stage at which animals arrested was variable, suggesting that the lethality of lin-38 was
not caused by a specific cell-fate defect. Rather, we propose that lin-38 is generally
required for growth and viability. The specific stage at which animals lacking lin-38
arrest might reflect the point at which the maternal stores of mRNA and protein are
exhausted.
The role of lin-38 in promoting viability is likely to be distinct from any role in
regulating expression of lin-3. Complete loss of EGF signaling causes lethality at the L1
larval stage, but this lethality involves a highly distinctive rod-like phenotype (Ferguson
and Horvitz, 1985) and does not resemble the lin-38 larval-arrest phenotype.
Overexpression of lin-3 can cause defects in vulval development as well as other
abnormalities but has not been observed to cause a larval-arrest phenotype like that of
lin-38. Therefore, the lethality of lin-38 is unlikely to be linked to any role in regulating
expression of lin-3, the only major target of the class A synMuv genes in vulval
development. Because lin-38 does not promote viability by repressing lin-3, lin-38 likely
regulates the transcription of a different gene or set of genes to promote viability.
The missense mutations lin-38(n751) and lin-38(n2727) recessively cause a
class A synMuv phenotype. However, RNAi of lin-38 suppressed the synMuv
phenotype of a lin-38(n751); lin-15B(n2245) strain. Therefore, the synMuv alleles of lin38 must cause altered function that is antagonized by wild-type lin-38 activity. It is
possible that lin-38 normally functions antagonistically to the synMuv genes in the class
A synMuv pathway, but it is also possible that wild-type lin-38 plays no role in vulval
development. It will be important to test if loss of lin-38 can suppress the synMuv
phenotype of other class A synMuv genes to distinguish between these possibilities.
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mcd-1 has two opposing functions in vulval development
mcd-1 can be mutated to cause a class A synMuv phenotype, indicating that
mcd-1 might act in the class A synMuv pathway to ensure proper vulval development by
repressing ectopic expression of the EGF ligand lin-3. However, RNAi of mcd-1 and the
mcd-1(n3376) mutation both suppress the synMuv phenotype, indicating that the net
effect of the wild-type mcd-1 locus is to oppose the synMuv genes in vulval
development. It is possible that the mcd-1 locus promotes vulval cell fates, and the mcd1(n4005) and mcd-1(n4418) mutations cause a gain-of-function, leading to a class A
synMuv phenotype. Arguing against this possibility is the fact that two dissimilar
classes of mutations, missense mutations that specifically affect the 3ʼ end of the mcd1L transcript and deletions that affect the 5ʼ end of both transcripts, can both cause a
class A synMuv phenotype. We prefer a model in which the mcd-1 locus has two
separable functions that promote and inhibit vulval cell fates, and the mcd-1(n4418) and
mcd-1(n4005) mutations preferentially remove the function that inhibits vulval cell fates.
Because mcd-1 encodes two transcripts and because different mcd-1
perturbations can cause opposite effects on vulval development, it is tempting to
speculate that the two mcd-1 transcripts have opposing activities. The strongest class A
synMuv alleles of mcd-1, mcd-1(n4418) and mcd-1(n2402), specifically affect the mcd1L transcript. Therefore, the model we favor is as follows (Figure 7B): The mcd-1L
transcript acts with other class A synMuv genes to inhibit vulval development by
repressing ectopic expression of lin-3. The mcd-1S transcript opposes mcd-1L and
other class A synMuv genes, perhaps by promoting lin-3 expression. The net effect of
the mcd-1 locus, including both transcripts, is to promote vulval development, such that
when mcd-1 activity is substantially reduced or eliminated by RNAi the synMuv
phenotype is suppressed. The mcd-1S transcript is responsible for the roles of mcd-1 in
cell-death and viability. Because mcd-1(n4418) affects only the mcd-1L transcript it has
no effect on those processes. The mcd-1(n4005) mutation, which caused a weak class
A synMuv phenotype, removes part of exon three and causes exon two to splice to the
middle of exon three. Because this mutation affects the splicing of the mcd-1 locus, it
could either affect the balance of mcd-1L and mcd-1S transcripts or it could have a
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stronger destabilizing effect on the MCD-1L protein than on the MCD-1S protein,
resulting in the weak class A synMuv phenotype.
mcd-1 controls multiple distinct aspects of development
The mcd-1(n3376) mutation causes a cell-death defect but not other obvious
abnormalities (Reddien et al., 2007). The mcd-1(n4005) mutation causes a cell-death
defect, a weak class A synMuv phenotype, and is synthetically lethal in combination
with some class B synMuv mutations (Reddien et al., 2007). Both the cell-death and
synthetic lethality defects are phenocopied by mcd-1 RNAi, indicating that those defects
are caused by loss-of-function of mcd-1 (Reddien et al., 2007). The mcd-1(n4418)
mutation caused a stronger class A synMuv defect than did mcd-1(n4005), but did not
cause cell-death defects or exhibit synthetic lethality with class B synMuv genes.
Therefore, the function of mcd-1 in promoting cell-death and viability is separable from
the function of mcd-1 in the class A synMuv pathway. The effects of the mcd-1(n4418)
and mcd-1(n4005) mutations cannot be explained by a model in which mcd-1 has a
single function with different thresholds of activity required to promote cell-death and
inhibit vulval development. Because mcd-1 has separable roles in cell-death and vulval
development, mcd-1 likely regulates the transcription of multiple target genes; lin-3 in
vulval development, and a different gene or group of genes to promote cell-death and
viability.
RNAi of mcd-1 caused severe developmental abnormalities and sterility,
suggesting that mcd-1 is an essential gene. The synthetic lethality between
mcd-1(n4005) and some class B synMuv mutations could reflect a distinct role for
mcd-1. Alternatively, mcd-1(n4005) might weakly impair the general role of mcd-1
promoting viability. Many class B synMuv single mutants are sickly and slow-growing,
and the mcd-1(n4005) mutation could cause a slight effect on viability that synergizes
with the sickness caused by those class B synMuv mutations.
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Conclusions
LIN-38 and MCD-1 are putative transcription factors that control multiple targets
to promote viability and proper development of C. elegans. In particular, lin-38 and
mcd-1 both likely function in the class A synMuv pathway to regulate expression of the
EGF ligand lin-3. In mammals, inappropriate activation of EGF/Ras signaling can lead
to excessive growth and to cancer (Normanno et al., 2006). Therefore, tight spatial and
temporal regulation of EGF/Ras signaling is essential, and genes that inhibit EGF/Ras
signaling might function as tumor suppressor genes. In C. elegans, lin-38, mcd-1, and
perhaps additional as yet unidentified essential genes that function in the class A
synMuv pathway might precisely regulate EGF signaling to ensure proper development.
An analogous class of genes in mammals might function as oncogenes or tumor
suppressor genes by regulating the expression of EGF-like ligands.
Acknowledgments
We thank Shohei Mitani of Tokyo Womenʼs Medical University for the lin-38(tm736)
deletion. We thank Daniel Denning for helpful comments concerning this manuscript.
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Table 1: lin-38 class A synMuv mutations
genotype
lin-38(n751)
lin-38(n2727)
lin-38(n751); lin-15B(n744)
lin-38(n751)/+; lin-15B(n744)c
lin-38(n2727); lin-15B(n744)
lin-38(n2727)/+; lin-15B(n744)d
lin-38(n751); lin-15B(n2245)
lin-38(n2727); lin-15B(n2245)
a
% multivulva (n)a
15ºC
20˚C
25˚C
0% (263)
0% (385 )
0.3% (344)
0% (199)
0% (379)
0% (179)
100% (224) 100% (367)
Lva b
N.D.
0% (118)
4% (24)
100% (231) 100% (180) 100% (210)
N.D.
1% (126)
N.D.
73% (564)
100% (128) 100% (221)
12.8% (453) 99.3% (292) 100% (242)
Animals were scored as Muv if any ventral ectopic protrusions were observed. n, total
number of animals scored.
b
Lva, larval arrest. Animals arrested as larvae, precluding the assaying of vulval
development.
c
Animals descended from dpy-5(e61); lin-38(n751); lin-15B(n744) hermaphrodites and
were also heterozygous for dpy-5(e61).
d
Animals descended from dpy-5(e61); lin-38(n2727); lin-15B(n744) hermaphrodites and
were also heterozygous for dpy-5(e61).
124
Table 2: RNAi of lin-38 causes lethality
Background
RNAi methoda
wild-type
lin-15B(n744)
lin-15B(n744)
lin-15B(n744)
lin-15B(n744)
wild-type
lin-52(n771)
lin-15B(n744)
injection
injection
injection
injection/zygotic RNAi
injection/zygotic RNAi
Feeding RNAi
Feeding RNAi
Feeding RNAi
a
dsRNA
Phenotype
exon 4
exon 4
exon 3
exon 3
exon 4
exons 2-6
exons 2-6
exons 2-6
larval arrest
larval arrest
larval arrestb
wild-type
wild-type
low penetrance larval arrestb
low penetrance larval arrestb
high penetrance larval arrestb
Injection RNAi was performed by injecting double-stranded RNA (dsRNA) into the
gonads of hermaphrodites and assaying the progeny. To attenuate the effects of lin-38
RNAi, zygotic RNAi (Herman, 2001) was performed to specifically target the zygotic
contribution of lin-38 while leaving the maternal contribution intact. For feeding RNAi,
worms were grown on bacteria expressing a fragment of the lin-38 cDNA between two
T7 promoters to produce dsRNA.
b
Animals that survived the RNAi treatment appeared wild-type and did not exhibit
abnormal vulval development.
125
Table 3: Only some mcd-1 mutations cause a class A synMuv phenotype
genotype
wild-type
mcd-1(n3376)b
mcd-1(n4005)
mcd-1(n4418)
mcd-1(n761)
mcd-1(n2402)
lin-8(n2731) mcd-1(n4005)c
lin-8(n2731) mcd-1(n4418)
lin-56(n2728) mcd-1(n4005)
lin-56(n2728) mcd-1(n4418)
mcd-1(n4005); lin-15A(n767)
mcd-1(n4418); lin-15A(n767)
lin-38(n751); mcd-1(n4005)
lin-38(n751) mcd-1(n4418)
mcd-1(n3376); lin-52(n771)
mcd-1(n4005); lin-52(n771)
mcd-1(n761); lin-52(n771)
mcd-1(n2402); lin-52(n771)
mcd-1(n4418); lin-52(n771)
mcd-1(n4418)/+; lin-52(n771)e
met-1(n4337); mcd-1(n4005)
met-1(n4337); mcd-1(n4418)
lin-35(n745); mcd-1(n4005)f
lin-35(n745); mcd-1(n4418)
mcd-1(n4005); lin-37(n758)g
mcd-1(n4418); lin-37(n758)
mcd-1(n4005); lin-36(n766)
mcd-1(n4140); lin-36(n766)
mcd-1(n761); lin-15B(n744)
mcd-1(n761)/+; lin-15B(n744)h
mcd-1(n2402); lin-15B(n744)
mcd-1(n2402)/+; lin-15B(n744)h
mcd-1(n4418); lin-15B(n744)
mcd-1(n4418)/+; lin-15B(n744)h
a
% multivulva (n)a
20ºC
22.5˚C
0% (many)
0% (many)
0% (710)
0% (631)
0.1% (869)
0.1% (685)
0% (910)
0% (795)
0% (452)
N.D.
0% (434)
N.D.
0% (699)
0% (538)
0% (560)
0% (467)
0% (644)
0% (526)
0% (588)
0% (893)
0.4% (780)
0.4% (523)
0% (1023)
0% (877)
0% (342)
0.5% (441)
0% (772)
0.1% (823)
0% (618)
0% (577)
0.6% (677)
4.2% (810)
0% (508)
0% (857)
100% (756)
100% (787)
99.9% (1065)
100% (1278)
0% (161)
0% (143)
21% (357)
80% (391)
83% (474)
98% (1020)
44% (116)
66% (105)
100% (710)
100% (305)
34% (102)
75% (65)
100% (317)
100% (486)
8.9% (583)
59% (630)
0.6% (621)
7.6% (632)
8.4% (273)
N.D.
N.D.
N.D.
100% (601)
N.D.
34% (99)
N.D.
100% (418)
100% (764)
55% (134)
65% (74)
Animals were scored as Muv if any ventral ectopic protrusions were observed. n, total
number of animals scored.
b
25˚C
0% (many)
0% (731)
0.6% (666)
0.6% (840)
0% (248)
0% (330)
0.3 % (327)
0.8% (517)
0% (344)
0.1% (1032)
12% (574)
1.2% (860)
4.9% (123)
1.7% (783)
0% (566)
74% (420)
46% (940)
100% (1042)
100% (1108)
0% (266)
99.7% (361)
99.9% (901)
91% (112)
100% (258)
98% (54)
100% (261)
98% (541)
84% (350)
100% (105)
0% (66)
lethal
N.D.
lethal
83% (18)
Strain is also homozygous for nIs106 (lin-11::gfp).
126
c
Strain is also homozygous for nIs133 (pkd-2::gfp).
d
Strain is also homozygous for rol-1(e91).
e
Animals descended from rol-6(e187) mcd-1(n4418); lin-52(n771) hermaphrodites and
were also heterozygous for rol-6(e187).
f
Animals were descended from lin-35(n745)/hT2[qIs48]; mcd-1(n4005) parents. qIS48
is a GFP-expressing transgene integrated onto the hT2 translocation (Mathies et al.,
2003).
g
Animals were descended from mcd-1(n4005); lin-37(n758)/hT2[qIs48] parents.
h
mcd-1/+; lin-15B(n744) animals were descended from dpy-5(e61); mcd-1; lin-
15B(n744) hermaphrodites and were also heterozygous for dpy-5(e61).
127
Table 4: Non-complementation between lin-38 and mcd-1 alleles
genotype
lin-38(n751)/lin-38(n2727); lin-15B(n744)
lin-38(n751) mcd-1(+)/lin-38(+) mcd-1(n4418); lin-15B(n744)
lin-38(n751) mcd-1(+)/lin-38(+) mcd-1(n761); lin-15B(n744)
lin-38(n751) mcd-1(+)/lin-38(+) mcd-1(n2402); lin-15B(n744)
mcd-1(n4418)/mcd-1(n761); lin-15B(n744)
mcd-1(n4418)/mcd-1(n2402); lin-15B(n744)
a
% multivulva (n)a
100% (175)
100% (254)
34% (47)
100% (261)
100% (74)
100% (83)
Animals were scored as Muv if any ventral ectopic protrusions were observed. n, total
number of animals scored.
128
Table 5: Recombination between lin-38 and mcd-1 alleles
genotype
lin-38(n751)/lin-38(n2727); lin-15B(n744)
lin-38(n751)/mcd-1(n4418); lin-15B(n744)
lin-38(n751)/mcd-1(n2402); lin-15B(n744)
mcd-1(n4418)/mcd-1(n761); lin-15B(n744)
mcd-1(n4418)/mcd-1(n2402); lin-15B(n744)
mcd-1(n4418)/lin-38(n2727); lin-15B(n744)
recombinant progeny
0 / 4913
31 / 1220
20 / 858
0 / 670
0 / 1560
17 / 492
Animals doubly heterozygous for different combinations of lin-38 and mcd-1 alleles were
generated in a lin-15B(n744) background and allowed to self-fertilize. Progeny that
displayed a Muv phenotype were classified as non-recombinant. Progeny that were
non-Muv were transferred to individual plates. Some non-Muv progeny produced nearly
100% Muv offspring and were probably non-recombinant animals that were non-Muv as
a result of incomplete penetrance, so these animals were classified as nonrecombinant. Non-Muv animals that produced mostly non-Muv offspring represent
recombination between the mcd-1 and lin-38 alleles and were classified as recombinant
progeny. Because some mcd-1 class A synMuv mutations are semi-dominant (Table
3), some of the recombinant progeny may have been included in the non-recombinant
class for genotypes including mcd-1(n4418) or mcd-1(n2402). Recombination was seen
between lin-38 and mcd-1 mutations, but not between two lin-38 mutations or between
two mcd-1 mutations.
129
Figure 1: Y48E1B.7 is lin-38
lin-38 had previously been mapped between rol-1 and unc-52 (Ferguson and Horvitz,
1989). We performed SNP mapping using polymorphisms between the N2 and CB4856
strains to further localize lin-38(n751) to an 8.9 kb interval between SNPs at nucleotides
52914 and 61805 of Y48E1B. Plasmids containing different regions of Y48E1B were
tested for their abilities to rescue the synMuv phenotype of lin-38(n751); lin-15(n2245)
animals. The number of lines that rescued the lin-38 synMuv phenotype and the total
number of lines obtained are shown. The plasmid pAS3, containing a subfragment of
Y48E1B between 50190 and 68865, was able to rescue the synMuv phenotype of
lin-38(n751); lin-15B(n2245) animals. The plasmid pAS2, with the sequences between
51286 and 62275 removed, did not rescue the lin-38(n751); lin-15B(n2245) synMuv
phenotype. The plasmid pAS3, containing a fragment of Y48E1B between 50190 and
61400, rescued the synMuv phenotype of lin-38(n751); lin-15B(n2245) animals. When a
frameshift mutation was introduced into Y48E1B.7 in pAS3 to produce pAS13, rescue
was abrogated.
130
Figure 1
unc-52
rol-1
LG II
n751
1 kb
Y48E1B
52914
Y48E1B
61805
Plasmid
Frameshift
Rescue
pAS1
8/8
pAS2
0/3
pAS3
5/5
pAS13
0/7
Figure 2: LIN-38 is a zinc-finger protein
(A) The exon-intron structure of lin-38, as determined by 5ʼ RACE and RT-PCR (exons,
filled boxes; introns, connecting lines; UTRs, open boxes). The extent of the deletion
lin-38(tm736), which causes a recessive larval arrest-phenotype, is indicated with a
horizontal line. lin-38(tm736) deletes the last four nucleotide of the fifth exon of lin-38
and the first 628 nucleotide of the fifth intron. The positions of the lin-38(n751) and
lin-38(n2727) missense mutations are indicated by arrowheads. The lin-38(n761) and
lin-38(n2402) mutants do not have any mutations in lin-38 exons.
(B) The predicted LIN-38 protein. The beginning of the region affected by the lin38(tm736) deletion is indicated by a vertical line: all amino acids after that point are out
of frame and the LIN-38(tm736) protein terminates 42 amino acids after the first
frameshifted residue.
132
Figure 2
n2727
n751 500 bp
tm736!
A
SL1
B
MSLQTIEEMDAYPAVDDSGDHLLDPNMIPTAATEVVMSGDGIERQFGDEEDTY
QYEYTYEDDPQLEIGEEEVVVTDEAEYQEHWKDEEKKMNLYDVLGSKMYLEDD
EKGGPKRSRIDDDEASFSDVYHHHQPPPPPHPRAGAPGDYRYAVDDYREYTVY
PTSGGPKQQHGGAHNSVDRTCQGCGMMLRRSVFYHHARMIREKGACNLFTPQR
FPCTQCDARIGTLEKLCQHMEQIHQAPTQIKTMVFTNEEDFKQFRIELEGKGG
NFRMARGNKKNKKGMVQYFRCNRLQTLSRSQTFRPVDNPSLEELPTNRKRGRL
tm736!
HQQELRQQSAKQVIRTENSCTAFYNKAYLDNGTIEVRFCDHHLHDDEKLRLPE
AVRARVIELARKNLPHVVILMIVKDERFKYCERNSANDRRIQDMKTQDIRQVL
AGNNRSVKTRISRGEQVEQFDRFAHLGHVDEDPTRPWNDVRPSAKVDRLSLSH
n2727 M
I
n751 R
C
SELRYLDLFDTNREEIMAKLNERSRVEQSKKMLYDSFIHRLSSSNEVIKQIDY
RDLIPNESTLLKLKKAYNYLAKIENELLNPRREQINPIRVTSYIRMMEEEARL
IERTRQAASAPGAAAGDEIDVVGIDEEDYHHHHHMDLHEVVEEEEVGYHQEDL
ELEQELQEHVEEDYLPVEEELVEEELVVEEEALPVEQHQEEEEDGEPTVTRAG
RVVKKKPQFDS!
Figure 3: LIN-38 and MCD-1 are paralogs
Alignment of LIN-38 and MCD-1L is shown. The dark boxes indicate identities, and the
light boxes indicate similarities. The zinc-finger domains of LIN-38 and MCD-1 are
highlighted in red. Arrowheads indicate the locations of the lin-38(n2727), lin-38(n751),
mcd-1(n761), mcd-1(n2402) mcd-1(n3376), and mcd-1(n4418) mutations.
134
Figure 3
n3376
n2727
n4418
n761
n2402
n751
Figure 4: mcd-1 gene structure
The mcd-1S isoform has been described previously (Reddien et al., 2007). The mcd-1L
isoform lacks the last 47 bases of the mcd-1S isoform and instead contains two
additional exons, which had been predicted to be a separate gene, Y51H1A.7. The
extent of the mcd-1(n4005) and mcd-1(n4140) deletions are indicated by horizontal
lines. The mcd-1(n761), mcd-1(n2402), and mcd-1(n4418) missense mutations, which
affects only the mcd-1L transcript, are indicated by arrowheads The zinc-finger domain
is colored red.
136
Figure 4
1 kb
n3376
mcd-1S
n3376
n4418
mcd-1L
n4005!
n4140!
n761 n2402
Figure 5: mcd-1(n4418) does not block programmed cell death
In wild-type animals six ventral cord (VC) neurons express lin-11::gfp, and seven
additional VC neuron-like cells undergo programmed cell-death. If programmed celldeath is completely blocked, then those seven VC neuron-like cells survive, and five of
them reliably express the reporter lin-11::gfp from the integrated nIs106 transgene. n,
number of animals scored. The histograms show the percentage of animals with zero
through five extra GFP-expressing VC neuron-like cells for each genotype.
(A) wild-type animals do not exhibit any inappropriate survival of undead VC neurons.
(B) ced-3(n2427) animals have an average of 1.7 extra undead VC neurons that
survive.
(C) mcd-1(n3376) occasionally causes inappropriate survival of undead VC neurons.
(D) mcd-1(n3376) strongly enhances the cell-death defect of ced-3(n2427) animals.
(E) mcd-1(n4418) does not cause any inappropriate survival of undead VC neurons.
(F) mcd-1(n4418) does not enhance the cell-death defect of ced-3(n2427) animals
(p=0.56 by the Mann-Whitney test).
138
Figure 5
A
B
C
D
E
F
Figure 6: Loss of mcd-1 suppresses the synMuv phenotype
(A) mcd-1(n3376) suppresses the synMuv phenotype of lin-15AB(n765). Animals were
scored as Muv if any ventral ectopic protrusions were observed. n, total number of
animals scored.
(B) A wild-type animal does not exhibit a Muv phenotype.
(C) An mcd-1(n4418); lin-52(n771) mutant exhibits a Muv phenotype
(D) RNAi of mcd-1 in a mcd-1(n4418); lin-52(n771) animal suppresses the Muv
phenotype.
140
Figure 6
A
%Muv (n)!
Genotype!
17.5˚C!
20˚C!
lin-15AB(n765)!
93% (843)!
100% (1117)!
mcd-1(n3376); lin-15AB(n765)!
26% (782)!
96% (660)!
B
wild-type
C
mcd-1(n4418); lin-52(n771)
D
mcd-1(n4418); lin-52(n771) + mcd-1(RNAi)
Figure 7: Summary of mcd-1 defects and model for mcd-1 function
(A) The defects associated with each mcd-1 allele or RNAi are shown. synLet, the
synthetic lethality observed in combination with some class B synMuv mutations. Let,
the sterility and dead eggs observed after mcd-1 RNAi.
(B) mcd-1S promotes programmed cell death by acting through targets other than lin-3.
mcd-1L represses expression of lin-3 to prevent excessive vulval cell fates, while mcd1S promotes vulval cell fates, perhaps through regulation of lin-3 expression. mcd-1S
has a stronger input into vulval development than mcd-1L. mcd-1(n3376), mcd1(n4005), and mcd-1 RNAi each cause a loss-of-function of the mcd-1S isoform and
therefore affect cell-death. mcd-1(n4005) and mcd-1 RNAi, but not mcd-1(n3376),
impair mcd-1S function sufficiently to affect viability. mcd-1(n4418) does not alter mcd1S and therefore plays no role in cell death or viability. mcd-1(n3376) does not impair
either mcd-1S or mcd-1L sufficiently to affect vulval development. mcd-1(n4005)
impairs mcd-1L more strongly than mcd-1S, causing derepression of lin-3 and a weak
class A synMuv phenotype, while mcd-1(n4418) affects only mcd-1L and therefore
causes a strong class A synMuv phenotype. RNAi of mcd-1 strongly impairs both mcd1S and mcd-1L and causes a synMuv suppression phenotype since mcd-1S has a
stronger input into vulval development than mcd-1L.
142
Figure 7
A
Allele
Isoform(s)
affected
Cell-death
defect?
synLet?
Let?
synMuv
n3376
mcd-1S+L
yes
no
no
synMuv suppressor
n4005
mcd-1S+L
yes
yes
no
weak class A
n4418
mcd-1L
no
no
no
strong class A
n761
mcd-1L
N.D.
no
no
weak class A
n2402
mcd-1L
N.D.
no
no
strong class A
RNAi
mcd-1S+L
yes
yes
yes
synMuv suppressor
B
mcd-1S
unknown
targets
cell death
viability
mcd-1S
mcd-1L
lin-38
lin-3
vulval cell fates
Chapter Five
Two putative C. elegans transcription factors, LIN-15A and LIN-56, interact and
function redundantly with an Rb pathway to regulate vulval development
Ewa M. Davison, Adam M. Saffer, Linda S. Huang, John DeModena, Paul W.
Sternberg, and H. Robert Horvitz
Chapter Five: Two putative C. elegans transcription factors, LIN-15A and LIN-56,
interact and function redundantly with an Rb pathway to regulate vulval
development
My contributions to this manuscript include Table 2 and RNAi of lin-3. Ewa Davison
wrote the manuscript, and I edited it to take into account reviewerʼs comments and new
literature. This manuscript is being prepared for submission.
144
Summary
The C. elegans class A and B synthetic multivulva (synMuv) genes redundantly
repress expression of lin-3 EGF to negatively regulate Ras-mediated vulval
development. The class B synMuv genes encode proteins homologous to components
of the NuRD and Myb-MuvB/dREAM transcriptional repressor complexes, indicating that
they likely silence lin-3 through chromatin remodeling. The two class A synMuv genes
cloned thus far, lin-8 and lin-15A, both encode novel proteins. The LIN-8 protein is
nuclear. We have characterized the class A synMuv gene lin-56 and found it to encode
a novel protein that shares a THAP-like C2CH motif with LIN-15A. Both the LIN-56 and
LIN-15A proteins localize to nuclei. Wild-type levels of LIN-56 require LIN-15A, and wildtype levels of LIN-15A require LIN-56. Furthermore, LIN-56 and LIN-15A interact in the
yeast two-hybrid system. We propose that LIN-56 and LIN-15A normally associate in a
nuclear complex that inhibits vulval specification by repressing lin-3 expression.
145
Introduction
Tumorigenesis requires deregulation of pathways controlling cell proliferation,
differentiation, and apoptosis and likely involves multiple mutations that result in the
activation of proto-oncogenes and the inactivation of tumor-suppressor genes. A
particularly frequent target of misregulation in human cancers is the epidermal growth
factor (EGF) and Ras signaling pathway that controls cell proliferation. The EGF/Ras
pathway can be overactivated by misexpression of EGF-like ligands, mutation or
overexpression of EGF receptors, or by constitutively active Ras mutations (reviewed by
Downward, 2003; Normanno et al., 2001; Normanno et al., 2006).
In C. elegans, an EGF/Ras pathway plays a central role in vulval development
(reviewed by Kornfeld, 1997; Moghal and Sternberg, 2003). Six multipotent cells, P(38).p, of the ventral ectoderm each can express either the 1° or 2° vulval fates or the 3°
non-vulval fate. The LIN-3 EGF-like ligand is expressed in the anchor cell of the somatic
gonad and activates the LET-23 EGF receptor (EGFR) in the closest P(3-8).p cells.
LET-23 EGFR subsequently signals through a Ras/MAP kinase pathway to specify
vulval fates (Aroian et al., 1990; Han and Sternberg, 1990; Hill and Sternberg, 1992;
Kornfeld et al., 1995a; Lackner et al., 1994; Wu et al., 1995). In the wild type, P6.p
assumes the 1° vulval fate, dividing three times to produce eight descendants, and P5.p
and P7.p assume the 2° vulval fate, generating seven descendants each. The 22
progeny of P(5-7).p undergo morphogenesis to generate the adult vulva. P3.p, P4.p,
and P8.p assume the non-vulval 3° fate and divide once and fuse with a multinucleate
hypodermal cell, hyp7. Loss-of-function mutations in components of the EGF/Ras
pathway cause P(5-7).p to adopt the non-vulval 3° fate and result in a vulvaless (Vul)
phenotype. Gain-of-function mutations in let-23 EGFR or let-60 Ras or overexpression
of lin-3 EGF cause P3.p, P4.p, and P8.p to adopt vulval 1° or 2° fates and result in a
multivulva (Muv) phenotype (Beitel et al., 1990; Han and Sternberg, 1990; Hill and
Sternberg, 1992; Katz et al., 1996). Muv animals produce extra vulval tissue that forms
ectopic ventral protrusions.
The EGF/Ras pathway, which is essential for C. elegans vulval induction, is
antagonized by the functionally redundant class A and B synthetic multivulva (synMuv)
146
genes (Ferguson and Horvitz, 1989). Hermaphrodites carrying only a single synMuv
mutation generally appear wild-type for vulval induction, while hermaphrodites carrying
mutations in both a class A and a class B synMuv gene exhibit a Muv phenotype. Four
class A synMuv genes and at least 25 class B genes have been identified (Andersen
and Horvitz, 2007; Ceol and Horvitz, 2001, 2004; Couteau et al., 2002; Davison et al.,
2005; Ferguson and Horvitz, 1985, 1989; Harrison et al., 2006; Harrison et al., 2007a;
Horvitz and Sulston, 1980; Hsieh et al., 1999; Lu and Horvitz, 1998; Poulin et al., 2005;
Solari and Ahringer, 2000; Thomas et al., 2003; Unhavaithaya et al., 2002; von
Zelewsky et al., 2000). The synMuv genes likely act upstream of EGF/Ras signaling, as
loss-of-function of components of the EGF/Ras pathway can suppress the synMuv
phenotype (Ceol and Horvitz, 2001, 2004; Cui et al., 2006a; Ferguson et al., 1987;
Huang et al., 1994; Lu and Horvitz, 1998). In class AB synMuv double mutants, lin-3
EGF is overexpressed compared to wild-type animals, indicating that the synMuv gene
negatively regulate EGF/Ras signaling by repressing expression of lin-3 EGF (Cui et al.,
2006a). All synMuv genes tested, including all four class A synMuv genes, repress lin-3
EGF expression (Andersen et al., 2008; Cui et al., 2006a).
The class B synMuv genes likely repress lin-3 via chromatin remodeling given
their molecular identities. lin-35, efl-1, dpl-1, lin-53, hda-1, let-418, met-2, and hpl-2
encode C. elegans counterparts of Rb, E2F, DP, the Rb-associated protein RbAp48,
histone deacetylase (HDAC), the Mi-2 chromatin remodeling enzyme, a histone H3
lysine-9 methyltransferase, and the histone H3 methyl-lysine-9 binding protein HP1,
respectively (Andersen and Horvitz, 2007; Ceol and Horvitz, 2001; Couteau et al., 2002;
Lu and Horvitz, 1998; Solari and Ahringer, 2000; von Zelewsky et al., 2000). Studies of
these mammalian proteins strongly suggest that an Rb/E2F/DP complex represses
transcription of target genes by recruiting HDAC, RbAp48, histone H3 lysine-9
methyltransferase and HP1 (reviewed by Nielsen et al., 2001; Vandel et al., 2001;
Zhang and Dean, 2001). RbAp48, HDAC and Mi-2 are components of the histone
deacetylase and chromatin remodeling NuRD complex (reviewed by Knoepfler and
Eisenman, 1999) and might be involved in transcriptional repression (reviewed by
Richards and Elgin, 2002). Furthermore, LIN-35 Rb, LIN-53RbAp48, DPL-1 DP, and
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additional class B synMuv proteins form a complex in vivo that resembles the
Drosophila Myb-MuvB and dREAM complexes (Harrison et al., 2006), both of which are
able to mediate transcriptional repression of many E2F-target genes (Korenjak et al.,
2004; Lewis et al., 2004).
Although the class A synMuv genes function redundantly with the class B
synMuv genes, the mechanism by which the class A synMuv genes repress lin-3 EGF
expression to inhibit Ras-mediated vulval development is not known. Of the four class A
synMuv genes, the lin-8 and lin-15A loci were cloned previously, and both encode novel
proteins (Clark et al., 1994; Davison et al., 2005; Huang et al., 1994). Nonetheless, it is
likely that the class A synMuv genes also act through regulation of gene expression.
First, although novel in sequence, LIN-8 is a nuclear protein that can interact physically
with LIN-35 Rb in vitro, suggesting that it might be present in vivo at the sites of
transcriptional repressor complexes (Davison et al., 2005). Second, the class A synMuv
genes either directly or indirectly repress the transcription of lin-3 EGF (Cui et al.,
2006a). We report here the cloning and characterization of the class A synMuv gene
lin-56 and propose that LIN-56 and LIN-15A normally associate in a nuclear complex
that affects cell-fate determination through the regulation of gene expression.
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Materials and Methods
Strains
C. elegans strains were cultivated as described (Brenner, 1974) and were grown
at 20°C unless otherwise indicated. Bristol N2 was the wild-type strain. The mutant
alleles used in this study are listed below, and a description is presented by Riddle et al.
(1997) unless noted otherwise: LGII dpy-10(e128), lin-8(n2731) (Thomas et al., 2003),
lin-38(n751), lin-56(n2728) (Thomas et al., 2003); LGIII lin-36(n766); LGIV let-60(n1876)
(Beitel et al., 1990); LGX lin-15A(n433, n767, n749), lin-15B(n744), lin-15AB(e1763).
Also used was the chromosomal rearrangement mnC1[dpy-10(e128) unc-52(e444)] and
the following deficiencies: eDf21, mnDf16, mnDf29, mnDf57, mnDf61, mnDf62, mnDf71,
and mnDf90 (Sigurdson et al., 1984). lin-56(n3355) was isolated in a noncomplementation screen using lin-56(n2728) (see below).
Non-complementation Screen for lin-56 Alleles
lin-15B(n744) L4 males were mutagenized using ethylmethane sulphonate (EMS)
(Brenner, 1974) and mated with dpy-10(e128) lin-56(n2728); lin-15B(n744)
hermaphrodites. Muv non-Dpy F1 progeny were used to establish homozygous non-Dpy
lines. Approximately 6550 mutagenized haploid genomes were screened. We isolated
one new lin-56 allele, designated n3355.
Transgenic Animals
Germline transformation by microinjection was performed as previously
described (Mello et al., 1991). The transformation markers pRF4 and myo-3::gfp were
co-injected with experimental constructs at 80 ng/µl and 50 ng/µl, respectively.
Transgenic animals were identified using either the Roller phenotype generated by
expression of the rol-6(su1006) dominant allele from pRF4 or GFP expression from
myo-3::gfp. Experimental constructs were injected at 10-100 ng/µl. Stable transformants
were analyzed.
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Molecular Biology
Standard molecular biology procedures were followed (Sambrook et al., 1989).
To disrupt ZK673.3 in pEMD4, an oligonucleotide linker containing an opal stop codon
was ligated into an SphI site within the second exon. The resulting construct, pEMD5,
contains an insertion of the sequence TGAGACTAGTGCATGC and is predicted to
produce a truncated protein containing only the first 120 amino acids of the wild-type
322 amino acids of ZK673.3. The disrupted ZK673.3 open reading frame was
transferred to pEMD1 by amplification using the polymerase chain reaction (PCR) of the
region surrounding the engineered insertion from pEMD5, followed by substitution of the
wild-type sequence in pEMD1 with the PCR product, generating pEMD14. To ensure
that loss of rescuing activity was caused by the engineered disruption of the ZK673.3
open reading frame, the latter was restored by digestion of pEMD14 with SphI followed
by religation, generating pEMD15.
RNA interference
RNAi was performed by feeding animals bacteria expressing double-stranded
RNA, essentially as previously described (Kamath et al., 2001; Timmons et al., 2001).
The bacterial strain expressing double-stranded lin-3 RNA was from Kamath et al.
(2003), and the DNA sequence of the insert was determined to ensure that it was
correct.
Antibody Preparation and Immunocytochemistry
Rabbit anti-LIN-56 antisera were generated using purified GST-LIN-56(aa 1-322)
as the immunogen, affinity purified against MBP-LIN-56(aa 1-322), as described by
Koelle and Horvitz (1996), and pre-adsorbed against an acetone precipitate of proteins
prepared from lin-56(n2728) mixed-stage worms, essentially as described by Harlow
and Lane (1988). Rabbit anti-LIN-15A antibodies were generated using purified
6His-LIN-15A(aa 77-324) as immunogen and were purified as with the anti-LIN-56
antisera, using 6His-LIN-15A(aa 77-324) protein for affinity purification and
lin-15AB(e1763) mixed-stage worms for pre-adsorption. Affinity-purified anti-LIN-56
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antibodies were used at 1:2000 for immunoblots and, following pre-adsorption, at 1:100
for immunocytochemistry. Affinity-purified and pre-adsorbed anti-LIN-15A antibodies
were used at 1:25 for immunocytochemistry. Anti-a-tubulin monoclonal antibody DM1A
(Sigma, St. Louis, MO) and MH27 (Francis and Waterston, 1991), which recognizes the
apical borders of C. elegans epithelial cells, were used as positive controls for
immunocytochemistry at 1:100 and 1:1000, respectively. Embryos were fixed in 0.8%
paraformaldehyde for 20 minutes, as described by Guenther and Garriga (1996). Larvae
and adults were fixed in 2% paraformaldehyde for 15 minutes, essentially as described
by Finney and Ruvkun (1990). Images were obtained using a Zeiss LSM510 laser
confocal microscope and software and processed with Adobe Photoshop.
Subcellular Fractionation of Embryos
Embryos were collected by bleaching gravid hermaphrodites in a 0.8 N NaOH,
8% hypochlorite solution. Fractionation into nuclear and cytosolic fractions was
performed as described by Chen et al. (2000). The protein content of each fraction was
determined using Bradford reagent, and 10 µg of each were used for immunoblots.
Rabbit polyclonal antiserum 3930 generated against C. elegans lamin (Gruenbaum et
al., 2002) was used at 1:5000 on immunoblots as a control for nuclear fractionation
quality. Anti-SQV-4 antibodies (Hwang and Horvitz, 2002) were used at 1:3000 on
immunoblots as a control for cytosolic fractionation quality.
RT-PCR Analysis
Total RNA was isolated from mixed-stage worms using TRIZOL (Invitrogen,
Carlsbad, CA). Standard RT-PCR (Titan One Tube RT-PCR System, Roche Applied
Science, Indianapolis, IN) was used to qualitatively compare levels of lin-56 and lin-15A
RNA in total RNA samples derived from wild-type, lin-56(n2728), lin-15A(n767), and
lin-15AB(e1763) animals. RT-PCR of hexokinase (H25P06.1) was performed as a
control. Primers used were: lin-15A Fwd9, 5'-CGAATGTCAAGCTTGGCGAACG-3';
lin-15A Rev5, 5'-CGGTTTACTGAGAGACCC-3'; lin-56 Fwd2, 5'-AGACTGGGCAGAATGCG-3'; lin-56 Rev2, 5'-GCTCCACTTTTTCAGGAAAAC-3'; hexokinase Fwd1A, 5'-GA-
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GCTCGGCATTCAATATCG-3'; hexokinase Rev1B, 5'-GCTTCATATGCAGCTGCAACC3'.
Quantitative real-time RT-PCR (Heid et al., 1996) was used to measure the
amount of lin-56 mRNA in poly(A)+ mRNA samples derived from wild-type,
lin-15A(n767) and lin-15AB(e1763) animals. poly(A)+ mRNA was purified from total RNA
using Oligotex resin (Qiagen, Valencia, CA). cDNA was produced from the purified
poly(A)+ mRNA using oligo-dT primer and SuperScript II Rnase H- Reverse
Transcriptase (Invitrogen). Using an ABI PRISM 7000 Sequence Detection System
(Applied Biosystems, Foster City, CA), absolute lin-56 and hexokinase mRNA levels
were determined relative to a genomic DNA dilution series, and lin-56 mRNA levels
were then normalized to hexokinase mRNA levels. 50 ng and 100 ng mRNA equivalents
were analyzed in triplicate for each genotype. lin-56 and hexokinase reactions were
performed in separate tubes. No significant amplification was observed when reverse
transcriptase was omitted from the initial RT reaction. Primers and probes used were:
lin-56 Fwd, 5'-TTGGTGCAAAGTCTACACGATGA-3'; lin-56 Rev, 5'-TTGCGCACATCGAACTTTGT-3'; lin-56 probe, 5'-6-FAM-TCGATCTTCCCTGGGCGAGCAGT-TAMRA-3';
hexokinase Fwd, 5'-CGTGGAGCCGCACTCATC-3'; hexokinase Rev, 5'-CAGATCCTTCAGCCGCTTCT-3'; hexokinase probe, 5'-VIC-TCGCTTGACTCTCGAAACGATTGCGTAMRA-3'.
Yeast Two-Hybrid System
Full-length cDNAs for lin-15A, lin-37, lin-53, and lin-56 were cloned into Gateway
entry vector pDONR201 (Invitrogen) and then each transferred into two Gateway twohybrid destination vectors: pDEST-DB, encoding the Gal4 DNA-binding domain (DB),
and pDEST-AD, encoding the Gal4 activation domain (AD) (Walhout et al., 2000a;
Walhout et al., 2000b). Yeast strain Y190 (MATa gal4 gal80 his3 trp1-901 ade2-101
ura3-52 leu2-3 leu2-112 URA3::GAL1-lacZ LYS2::GAL1-HIS3 cyhr) was cotransformed
with DB- and AD-fusion constructs. Interaction in the two-hybrid system was assayed by
colony formation on SC-Trp-Leu-His medium containing 25 mM 3-aminotriazole (3-AT).
152
Results
lin-56 encodes a putative transcription factor containing a THAP-like domain
lin-56 was previously mapped close to unc-4 on chromosome II (Thomas et al.,
2003). Using deficiencies, we further mapped lin-56 to a region of 554 kb between
daf-19 and bli-1 (Figure 1A). Cosmid ZK673 and a 3.5 kb SnaBI-SnaBI subclone of
ZK673 (pEMD4) rescued the synMuv phenotype when injected into lin-56(n2728);
lin-15B(n744) animals (Figure 1B). The 3.5 kb minimal rescuing fragment contains a
single predicted gene, ZK673.3 (C. elegans Sequencing Consortium 1998). We found
the only existing lin-56 allele, n2728, to contain an 11.2 kb deletion that eliminates not
only ZK673.3 but also its downstream neighbor ZK673.4 and the 3'-end of its upstream
neighbor ZK673.2 (Figure 1B).
Since ZK673.3 and ZK673.4 share a small region of similarity (see below), we
were concerned that the class A synMuv phenotype of the n2728 mutant might be
caused not by loss of ZK673.3 as the rescue experiments suggest but either by loss of
ZK673.4 or by loss of both ZK673.3 and ZK673.4. Disruption of ZK673.3 in a 10.7 kb
rescuing subclone of ZK673 (pEMD1) that contains both ZK673.3 and ZK673.4 resulted
in loss of rescuing activity (pEMD14) (Figure 1B), and expression of a ZK673.3 cDNA
under the control of heat-shock promoters efficiently rescued the synMuv phenotype of
lin-56(n2728); lin-15B(n744) animals (Table 1). Furthermore, RNA-mediated
interference (RNAi) (Fire et al., 1998) of ZK673.3 but not of ZK673.4 resulted in a Muv
phenotype in lin-15B(n744) animals (data not shown). Finally, we isolated a second
lin-56 allele, n3355, in a non-complementation screen (see Materials and Methods) and
found n3355 to correspond to a glutamine-to-ochre nonsense mutation at amino acid 61
of ZK673.3 (Figure 1C). We conclude that ZK673.3 is lin-56.
Blumenthal et al. have suggested that ZK673.2 and ZK673.3 may be expressed
as an operon, because these two genes are separated by fewer than 500 basepairs and
because they detected the C. elegans SL2 trans-spliced leader sequence at the 5' end
of ZK673.3 mRNAs (Blumenthal et al., 2002). (The SL2 sequence has been found
primarily at the 5' ends of mRNAs that appear to be expressed from downstream genes
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in operons (Riddle, 1997). However, no sequence upstream of the ZK673.2 open
reading frame was required for rescue by ZK673.3 of the lin-56(n2728) class A synMuv
phenotype (Figure 1B), consistent with the hypothesis that ZK673.2 and ZK673.3 are at
least not always expressed as an operon.
LIN-56, a novel acidic protein of 322 amino acids, contains a domain similar to
the THAP domain (Figure 1C). Specifically, LIN-56 shares a C-X-[ILV]-C-X(33,38)-AX(11,13)-C-X(2)-H motif with several C. elegans proteins (Figure 1D), including the
class A synMuv protein LIN-15A, the class B synMuv protein LIN-15B, the protein
encoded by its downstream genomic neighbor ZK673.4, and HIM-17, a protein required
for meiotic recombination and histone H3 lysine-9 methylation in the germline (Reddy
and Villeneuve, 2004). The conservation of cysteine and histidine residues suggests
that this motif may bind a zinc ion and mediate binding to DNA, RNA, protein, or a small
molecule (reviewed by Laity et al., 2001). Although the interval between the two internal
cysteines is greater than that found in typical zinc fingers (Krishna et al., 2003), the
spacing of the cysteine and histidine residues within the C2CH motif described here
matches the spacing characteristic of the THAP domain, which can mediate sequencespecific DNA binding (Cayrol et al., 2007; Clouaire et al., 2005; Roussigne et al., 2003;
Sabogal et al., 2009). Additional features of the THAP domain, including an invariant
tryptophan between the second and third cysteine residues and a C-terminal AVPTIF
motif, are not conserved. This THAP-like motif is likely functionally significant, as two
lin-15A alleles contain missense mutations therein, and one of these mutations affects a
conserved alanine residue (Figure 1D).
The Muv phenotypes of many synMuv double mutants are suppressed by RNAi
of lin-3, suggesting that the synMuv genes act upstream of lin-3 in vulval development.
To determine if lin-56 also acts upstream of lin-3, we inactivated lin-3 by feeding
lin-56(n2728); lin-15B(n744) animals bacteria expressing double-stranded lin-3 RNA.
lin-56(n2728); lin-15B(n744) animals fed bacteria with an empty vector exhibited a Muv
phenotype with 100% penetrance (n=209). lin-56(n2728); lin-15B(n744) animals fed
bacteria expressing lin-3 dsRNA exhibited a Muv phenotype with 85% penetrance
(n=179), and the expressivity of the Muv animals, as determined by the number of
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ectopic pseudovulvae, was noticeably lower than animals fed control bacteria.
Therefore, like other class A synMuv genes, lin-56 might act upstream of lin-3.
LIN-56 is a ubiquitous nuclear protein
To determine the expression pattern of the LIN-56 protein, we generated antiLIN-56 antibodies, which recognized a protein of approximately 40 kD in wild-type but
not lin-56(n2728) protein extracts (Figure 2A); the predicted size of LIN-56 is 37 kD. The
anti-LIN-56 antibodies sometimes appear to recognize a doublet (data not shown),
which could represent alternative splice variants of lin-56 or products resulting from
distinct transcriptional initiation sites. Although the three lin-56 cDNA clones (courtesy of
Y. Kohara) analyzed are identical in sequence and lack any evidence of trans-splicing,
SL2 trans-splicing of the ZK673.3 mRNA has been observed by others (Blumenthal et
al., 2002; Hwang et al., 2004). Alternatively, LIN-56 may be post-translationally
modified.
The anti-LIN-56 antibodies were used for whole-mount staining of worms.
Starting at the late one-cell or early two-cell stage, LIN-56 staining was observed in the
nuclei of most if not all somatic cells throughout embryonic and larval development as
well as adulthood (Figure 2B,C,E; data not shown). Nuclear staining was never
observed with the anti-LIN-56 antibodies in the somatic cells of lin-56(n2728) animals of
any stage (Figure 2D; data not shown), demonstrating specificity. LIN-56 is present in
the P(3-8).p vulval equivalence group and their descendants throughout vulval
development (Figure 2E). LIN-56 did not co-localize with chromatin during metaphase or
anaphase (Figure 2F,G). Germline staining was observed in both wild-type and
lin-56(n2728) animals and thus was not specific.
To confirm that LIN-56 is localized to nuclei, we performed subcellular
fractionation of embryo lysates. We used lamin as a marker for nuclear fractions
(Gruenbaum et al., 2002; Liu et al., 2000) and SQV-4 as a marker for cytosolic material
(Hwang and Horvitz, 2002). The majority of the LIN-56 protein co-fractionated with
nuclear lamin; LIN-56 protein was barely detectable in the SQV-4-containing cytosolic
fraction (see Figure 3B below). LIN-56 is thus stably associated with nuclei.
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LIN-56 protein, but not lin-56 mRNA, is reduced in lin-15A(lf) mutants
Since lin-56, lin-8, lin-15A, and lin-38 all have a class A synMuv loss-of-function
phenotype (Ferguson and Horvitz, 1989; Thomas et al., 2003), we analyzed the impact
of loss-of-function mutations in these other class A synMuv genes on the expression of
LIN-56. The LIN-56 staining pattern of lin-8(n2731) and lin-38(n751) animals we
observed after staining with anti-LIN-56 antibodies was not different from that of the wild
type (Figure 3A; data not shown). lin-8(n2731) causes an early nonsense mutation and
is likely a protein null allele (Davison et al., 2005). lin-38(n751) is a partial loss-offunction allele, as null mutations in lin-38 are inviable (Saffer and Horvitz, unpublished
results). By contrast, LIN-56 nuclear staining was greatly reduced in lin-15A(n767) and
lin-15AB(e1763) individuals at all stages (Figure 3A; data not shown). The lin-15A(n767)
allele is a small deletion predicted to result in a truncated LIN-15A protein (Huang et al.,
1994). The lin-15AB(e1763) allele is a large deletion that eliminates lin-15A as well as
its upstream genomic neighbor, lin-15B (Clark et al., 1994; Huang et al., 1994).
Fractionation experiments and immunoblot analyses confirmed that LIN-56 protein was
barely detectable in the nuclear fractions of lin-15A(n767) and lin-15AB(e1763) embryos
(Figure 3B). Also, the cytosolic fractions of these lin-15A(lf) mutants contained no more
LIN-56 protein than the cytosolic fraction of wild-type embryos (Figure 3B), indicating
that the reduced amount of LIN-56 in the nuclei of lin-15A(lf) mutants was not the result
of mislocalization of this protein to the cytosol. Instead, the total amount of LIN-56
protein in lin-15A(lf) animals was greatly reduced in comparison to that in the wild type,
suggesting that lin-15A is required for the expression or stability of lin-56 mRNA or
LIN-56 protein.
To determine if the low level of LIN-56 protein in lin-15A(lf) animals was the result
of decreased transcription or stability of lin-56 mRNA, we used quantitative real-time
RT-PCR (Heid et al., 1996) to measure lin-56 mRNA levels in wild-type, lin-15A(n767)
and lin-15AB(e1763) animals relative to hexokinase mRNA levels. Relative lin-56 mRNA
levels appeared to be somewhat higher in lin-15A(n767) and lin-15AB(e1763) animals
than in wild-type animals (Figure 3C; see also Figure 4B). The reason for the apparent
increase in lin-56 mRNA levels in the two lin-15A(lf) mutants analyzed was not
156
determined. The decreased amount of LIN-56 protein in lin-15A(lf) as compared to wildtype animals thus likely results from a post-transcriptional event, suggesting that
LIN-15A is required for either translation or stability of LIN-56 protein.
LIN-15A protein, but not lin-15A RNA, is reduced in lin-56(lf) mutants
Antisera directed against the LIN-15A protein revealed broad nuclear staining
(Figure 4A and data not shown). Since LIN-56 protein levels are reduced in lin-15A(lf)
mutants, we examined whether LIN-15A protein levels are reduced in lin-56(lf) mutants.
LIN-15A nuclear staining was greatly reduced in lin-56(n2728) and lin-56(n3355)
embryos (Figure 4A), indicating that lin-56 is likely required for expression or stability of
lin-15A RNA or LIN-15A protein. Alternatively, it is possible that lin-56 is required for the
nuclear localization of LIN-15A. Loss of lin-56 function reduced LIN-15A levels more in
late than in early embryos. LIN-15A levels appeared greater in early lin-56(n3355)
embryos than in early lin-56(n2728) embryos (data not shown), suggesting that n3355
may not be as strong an allele of lin-56 as is n2728 or that another gene deleted by
n2728 may also have an effect on LIN-15A levels. LIN-15A expression and localization
in lin-8(n2731) and lin-38(n751) embryos were indistinguishable from those in wild-type
(data not shown).
We used RT-PCR to measure the amount of lin-15A RNA in wild-type,
lin-56(n2728), lin-15A(n767), and lin-15AB(e1763) animals. lin-15A RNA was amplified
from both the wild-type and lin-56(lf) RNA samples but not from the lin-15A(lf) RNA
samples (Figure 4B). The decreased amount of LIN-15A protein in lin-56(lf) as
compared to wild-type animals thus likely results from a post-transcriptional event,
consistent with the hypothesis that LIN-56 is required for either translation or stability of
LIN-15A protein.
Overexpression of lin-56 does not rescue the lin-15AB(lf) synMuv phenotype, nor
does overexpression of lin-15A rescue the lin-56(lf) synMuv phenotype
If lin-15A acts only to control the translation or stability of LIN-56 protein, then
restoring the level of LIN-56 protein should rescue the lin-15A(lf); synMuvB(lf) synMuv
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phenotype. To test this possibility, we induced a lin-56 cDNA under the control of heatshock promoters shortly after L1 lethargus in lin-56(n2728); lin-15B(n744) animals and
rescued the synMuv phenotype (Table 1). The same treatment failed to rescue the
synMuv phenotype of lin-15AB(e1763) animals (Table 1). As assayed by immunoblot,
LIN-56 protein was produced under these conditions at levels at least ten-fold greater
than those of endogenous LIN-56 in the wild type (Supplemental Figure 1). The
mechanism that normally prevents LIN-56 protein accumulation in lin-15AB(e1763)
animals is likely overwhelmed by the level of LIN-56 produced by heat-shock
overexpression. Also, LIN-56 produced from the heat-shock promoters in
lin-15AB(e1763) animals did not obviously differ in electrophoretic mobility from that in
lin-56(n2728); lin-15B(n744) animals by immunoblot (Supplemental Figure 1). Since
overexpression of LIN-56 failed to rescue the synMuv phenotype of a lin-15A(lf);
synMuvB(lf) mutant, lin-15A cannot function only to positively regulate the translation or
stability of LIN-56 protein.
Likewise, if lin-56 acts only to control the translation or stability of LIN-15A
protein, then restoring the level of LIN-15A protein should rescue the lin-56(lf);
synMuvB(lf) synMuv phenotype. To test this possibility, we induced a lin-15A cDNA
under the control of heat-shock promoters shortly after L1 lethargus in lin-36(n766);
lin-15A(n767) animals and rescued the synMuv phenotype (Table 2). The same
treatment failed to rescue the synMuv phenotype of lin-56(n2728); lin-36(n766) animals
(Table 2). Since overexpression of LIN-15A failed to rescue the synMuv phenotype of a
lin-56(lf); synMuvB(lf) mutant, lin-56 cannot function only to positively regulate the
translation or stability of LIN-15A protein.
LIN-56 and LIN-15A physically interact
Since LIN-56 protein levels are reduced in a lin-15A mutant background and
LIN-15A protein levels are reduced in a lin-56 mutant background, we hypothesized that
LIN-56 and LIN-15A might normally be stabilized by association with each other in a
functional complex. To investigate if LIN-56 and LIN-15A can interact, we used the
GAL4-based yeast two-hybrid system (Fields and Song, 1989). The class B synMuv
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proteins LIN-37 and LIN-53 were previously shown to interact using this approach
(Walhout et al., 2000a; see also Figure 5). Class A synMuv proteins LIN-56 and
LIN-15A interacted specifically with each other, as detected using yeast cotransformed
with either constructs DB-LIN-15A and AD-LIN-56, or DB-LIN-56 and AD-LIN-15A
(Figure 5). By contrast, neither LIN-56 nor LIN-15A appeared to interact specifically with
either LIN-37 or LIN-53 (Figure 5).
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Discussion
LIN-56 and LIN-15A might function as a complex in vivo
lin-56 and lin-15A encode putative transcription factors that are present in most
or all cells during C. elegans development and in adulthood. The LIN-56 and LIN-15A
proteins share a THAP-like C2CH motif and are dependent on each other for wild-type
levels. lin-15A is required at the post-transcriptional level for LIN-56 protein expression
or stability, and vice versa. As overexpression of LIN-56 did not rescue the lin-15A(lf)
synMuv phenotype, it seems unlikely that lin-15A functions only to regulate LIN-56
protein levels. Likewise, expression of lin-15A was unable to rescue the lin-56(lf)
synMuv phenotype, suggesting that lin-56 does not function only to regulate LIN-15A
levels. Rather, given the ability of LIN-56 and LIN-15A to interact in the yeast twohybrid system, we favor a model in which LIN-56 and LIN-15A normally associate in a
functional complex required for the inhibition of vulval cell fates, with the absence of one
of the complex subunits resulting in the instability of the other(s). This model is
supported by genetic evidence showing that lin-15A and lin-56 are the only pair of class
A synMuv genes that do not act in parallel to each other (Andersen et al., 2008). The
function of neither lin-8 nor lin-38 is required for the normal expression or localization of
the LIN-56 and LIN-15A proteins, so lin-8 and lin-38 function cannot be required for an
association of the LIN-56 and LIN-15A proteins in vivo.
The class A synMuv genes likely directly regulate gene expression
The THAP C2CH domain is conserved from C. elegans to humans and was
initially proposed to mediate DNA binding on the basis of its similarity to a region of the
Drosophila P element transposase (Roussigne et al., 2003). The THAP domain of the
human protein THAP1 has since been shown to possess zinc-dependent sequencespecific DNA binding activity in vitro (Clouaire et al., 2005). Furthermore,
coimmunoprecipitation studies revealed that THAP1 associates in vivo with the
promoter of a pRB/E2F cell-cycle target gene (Cayrol et al., 2007).
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Several C. elegans proteins contain THAP domains, THAP-like domains, or both
(Clouaire et al., 2005; Reddy and Villeneuve, 2004). Analysis of a subset of the THAP
domain-containing proteins in C. elegans suggests that they might mediate access to
chromatin remodeling and chromatin-modifying complexes (Reddy and Villeneuve,
2004). Specifically, HIM-17 and the class B synMuv proteins LIN-15B and LIN-36
contain one or more THAP domains. HIM-17 is required both for accumulation of
histone H3 methyl-lysine 9 in the germline and for meiotic recombination, suggesting a
link between these two processes (Reddy and Villeneuve, 2004). The class B synMuv
proteins are thought to silence genes required for vulval cell-fate specification through
chromatin remodeling, most likely including histone H3 lysine-9 methylation (Andersen
and Horvitz, 2007; Ceol and Horvitz, 2001; Couteau et al., 2002; Lu and Horvitz, 1998;
Solari and Ahringer, 2000; von Zelewsky et al., 2000). Furthermore, the class B synMuv
protein LIN-35 Rb acts with HIM-17 in meiotic recombination (Reddy and Villeneuve,
2004).
The region of similarity between the class A synMuv proteins LIN-56 and LIN-15A
shares the C2CH signature of the THAP domain but lacks additional conserved
residues, such as an invariant tryptophan and a C-terminal AVPTIF motif (Clouaire et
al., 2005). This variant THAP-like domain is found also in HIM-17 and LIN-15B (Reddy
and Villeneuve, 2004). Given the nuclear localization of LIN-56 and LIN-15A, we
suggest that this variant C2CH motif is also likely to mediate interaction with DNA,
chromatin, or chromatin-associated proteins. Together with the nuclear localization of
LIN-8 and its physical interaction with LIN-35 Rb (Davison et al., 2005), these results
strongly suggest that class A synMuv proteins generally inhibit vulval cell-fate
specification through the regulation of transcription.
The class A synMuv genes, including both lin-15A and lin-56, repress expression
of lin-3 EGF redundantly with the class B synMuv genes (Andersen et al., 2008; Cui et
al., 2006a). It was previously shown that RNAi of lin-3 can suppress the lin-15A(lf);
synMuvB(lf) synMuv phenotype (Cui et al., 2006a), and we have shown that RNAi of
lin-3 can also suppress the lin-56(lf); synMuvB(lf) synMuv phenotype, indicating that
both lin-15A and lin-56 could act upstream of lin-3. Given their molecular identities, the
161
class B synMuv genes likely repress gene expression by chromatin remodeling. Based
on their potential DNA-binding THAP-like domains and nuclear localization, we propose
that the class A synMuv genes also regulate transcription. The class A and B synMuv
proteins might both directly repress expression on lin-3. Alternatively, either one or both
classes of synMuv proteins could directly regulate the expression of another protein,
which subsequently represses lin-3.
Class A synMuv genes might have functions beyond those in vulval development
Mutation of the class A synMuv genes, unlike mutation of the class B synMuv
genes, has not been reported to result in defects other than those associated with vulval
development. Specifically, mutation of the class A synMuv genes does not appear to
cause cell-cycle defects (Boxem and van den Heuvel, 2002; Fay et al., 2002), reduced
gene expression from repetitive transgene arrays (Hsieh et al., 1999), or sterility or
lethality (Beitel et al., 2000; Belfiore et al., 2002; Ceol and Horvitz, 2001; Couteau et al.,
2002; Dufourcq et al., 2002; Lu and Horvitz, 1998; Melendez and Greenwald, 2000;
Unhavaithaya et al., 2002; von Zelewsky et al., 2000). Nonetheless, the broad
expression patterns of the LIN-8 (Davison et al., 2005), LIN-15A, and LIN-56 proteins
suggest that additional roles might exist for the class A synMuv genes. Perhaps the
class A synMuv genes function redundantly with loci other than the class B synMuv
genes to regulate biological processes other than the vulval cell-fate decision. This
possibility seems particularly likely for lin-8, which is a member of a novel C. elegans
gene family with 16 other genes (Davison et al., 2005).
Implications for mammalian tumorigenesis
The inhibition of vulval development in C. elegans involves multiple redundant
pathways – both a class A and a class B synMuv gene must be inactivated for ectopic
vulval development to occur (Ferguson and Horvitz, 1989). The class B synMuv genes
include counterparts of the mammalian tumor-suppressor gene Rb and genes that
interact with Rb (Ceol and Horvitz, 2001; Lu and Horvitz, 1998). pRb and the related
proteins p107 and p130 play critical roles in mammalian cell-cycle regulation, apoptosis,
162
development, and differentiation (Classon and Harlow, 2002), and Rb is often mutated
in human cancers (Nevins, 2001). Inappropriate activation of EGF/Ras signaling is also
a common event in cancers (Normanno et al., 2006). Because the class A synMuv
genes function redundantly with an Rb pathway to repress transcription of an EGF
ligand and inhibit Ras-mediated vulval cell-fate specification, we propose that analogous
THAP domain proteins in mammals act as tumor suppressor genes by repressing the
expression of EGF-like ligands.
Acknowledgments
We thank Erik Andersen, Craig Ceol, Alison Frand, and Niels Ringstad for editorial
comments, Melissa Harrison for help with yeast two-hybrid analysis, Erik Andersen for
construction of lin-15A and lin-56 Gateway entry clones, Beth Castor for help with DNA
sequence determination, Na An for strain management, Yuji Kohara for EST cDNA
clones, Alan Coulson and the C. elegans Sequencing Consortium for cosmid clones and
sequences, and Steve Bell for use of the ABI PRISM 7000 Sequence Detection System.
The Deficiency strains were provided by Theresa Stiernagle of the Caenorhabditis
Genetics Center, which is supported by the NIH National Center for Research
Resources. This work was supported by NIH grant GM24663 to H.R.H.. E.M.D. was
supported by a Howard Hughes Medical Institute predoctoral fellowship. L. S. H. was
supported by a March of Dimes Birth Defects Foundation to P.W.S. H.R.H. and P.W.S.
are Investigators of the Howard Hughes Medical Institute.
163
Table 1: lin-56 overexpression rescues the lin-56(lf); lin-15B(lf) but not the
lin-15AB(lf) synMuv phenotype
Heat
Genotype
Transgene Line
% Muva ± s.d. (n)b
shock
lin-56(n2728); lin-15B(n744)
Phs-lin-56
1
2
lin-56(n2728); lin-15B(n744)
lin-15AB(e1763)
97.9% ± 0.9% (490)
16.5% ± 6.8% (340)
99.7% ± 0.3% (608)
17.5% ±15.3% (244)
Phs-gfp
1
+
100% ± 0% (319)
100% ± 0% (365)
Phs-lin-56
1
+
+
+
+
+
100% ± 0% (280)
100% ± 0% (211)
100% ± 0% (117)
100% ± 0% (112)
100% ± 0% (172)
100% ± 0% (319)
100% ± 0% (140)
100% ± 0% (226)
100% ± 0% (153)
100% ± 0% (107)
+
100% ± 0% (301)
100% ± 0% (158)
2
3
4
5
lin-15AB(e1763)
+
+
Phs-gfp
1
lin-56(n2728); lin-15B(n744) or lin-15AB(e1763) hermaphrodites carrying an
extrachromosomal array of either a lin-56 cDNA (Phs-lin-56) or the gfp coding
sequence (Phs-gfp) fused to the C. elegans heat-shock promoters and a dominant
Rol marker were synchronized by bleaching and starvation-induced L1 arrest.
Expression from the heat-shock promoters was induced at the early L2 stage (2425 hrs post starvation) by incubation at 33°C for 1 hr. Animals were assayed in at
least three individual batches, with at least 100 Rol worms analyzed in total for
each line.
a
% Muv, percentage of Rol animals that were Muv
b
n, number of animals examined
164
Table 2: lin-15A overexpression rescues the lin-36(lf); lin-15A(lf) but not the
lin-56(lf); lin-36(lf) synMuv phenotype
Heat
Genotype
Transgene Line
% Muva (n)b
shock
lin-36(n766); lin-15A(n767)
none
99.6% (251)
lin-36(n766); lin-15A(n767)
Phs-lin-15A
1
2
lin-56(n2728); lin-36(n766)
none
lin-56(n2728); lin-36(n766)
Phs-lin-15A
100% (39)
16% (96)
99% (86)
12% (93)
-
99.6% (234)
100% (177)
+
99.8% (605)
2
99.6% (227)
+
100% (675)
lin-36(n766); lin-15A(n766) or lin-56(n2728); lin-15A(n766) hermaphrodites with
or without an extrachromosomal array of lin-15A cDNA (Phs-lin-56) fused to the C.
elegans heat-shock promoters and a GFP+ marker were synchronized by
bleaching and starvation-induced L1 arrest. Expression from the heat-shock
promoters was induced at the early L2 stage (24-25 hrs post starvation) by
incubation at 33°C for 1 hr.
a
b
1
+
+
% Muv, percentage of animals that were Muv
n, number of animals examined
165
Figure 1: Cloning of lin-56
(A) Genetic map of the lin-56 region. Deficiency mapping placed lin-56 between the right
endpoint of mnDf29 and the left endpoint of mnDf90, as defined by the markers daf-19
and bli-1, respectively (shaded region Cloned genes are in black; genes not cloned are
in grey. Shaded rectangles indicate regions known to be deleted by each deficiency,
and open rectangles indicate regions that contain a deficiency endpoint and might be
deleted by each deficiency.
(B) Transformation rescue of lin-56. Predicted genes contained within the rescuing
cosmid ZK673 (top) and subclones derived from this cosmid (bottom) are shown. The
region deleted in lin-56(n2728) mutants is indicated by the solid bar. The sequences
flanking the lin-56(n2728) deletion are ACCAAAGGAGGAGGTCAGCC [11190 bp
deletion] CCTTGTGGGGGAACAATGCG. Insertion of a TGA stop codon is indicated by
a closed arrowhead.
(C) Sequence of the LIN-56 protein. The glutamine mutated to an ochre stop codon in
the n3355 allele is shaded. The region aligned in (D) is underlined.
(D) Alignment of the novel C2CH motif found in LIN-56, LIN-15A, LIN-15B, HIM-17, and
three additional uncharacterized C. elegans proteins. Y32B12B.4 contains five repeats,
denoted r1-r5, of the motif. Conserved cysteines and histidine are in red. Solid and
shaded boxes indicate identities and similarities, respectively, with LIN-56. Positions of
missense mutations found in lin-15A(n433) and lin-15A(n749) mutants are marked with
arrowheads.
166
C
MDHHAMYRTAEFNKTTVRLLAEFIEKTGQNATIVNMDSFLEFFAYLNPTA
50
PIPTVPEIEKQLLLKSPIRCIVCGMETESDSAVTLSIDNASIILTATVIG
100
YCRDPSDAVNQIRKESLRACTKHFNSIFHVIFEGLQIENTYCAHHAKYSL
150
ANRWCKVYTMIRSSLGEQFTKFDVRNFKSILQSFLDTFGEIDDDKKDKES
200
SHFDECFEEMDSENVEIKMESPQEEAAEKSKFSENLVEVKLEPIETHELD
250
KTISDFSSSDIIDSSQKLQQNGFPEKVEQMDKYSNKLKDEASDKKYEKPG
300
KKDYVEEEGYWAPITDSEDDEA
322
n3355stop
D
LIN-56
ZK673.4
LIN-15A
LIN-15B
T25B9.8
HIM-17
Y32B12B.4 r1
Y32B12B.4 r2
Y32B12B.4 r3
Y32B12B.4 r4
Y32B12B.4 r5
R
P
P
R
P
K
Q
R
R
R
R
C
C
C
C
C
C
C
C
C
C
C
I
I
I
A
L
V
S
F
F
F
A
V
I
L
V
V
L
L
L
L
L
V
C
C
C
C
C
C
C
C
C
C
C
G
G
E
G
N
D
P
P
A
P
C
M
N
K
H
Q
D
N
N
T
N
Q
E
E
A
L
Q
W
K
K
K
K
S
T
V
L
E
M
K
N
N
N
I
V
E
P
L
I
E
K
C
C
C
C
K
S
G
M
H
M
V
I
F
S
S
P
D
H
R
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T
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M
M
V
T
R
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R
K
D
K
K
K
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S
S
L
V
M
Q
H
I
K
M
V
I
I
K
R
V
V
V
V
V
L
lin-15A(n749)Q
T
R
A
N
S
P
V
F
F
F
K
L
V
M
V
V
F
L
K
I
M
V
R
-
A
-
P
-
S
T
H
V
I
S
T
I
N
S
D
D
D
D
E
N
E
T
E
E
E
E
N
D
N
N
T
A
S
Y
Y
L
N
D
E
E
D
E
E
E
E
E
E
A
A
A
K
A
R
K
K
K
K
K
S
A
V
L
Y
S
Y
Y
Y
C
F
I
I
K
L
I
F
I
I
I
I
L
I
F
V
I
M
L
I
I
I
I
L
L
L
L
M
I
I
A
V
A
A
A
T
T
M
L
Y
D
I
I
I
I
F
A
A
A
G
V
I
G
G
G
G
A
T
A
A
C
C
V
L
L
L
L
L
V
V
V
I
V
I
S
S
S
S
V
I
L
M
Y
M
H
L
L
L
L
L
S
S
-
S
S
-
I
I
A
A
-
G
T
S
R
N
S
S
S
A
A
R
Y
D
G
G
D
D
D
D
T
T
A
C
Q
H
E
K
K
F
F
D
D
K
R
K
F
F
Y
M
D
F
F
Y
D
T
R
T
D
T
D
D
D
D
D
P
I
M
L
M
I
A
A
P
A
H
S
R
A
G
D
K
G
G
E
G
E
D
Q
T
Q
K
K
L
L
L
L
-
A
A
A
A
A
A
A
A
A
A
A
V
K
E
Q
K
L
L
L
V
L
H
N
R
K
L
E
A
S
S
N
T
L
Q
D
A
F
L
S
F
F
F
F
N
lin-15A(n433)V
M
L
A
A
A
A
-
I
I
I
A
A
A
D
D
D
D
L
R
L
R
R
R
K
S
S
S
S
V
K
S
H
M
T
Y
G
G
G
N
E
E
E
E
Q
N
K
R
R
T
R
S
Y
R
S
R
R
N
Q
H
Q
C
L
L
L
K
F
P
K
R
R
M
M
T
A
L
I
I
I
R
R
T
R
Y
K
L
T
Y
Y
Y
N
A
V
M
I
C
I
V
I
I
I
V
C
C
C
C
C
C
C
C
C
C
C
T
L
Y
R
V
N
T
T
T
T
K
K
R
D
L
S
I
Q
Q
Q
Q
T
H
H
H
H
H
H
H
H
H
H
H
F
S
V
F
L
F
I
V
I
I
F
124
231
266
996
344
695
121
384
704
947
1187
Figure 2: LIN-56 is broadly expressed and localized to nuclei
(A) Immunoblot of wild-type and lin-56(n2728) protein extracts probed with anti-LIN-56
antibodies. The arrow indicates the band that corresponds to LIN-56. Molecular weights
of marker proteins are indicated at left in kilodaltons (kD).
(B-E) Whole-mount staining with anti-LIN-56 antibodies (red), DAPI (blue), and either
anti-tubulin antibody (green) in embryos or MH27 antibody (green) in larvae as a fixation
control. Scale bars, 5 µm. Anterior is to the left in all images. pb, polar body.
(B,C) LIN-56 staining is observed in the nuclei of most if not all cells throughout
embryonic development. Shown are (B) 4-cell and (C) late-stage wild-type embryos.
(D) Nuclear staining is never observed with anti-LIN-56 antisera in lin-56(n2728)
animals. Shown is a 4-cell lin-56(n2728) embryo.
(E) LIN-56 is present in the P(3-8).p vulval equivalence group and their descendents
during wild-type vulval development. Shown is the midbody of a wild-type L2 larva.
Arrowheads point to nuclei of P(3-8).p. MH27 stains the apical borders between the
Pn.p cells.
(F,G) LIN-56 does not co-localize with chromatin during metaphase or anaphase. (F)
and (G) show two-cell wild-type embryos. ana, anaphase; meta, metaphase; pro,
prophase; telo, telophase.
169
250
150
100
75
50
37
ild
(n
2
ty
pe
lin
-5
6
w
72
A
8)
anti-LIN-56
DAPI
triple with anti-tubulin
DAPI
triple with anti-tubulin
DAPI
triple with anti-tubulin
pb
B wild-type embryo
anti-LIN-56
C wild-type embryo
anti-LIN-56
pb
D lin-56(n2728) embryo
anti-LIN-56
E wild-type developing vulva
DAPI
triple with MH27
anti-LIN-56
DAPI
triple with anti-tubulin
meta
pro
F wild-type embryo
telo
ana
G wild-type embryo
Figure 3: LIN-56 protein but not lin-56 mRNA levels are greatly reduced in
lin-15A(lf) mutants
(A) LIN-56 nuclear staining is greatly reduced in lin-15A(n767) and lin-15AB(e1763)
mutants, but not in lin-8(n2731) or lin-38(n751) mutants. Shown are 4-cell embryos
stained with anti-LIN-56 antibody (red), DAPI (blue), and anti-tubulin antibody (green).
Scale bars, 5 µm. Anterior is to the left in all images.
(B) Immunoblot of nuclear and S-100 cytosolic fractions derived from wild-type,
lin-56(n2728), lin-15AB(e1763), and lin-15A(n767) embryos probed with anti-LIN-56
antibody. Shown for reference are the same immunoblot probed with anti-lamin and
anti-SQV-4 antibodies as markers for nuclear and cytosolic proteins, respectively.
(C) lin-56 mRNA levels are not reduced in lin-15A(lf) mutants as compared to the wild
type. Quantitative real-time RT-PCR (Heid et al., 1996) was used to measure lin-56
mRNA levels relative to hexokinase mRNA levels. Results for each genotype are shown
normalized to those for the wild type. Error bars, s.d.
173
!
lin-15A(n767)
lin-15AB(e1763)
lin-8(n2731)
lin-38(n751)
./!#14-5167
!"#$%&'(#!)'*+,-'./0'./!#1!232%#/
AB
(e
17
63
)
-1
5
lin
pe
ty
76
7)
A(
n
-1
5
lin
ild
w
C
Relative lin-56 mRNA levels + SD
Nuclei
5.0
4.0
3.0
2.0
1.0
lin
-
lin
-
15
15
27
(n
A(
n
7)
63
)
17
76
AB
(e
pe
63
7)
28
76
27
A(
n
)
17
28
AB
(e
pe
ty
56
15
15
w
ild
lin
-
lin
-
lin
-
(n
ty
56
w
ild
lin
-
B
S-100
anti-LIN-56
anti-lamin
anti-SQV-4
)
)
Figure 4: LIN-15A protein but not lin-15A RNA levels are reduced in lin-56(lf)
mutants
(A) LIN-15A nuclear staining is greatly reduced in lin-56(n2728) and lin-56(n3355)
mutants. Shown are multicellular embryos stained with anti-LIN-15A antibodies (green),
anti-tubulin antibody (red), and DAPI (blue). The genotype of each embryo is indicated
on the left. Scale bars, 5 µm. Anterior is to the left in all images.
(B) lin-15A RNA levels are not reduced in lin-56(n2728) mutants compared to the wild
type. RT-PCR was used to determine levels of lin-15A, lin-56, and hexokinase RNA in
total RNA derived from wild-type, lin-56(n2728), lin-15A(n767), and lin-15AB(e1763)
animals. Marker DNA fragments are indicated at left. The number of PCR cycles is
indicated at right.
176
A
anti-LIN-15A
wild type
lin-15AB(e1763)
lin-56(n2728)
lin-56(n3355)
DAPI
triple with anti-tubulin
Genotype:
Amplicon:
1000 bp
850 bp
1000 bp
850 bp
1000 bp
850 bp
650 bp
lin-15A
lin-56
15
lin
-
15
lin
-
27
27
AB
(e
17
63
)
)
67
28
n7
A(
(n
63
)
17
67
(e
e
AB
63
)
17
28
n7
A(
(n
e
(e
)
)
67
28
n7
AB
ty
p
56
lin
-
w
ild
15
lin
-
15
lin
-
27
A(
ty
p
56
lin
-
w
ild
15
lin
-
15
lin
-
e
(n
ty
p
56
lin
-
w
ild
)
)
)
B
hexokinase
25 cycles
650 bp
30 cycles
650 bp
35 cycles
Figure 5: LIN-15A and LIN-56 interact with each other in the yeast two-hybrid
system
Growth of cotransformant Y190 colonies on SC-Trp-Leu-His media in either the absence
or presence of 25 mM 3-AT. Interaction of the DB- and AD-fusion proteins results in
increased expression of a GAL1::HIS3 reporter gene, permitting colony formation in the
absence of histidine and presence of 3-aminotriazole (3-AT), a competitive inhibitor of
the enzyme encoded by HIS3. Plasmids cotransformed into each strain are indicated at
left. Interactions are evident between DB-LIN-15A and AD-LIN-56, DB-LIN-56 and ADLIN-15A, as well as DB-LIN-53 and AD-LIN-37, as previously reported (Walhout et al.,
2000a). DB, Gal4 DNA binding domain. AD, GAL4 activation domain.
179
SC-Trp-Leu-His + 0 mM 3-AT
Dilution
-2
-3
-4
-5
Factor: 10 10 10 10
DB-LIN-53 + AD-empty
DB-LIN-15A + AD-empty
DB-LIN-56 + AD-empty
DB-LIN-53 + AD-LIN-15A
DB-LIN-53 + AD-LIN-37
DB-LIN-53 + AD-LIN-53
DB-LIN-53 + AD-LIN-56
DB-LIN-15A + AD-LIN-15A
DB-LIN-15A + AD-LIN-37
DB-LIN-15A + AD-LIN-53
DB-LIN-15A + AD-LIN-56
DB-LIN-56 + AD-LIN-15A
DB-LIN-56 + AD-LIN-37
DB-LIN-56 + AD-LIN-53
DB-LIN-56 + AD-LIN-56
10-2 10-3 10-4 10-5
SC-Trp-Leu-His + 25 mM 3-AT
10-2 10-3 10-4 10-5
10-2 10-3 10-4 10-5
al
s)
al
im
an
00
0
w
ild
- ty
pe
(1
(5
pe
- ty
ild
w
s)
im
an
an
0
(1
pe
- ty
al
al
im
s)
im
an
0
ild
s)
im
al
0
(1
w
hs
-g
fp
56
P
n- li
hs
P
an
im
(1
2
e
l in
e
l in
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n- li
hs
P
an
(1
1
ed
ix
m
pe
- ty
ild
w
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ag
st
ed
ix
m
8)
72
n2
6(
-5
lin
e
st
ag
e
al
s)
s)
Supplemental Figure 1
250
150
100
75
50
37
Phs-lin-56 transgenic animals express LIN-56
at least 10-fold higher than wild-type animals.
Immunoblot of wild-type, lin-56(n2728), or
transgenic protein extracts probed with antiLIN-56 antibodies. The arrow indicates the
band that corresponds to LIN-56. Molecular
weights of marker proteins are indicated at
left in kilodaltons.
Chapter Six
Future Directions
Chapter Six: Future Directions
182
Where is lin-3 ectopically expressed in each synMuv mutant?
I have shown that lin-3 is ectopically expressed throughout the animal in the
class AB synMuv mutants lin-15AB(e1763) and lin-3(n4441); lin-15B(n744) (Chapter
Two). However, the expression pattern of lin-3 has not been determined for other class
AB synMuv double mutants. In Chapter Three, I showed that global lin-3 mRNA levels
are repressed by all synMuv genes that I assayed. Furthermore, the level of lin-3
overexpression correlated well with the strength of the synMuv phenotype for each
synMuv double mutant. Each class A and B synMuv gene might function ubiquitously to
prevent ectopic lin-3 expression. Consistent with that possibility, most synMuv genes
that have been examined are expressed ubiquitously (Ceol and Horvitz, 2001, 2004;
Couteau et al., 2002; Davison et al., 2005; Harrison et al., 2006; Harrison et al., 2007a;
Unhavaithaya et al., 2002; von Zelewsky et al., 2000). However, it is also possible that
some synMuv genes repress lin-3 only in specific tissues.
Using the same fluorescence in situ hybridization (FISH) technique as in Chapter
Two, the expression pattern of lin-3 in class AB synMuv double mutants including each
of the class A and B synMuv genes should be assayed. This will determine if all class A
and B synMuv genes repress lin-3, and where each synMuv gene represses lin-3. By
determining if all synMuv genes affect lin-3 expression, a framework can be established
for thinking about the synMuv genes as all functioning through lin-3, as opposed to
different synMuv genes regulating different targets. Also, if any synMuv genes repress
lin-3 in only some cells, this might have implications for the design of certain
biochemical experiments (see below).
Most class B synMuv mutations have wild-type vulval development (Andersen et
al., 2008). For example, lin-15B(n744) is not Muv at any temperature, and I showed in
Chapter Two that lin-15B(n744) animals express only about 10 ectopic copies of lin-3
mRNA. However, mutations in some class B synMuv genes, including let-418, lin-13,
and trr-1, exhibit a Muv phenotype as single mutants (Ceol and Horvitz, 2004; Melendez
and Greenwald, 2000; von Zelewsky et al., 2000). These class B synMuv genes could
have a greater role repressing lin-3 compared to other class B synMuv genes, such that
the single mutants have sufficient ectopic lin-3 expression to cause a Muv phenotype.
183
Alternatively, these class B synMuv genes could have the same input into lin-3
repression as other class B synMuv genes, but could also be regulating other targets
responsible for the weak Muv phenotype of the single mutants. Determining the number
of ectopically expressed lin-3 mRNA molecules in these Muv class B synMuv single
mutants can distinguish between these possibilities.
Do any synMuv suppressors promote germline lin-3 expression?
In Chapter Two, I showed that lin-3 is expressed in the germline. It is not known
what the biological function of lin-3 is in the germline, or how the transcription of lin-3 in
the germline is regulated. The class B synMuv genes might regulate vulval
development by preventing somatic cells from adopting a more germline-like fate, a fate
that includes lin-3 expression. If the germline-like fate did not cause lin-3 expression,
then the class B synMuv genes might have no role in regulating lin-3 expression, and
class AB synMuv double mutants would appear grossly wild-type. Therefore, a
mutation that specifically prevents lin-3 expression in the germline, such as a mutation
in either a lin-3 enhancer element or in a gene that normally promotes lin-3 germline
expression, might suppress the synMuv phenotype. Mutations and RNAi experiments
have shown that loss-of-function of many genes can suppress the synMuv phenotype,
(Andersen et al., 2006; Clark, 1992; Cui et al., 2006b). FISH should be performed on
each of these suppressor mutants to determine the expression pattern of lin-3. If any of
the suppressors lack lin-3 expression specifically in the germline, then the
corresponding gene normally promotes the germline expression of lin-3. Null alleles of
lin-3 cause lethality, which might complicate attempts to determine the function of lin-3
in the germline by studying lin-3 mutants. Identifying genes that promote germline
expression of lin-3 could reveal the role of lin-3 in the germline. Moreover, the finding
that a synMuv suppressor gene is required for lin-3 expression in the germline would
support the model that the class B synMuv genes prevent ectopic vulval induction by
preventing somatic cells from inappropriately adopting germline-like fates.
184
Why is the synMuv phenotype temperature-sensitive?
synMuv double mutants exhibit a stronger synMuv phenotype at higher
temperatures, as judged by either penetrance or expressivity (Ferguson and Horvitz,
1989). This temperature-sensitivity is an aspect of the synMuv phenotype itself, since
most or all synMuv mutants are temperature-sensitive, including null mutants. However,
nothing is known about the molecular basis of this temperature-sensitivity. The
temperature-sensitivity could reflect an effect of temperature on chromatin structure or
accessibility at the lin-3 locus, on the function of the synMuv proteins themselves, or on
some other aspect of EGF signaling in C. elegans. It will be useful to determine if the
temperature-sensitive process in vulval development is synMuv repression of lin-3, lin-3
transcription in general, or downstream of lin-3 transcription. Because the lin-15A(n767)
mutant causes a low but observable level of ectopic lin-3 expression, it is an excellent
background to study the effects of temperature on synMuv repression of lin-3.
lin-15A(n767) mutants grown at 20˚C typically have 40-60 ectopic copies of lin-3 mRNA
(Chapter Two), and lin-15A(n767) mutants exhibit a low penetrance Muv defect at 25˚C
but not at 20˚C (Chapter Three). lin-3 expression could be assayed in wild-type animals
and in lin-15A(n767) mutants at 20˚C and 25˚C using FISH, counting the exact number
of lin-3 mRNA molecules expressed in the anchor cell and ectopically. If the effects of
temperature on the synMuv phenotype are downstream of lin-3 expression, then lin-3
mRNA levels will be unaffected by temperature in wild-type and lin-15A(n767) animals.
If lin-3 transcription is affected by temperature independently of synMuv repression,
then expression of lin-3, both ectopically and in the anchor cell, might increase at 25˚C
compared to 20˚C in wild-type and lin-15A(n767) animals. Finally, if the synMuv
repression of lin-3 is temperature-sensitive, then ectopic lin-3 mRNA levels in
lin-15A(n767) animals would be higher at 25˚C than at 20˚C, but lin-3 mRNA levels in
the anchor cell would be unaffected by temperature. These experiments will indicate
what step of synMuv and EGF-mediated vulval development is temperature-sensitive,
which can then direct future experiments to understand the molecular basis of this
temperature-sensitivity.
185
What transcriptional targets are responsible for the lin-38(null) lethality?
RNAi or a deletion of lin-38 causes a larval arrest phenotype, with the stage of
arrest ranging from L1 to L3 larvae (Chapter Four). Because the stage of arrest is not
consistent, the lethality is likely not caused by a specific developmental cell-fate defect,
but rather by a general requirement of lin-38 for growth and viability. The specific stage
at which animals lacking lin-38 arrest might reflect the time at which the maternal
stockpiles of lin-38 mRNA and protein are exhausted. The larval arrest phenotype
caused by loss of lin-38 is not similar to any phenotype known to be caused by either
loss-of-function or overexpression of lin-3. Therefore, lin-38 is likely to be regulating
other genes to promote viability and growth.
One way to identify such lin-38 targets is by seeking mutations that suppress the
lin-38 lethality. This will only work if lin-38 lethality is caused by misregulation of one or
a few key target genes. If lin-38 is a transcriptional repressor, then loss of lin-38 might
cause overexpression of a target gene, and mutating that target would then suppress
the lin-38 lethality. I have performed a preliminary pilot screen seeking mutations that
suppress the lethality of lin-38 loss-of-function, but did not identify any suppressors.
More extensive screens for suppressors of lin-38 lethality might identify targets that
lin-38 regulates to promote viability.
It will also be useful to determine the cellular focus for the lethality caused by loss
of lin-38. This could be tested by either rescue experiments using heterologous
promoters, or by mosaic analysis. lin-38 expressed from an extrachromosomal array
can effectively rescue the lin-38 lethal phenotype, and could be used for mosaic
experiments.
Another approach to understand the role of lin-38 in promoting viability is to
identify direct and indirect targets of lin-38. Microarray analysis of lin-38(tm736) animals
could identify genes regulated either directly or indirectly by lin-38. To identify direct
targets of lin-38, chromatin immunoprecipitation (ChIP) experiments (Solomon et al.,
1988) could be performed using either antibodies that recognize LIN-38 or an epitopetagged lin-38 transgene. Following the ChIP experiment, high-throughput sequencing
can identify enriched genes that are likely to be direct lin-38 targets (Barski and Zhao,
186
2009). Misregulation of one or more of these targets might be responsible for the
lethality causes by loss of lin-38. This can be tested by determining if RNAi or
overexpression of the putative target can phenocopy or suppress the lin-38 lethality.
What are the cis-acting synMuv elements in the lin-3 promoter?
A site in the lin-3 promoter identified by the lin-3(n4441) mutation is required for
class A synMuv repression of lin-3. lin-3(n4441) is a point mutation, approximately 200
base pairs upstream of the start of lin-3 transcription. The extent of the class A synMuv
element defined by the lin-3(n4441) mutation is not known, nor is it not known if there
are other sites in the lin-3 promoter, introns, or 3ʼ regions required for synMuv-mediated
repression of lin-3. If other cis-acting elements are required for the class A synMuv
genes to repress lin-3, then one way to identify them is to seek mutations that cause a
dominant class A synMuv phenotype. I have performed several such screens since
isolating lin-3(n4441), but have not identified any additional dominant class A synMuv
mutations. However, the mutational target for this screen could be quite small, so it
might be useful to continue seeking more dominant class A synMuv mutants.
A similar approach could be used to identify elements in the lin-3 promoter
required for class B synMuv repression of lin-3. A dominant class B synMuv mutation
might identify a site in the promoter of lin-3 (or another class B synMuv target gene)
required for class B synMuv-mediated repression. Several screens have been
performed in a class A synMuv mutant background seeking dominant class B synMuv
mutations (A.M.S, Craig Ceol, Scott Valastyan, and H.R.H, data not shown). One
complication is that many class B synMuv genes are haploinsufficient, particularly at
higher temperatures (Erik Andersen, personal communication). Because there are a
large number of class B synMuv genes, and mutations that cause loss-of-function are
much more common that potentially rare non-coding gain-of-function mutations, the vast
majority of isolates from screens for dominant class B synMuv mutations are likely to be
loss-of-function mutations in class B synMuv genes. Therefore, such screens need to
either be designed to avoid the problem of haploinsufficiency, or a rapid method for
identifying the desired dominant class B synMuv mutations from the initial isolates is
187
needed. If the screen is performed at lower temperatures, such as 20˚ C, then loss of
one copy of most class B synMuv genes might not cause a synMuv phenotype. The
downside to screening at lower temperatures is that weaker dominant class B synMuv
mutations might be missed, although the lin-3(n4441) mutation causes a very strong
class A synMuv phenotype that could be easily identified at any temperature.
Alternatively, using a balancer chromosome that covers the lin-3 locus, it would be
possible to very quickly determine which isolates are linked to lin-3, and whether they
are strongly dominant or not. The downside to this approach is that half of the isolates
will be on the balancer chromosome and extremely difficult to manipulate, effectively
lowering the efficiency of screening by a factor of two.
Alternatively, transgenic animals carrying mutant versions of the lin-3 locus could
be used to dissect the lin-3 promoter. Ideally, single copy integration (Frokjaer-Jensen
et al., 2008) would be used, because other transgenic techniques have the
complications of high copy number and unusual transcriptional regulation, and I have
shown that traditional extrachromosomal arrays are not useful for studying synMuv
transcriptional regulation (Appendix Three). I have integrated a wild-type copy of the
lin-3 locus using this technique, and the resulting strain has normal vulval development.
In a class B synMuv mutant background, a transgenic lin-3 locus with a class A synMuv
mutation such as lin-3(n4441) should cause a Muv phenotype, while the wild-type lin-3
locus or a lin-3 locus carrying an unrelated mutation should not cause a Muv phenotype.
A series of small deletions or single nucleotide mutations, centered around the
lin-3(n4441) mutation, could be used to determine the extent of the class A synMuv
element in the lin-3 promoter. The same technique can be used to seek class B
synMuv elements in the lin-3 promoter. For example, any matches to the DP/E2F
consensus binding sequence (Chi and Reinke, 2006) in the lin-3 locus could be tested
for class B synMuv activity in such an assay.
What proteins physically interact with class A synMuv proteins?
Biochemical and genetic evidence indicate that the class A synMuv proteins
LIN-15A and LIN-56 form a complex, and that complex formation is important for their
188
function (Chapters Three and Five). It is not known if other class A synMuv proteins
form complexes. Additionally, the class A synMuv protein LIN-8 and the class B
synMuv protein LIN-35 interact in vitro and in a yeast two-hybrid assay (Davison et al.,
2005). However, that interaction has not been confirmed in vivo, nor has it been tested
if there are other proteins that interact with class A synMuv proteins.
Immunoprecipitation experiments could identify proteins that form complexes with class
A synMuv proteins. Antibodies have been raised that recognize the class A synMuv
proteins LIN-8, LIN-15A, and LIN-56 (Davison et al., 2005; Chapter Five). Some of
these antibodies might be usable for immunoprecipitation. Additionally, I have produced
a lin-15A::GFP fusion that can effectively rescue the class A synMuv phenotype of a
lin-15A mutant, and this fusion protein could be precipitated with anti-GFP antibodies.
Immunoprecipitation experiments followed by western blots with antibodies that
recognize class A synMuv proteins would determine if the class A synMuv proteins form
a complex together. Additionally, mass spectrometry can be used to identify other
proteins that coimmunoprecipitate with class A synMuv proteins. Strong loss-of-function
alleles of lin-38 are lethal, and there might be additional essential class A synMuv genes
that are refractory to genetic identification. Biochemical approaches may be able to
identify such essential class A synMuv genes.
What are the molecular functions of the class A synMuv proteins?
The class A synMuv genes either directly or indirectly repress lin-3, and based on
their domains and nuclear localization it is likely that the class A synMuv genes function
as transcriptional regulators. It should be tested if the class A synMuv proteins are
either transcriptional activators or repressors. One approach is to fuse fragments of
different class A synMuv proteins to a well-characterized DNA-binding domain, such as
the GAL4 DNA-binding domain. Using a reporter with some basal transcription and a
GAL4 binding site, the transcriptional effects of each class A synMuv proteins can then
be tested. If the class A synMuv proteins contain domains that are either repressors or
activators in vivo, then the expression of the reporter would be modulated. Ideally these
experiments would be performed in C. elegans, perhaps using a reporter integrated as a
189
single copy, with the different GAL4::class A synMuv protein fusions quickly expressed
using conventional transgenesis techniques.
A similar approach would be to fuse the transcriptional activation domain of VP16
to different synMuv genes in vivo. The VP16 activation domain is a strong activation
domain and is functional in a wide range of organisms, including C. elegans (Sze et al.,
1997; Triezenberg et al., 1988). If the class A synMuv proteins normally do not function
in transcription, then the addition of the VP16 activation domain to a class A synMuv
gene will either have no effect, or will cause a loss-of-function (which would appear wildtype). If the class A synMuv proteins are activators, then the addition of the VP16
activation domain might cause no defect, or it might cause a constitutive gain-offunction, possible resulting in a synMuv suppression phenotype. If the class A synMuv
proteins normally function at the lin-3 promoter as transcriptional repressors, then
transforming the class A synMuv proteins into activators with the addition of the VP16
activation domain might cause inappropriate transcriptional activation of lin-3 and a Muv
phenotype. This will be distinguishable from a dominant-negative class A synMuv
phenotype, which would only have an effect in a class B synMuv mutant background.
Therefore, if fusion of the VP16 activation domain to any class A synMuv protein causes
a Muv phenotype, then that class A synMuv protein is likely to normally function as a
transcriptional repressor.
What proteins are present at the lin-3 promoter?
The element in the lin-3 promoter defined by lin-3(n4441) could be a binding site
for one or more class A synMuv proteins. Alternatively, the class A synMuv proteins
could regulate another protein that binds to that site. Several of the class A synMuv
genes contain either a zinc-finger domain or a THAP domain, both of which can
potentially bind to DNA. The best way to determine if the class A synMuv proteins are
present at the lin-3 promoter is by using ChIP (Solomon et al., 1988). Antibodies have
been raised that recognize some class A synMuv proteins, and might be usable for
ChIP. A fusion between LIN-15 and GFP is functional, and fusions between GFP (or a
different epitope tag) and the other class A synMuv genes could be easily constructed.
190
If no class A synMuv proteins are present at the lin-3 promoter, then either yeast onehybrid experiments (Li and Herskowitz, 1993) or another approach should be used to
identify what proteins do bind to the lin-3 promoter at the site of the lin-3(n4441)
mutation. If the class A synMuv proteins are present at the lin-3 promoter near the site
of the lin-3(n4441) mutation, then it can be tested if that association is abrogated by the
lin-3(n4441) mutation by repeating the ChIP experiments with lin-3(n4441) mutants.
Furthermore, it might be possible to understand how the class A synMuv genes interact
with each other to repress lin-3 by testing if the association of certain class A synMuv
proteins with the lin-3 promoter is dependent on the function of other class A synMuv
proteins.
The class B synMuv genes partially repress germline fates in the soma
(Unhavaithaya et al., 2002; Wang et al., 2005). I have shown that lin-3 is expressed in
the germline, and that the effects of class B synMuv mutations on vulval development
potentially reflect the class B synMuv genes repressing germline fates, and hence lin-3
expression. The class B synMuv genes could repress the expression of a small number
of master regulators of the germline fate, which in turn can activate a large number of
genes that are normally expressed in the germline. Alternatively, the class B synMuv
genes might more directly prevent germline fates in the soma by directly repressing
genes that are restricted to the germline. To distinguish between these models, it could
be tested if class B synMuv proteins are present at the promoters of germline genes that
are repressed by class B synMuv genes. The mRNA levels of the germline genes lin-3
and gpd-1 are repressed by synMuv genes, and levels of the germline protein PGL-1
are increased in many class B synMuv mutants (Unhavaithaya et al., 2002; Wang et al.,
2005; data not shown). If the class B synMuv genes directly impart somatic fates by
repressing germline genes in the soma, then some or all class B synMuv genes should
be present at the lin-3, gpd-1, and pgl-1 promoters.
The source of the biological material for ChIP experiments is an important
consideration. If the synMuv genes are localized to the lin-3 promoter, but only in some
cells, then ChIP experiments might be meaningful only if those specific cells are used as
the starting material. I have shown that the class A and B synMuv genes function
191
throughout the animal to repress ectopic expression of lin-3 (Chapter Two). Therefore,
whole animals might be a reasonable source of starting material for biochemical
experiments. However, in the cells that normally express lin-3, the synMuv genes might
not be functional, and the state of the lin-3 promoter could be different than in the cells
that do not express lin-3. To study synMuv repression of lin-3, the ideal source of
biological material would be the cells that do not normally express lin-3. There are three
main sources of lin-3 in late L2 and early L3 larvae: the anchor cell, the germline, and
the pharynx. Ideally, DNA and protein would be isolated from all cells except for those
three tissues. Mutants in which the germline does not proliferate can be used to
eliminate germline expression. These animals will obviously be sterile, but large
numbers of animals can be obtained with either a temperature-sensitive allele, or by
GFP-based sorting. The anchor cell is only one cell out of hundreds, so even if the
state of the lin-3 promoter is different in the anchor cell than in other cells, that might not
be a concern for ChIP experiments. Alternatively, material could be harvested at the L2
larval stage or earlier, before the anchor cell is generated. The pharynx is required for
viability, and unfortunately there is no way to grow animals lacking a pharynx.
However, the fraction of the animal that is lin-3-expressing pharynx might be sufficiently
low to allow investigation of the effects of synMuv mutations on the state of the lin-3
promoter using whole animals.
Concluding remarks
Proper development of the C. elegans vulva requires that expression of lin-3 EGF
be tightly restricted. Numerous genetic approaches have identified a large number of
synMuv genes that prevent ectopic expression of lin-3. C. elegans can be a challenging
system for biochemical experiments, particularly because of the difficulties in obtaining
DNA and proteins from a homogeneous population of cells. However, because the
synMuv genes all function on a well-defined in vivo target with biological relevance, and
because vulval development in C. elegans is highly amenable to genetic analysis, lin-3
and the synMuv genes are an excellent system for studying chromatin remodeling and
transcriptional repression in development. Some synMuv genes are homologous to
192
genes with well-characterized roles in chromatin remodeling and transcriptional
repression, while the function of other synMuv genes is unknown. Future investigations
of the synMuv genes will identify the specific molecular function of each synMuv gene,
and will elucidate how the synMuv genes interact with each other to tightly repress
ectopic expression of the EGF ligand lin-3.
Acknowledgments
I thank Kostas Boulias for helpful comments about this chapter.
193
Appendix One
Identification of new class A synMuv mutations
Adam Saffer and H. Robert Horvitz
Appendix One: Identification of new class A synMuv mutations
194
Materials and Methods
Strains and genetics
C. elegans strains were cultured by standard methods on OP50 bacteria
(Brenner, 1974).
The following mutations were used in this study:
LGIII: lin-52(n771) (Ferguson and Horvitz, 1989)
LGV: mys-1(n3681) (Ceol and Horvitz, 2004)
LGX: lin-15B(n744) (Ferguson and Horvitz, 1989)
Screens
L4 stage animals were mutagenized with 47 mM ethyl methanesulfonate (EMS)
for four hours. Approximately five mutagenized P0 animals were placed on each plate
and grown for three to four days at 20˚C. L4 stage F1 animals, each representing two
mutagenized haploid genomes, were then picked individually to plates. Typically no
more than 30 F1 animals were picked from a single plate of P0 animals. lin-15B(n744)
F1 animals were placed at 20˚ C, mys-1(n3681) F1 animals were placed at 22.5˚ C, and
lin-52(n772) F1 animals were placed at 25˚ C. F2 progeny were then screened for
multivulva (Muv) animals three to five days later. Each plate was screened on two
consecutive days (either three and four days later, or four and five days later, depending
on the growth rate) to increase the chances of recovering slow growing mutants. From
plates with Muv animals, several Muv animals and several wild-type siblings were
transferred individually to plates.
195
Results
The synthetic multivulva (synMuv) genes are grouped into two redundant
classes, A and B. Mutations in both a class A and a class B synMuv gene are required
to cause a strong Muv phenotype (Andersen et al., 2008; Ferguson and Horvitz, 1989).
Therefore, by mutagenizing superficially wild-type animals carrying a class B synMuv
mutation, it is possible to isolate Muv animals with class A synMuv mutations. Several
genetic screens have been performed seeking class A synMuv mutations (Ferguson
and Horvitz, 1989; Thomas et al., 2003) (Summarized in Table 1). However, these
screens were unable to recover mutations that caused both a Muv phenotype and
sterility. Clonal screens allow the isolation of mutations that cause sterility from the
phenotypically wild-type heterozygous siblings. A clonal screen for class B synMuv
mutations identified several new genes that had been missed by prior screens (Ceol et
al., 2006).
We screened the progeny of approximately 5,350 mutagenized lin-15B(n744)
class B synMuv mutant animals (corresponding to 10,700 haploid genomes), and
isolated ten Muv mutants. We subsequently performed complementation tests to
determine which of these mutations were alleles of the previously identified class A
synMuv genes lin-8, lin-15A, lin-38, and lin-56. Four isolates were alleles of lin-8, three
were alleles of lin-15A, and two were alleles of lin-56 (Table 2). The allele n4377
caused a very low penetrance Muv defect in the lin-15B(n744) background and was not
studied further.
We screened the progeny of approximately 2,150 mutagenized mys-1(n3681)
class B synMuv mutant animals (corresponding to 4,300 haploid genomes), but did not
identify any Muv mutants. mys-1(n3681) causes a weak class B synMuv phenotype
(Andersen et al., 2008; Ceol and Horvitz, 2004), which could explain the failure to
identify any Muv animals in this screen.
We screened the progeny of approximately 4,750 mutagenized lin-52(n771) class
B synMuv mutant animals (corresponding to 9,500 haploid genomes), and isolated 17
Muv mutants. The n4441 mutation was discovered in the F1 generation in this screen.
One isolate, n4444, exhibited a very low penetrance Muv defect and was not pursued
196
further. By complementation tests, we determined that eight of the isolates were alleles
of lin-8, two were alleles of lin-15A, and one was an allele of lin-56. We also identified
an allele of the class A synMuv gene mcd-1 (Chapter Four), and a dominant class A
synMuv allele of the EGF ligand lin-3 (Chapter Two). In addition we isolated two alleles
of lin-1. lin-1 acts downstream of the Ras pathway in vulval development and loss-offunction of lin-1 causes a Muv phenotype (Ferguson and Horvitz, 1985). We also
isolated an allele of lin-13, which is a class B synMuv gene that can also be mutated to
cause a Muv phenotype as a single mutant (Melendez and Greenwald, 2000).
197
Table 1: Previous genetic screens for class A synMuv mutations
Source
Background
Screen Method
Coverage
Isolates
Ferguson and
Horvitz, 1989
lin-9(n112)
EMS, non-clonal
4,000
genomes
2 lin-15A
1 lin-38
1 mcd-1
EMS, non-clonal
10,000
genomes
3 lin-8
1 mcd-1
1 lin-15A
5 lin-8
7 lin-15A
1 lin-38
1 lin-56
no new
genes
Thomas et al.,
2003
lin-36(n766)
Thomas et al.,
2003
lin-15B(n744)
EMS, non-clonal
13,000
genomes
Poulin et al., 2005
lin-15B(n744)
RNAi
86% genome
198
Table 2: Class A synMuv screen isolates
Allele
Gene
Background
n4368
n4369
n4370
n4371
n4372
n4373
n4374
n4375
n4376
n4377
n4412
n4413
n4414
n4415
n4416
n4417
n4418
n4419
n4420
n4421
n4422
n4423
n4440
n4441
n4443
n4444
n4527
lin-15A
lin-56
lin-56
lin-8
lin-8
lin-8
lin-15A
lin-15A
lin-8
unknown
lin-1
lin-56
lin-8
lin-8
lin-8
lin-8
mcd-1
lin-15A
lin-8
lin-15A
lin-8
lin-8
lin-1
lin-3
lin-8
unknown
lin-13
lin-15B(n744)
lin-15B(n744)
lin-15B(n744)
lin-15B(n744)
lin-15B(n744)
lin-15B(n744)
lin-15B(n744)
lin-15B(n744)
lin-15B(n744)
lin-15B(n744)
lin-52(n771)
lin-52(n771)
lin-52(n771)
lin-52(n771)
lin-52(n771)
lin-52(n771)
lin-52(n771)
lin-52(n771)
lin-52(n771)
lin-52(n771)
lin-52(n771)
lin-52(n771)
lin-52(n771)
lin-52(n771)
lin-52(n771)
lin-52(n771)
lin-52(n771)
a
Molecular lesion
Q111ochre
R162opal
A129Ta
Q139ochrea
W79opala
Exon 1 splice donora
W147opala
Q139ochrea
-1 frameshift after amino acid 66a
Y115ochrea
R641Cb
Q235ochrea
W79opala
Q297ambera
point mutation in promoterc
R146Ca
Sequence determined by Hillel Schwartz (personal communication)
See chapter Four
c
See chapter Two
b
199
Appendix Two
The class A synMuv genes lin-15A and lin-56 function in multiple tissues
Adam M. Saffer and H. Robert Horvitz
Appendix Two: The class A synMuv genes lin-15A and lin-56 function in multiple
tissues
200
Introduction
The site(s) of action of the synMuv genes has been a long-standing question.
For components of the EGF/Ras pathway, the site of action is clear: lin-3 EGF acts in
the anchor cell, while let-23 EGFR and the Ras/MAPK pathway have been shown
convincingly by mosaic analysis to act cell-autonomously in the Pn.p cells (Hill and
Sternberg, 1992; Koga and Ohshima, 1995; Lackner et al., 1994; Miller et al., 1996;
Simske and Kim, 1995; Yochem et al., 1997). The synMuv phenotype is epistatic to
anchor cell ablation, indicating that the synMuv genes act outside of the anchor cell
(Ferguson et al., 1987; Sternberg and Horvitz, 1989). When interpreting site-of-action
data for synMuv genes, many authors have made the simplifying assumption that the
site of action was going to be the anchor cell, the hpy7 hypodermal synctytium cell, or
the Pn.p cells themselves and that there is a single site of action, as opposed to input
from multiple cell types and tissues. However, these assumptions need not be valid and
it has long been known that the inductive signal can act at a substantial distance and
that vulval development involves long-range signaling (Katz et al., 1995; Thomas et al.,
1990). Mosaic analyses of lin-15 and lin-37 did not identify a clear single site of action,
but both experiments clearly indicated that lin-15 and lin-37 do not act cellautonomously in the Pn.p cells (Hedgecock and Herman, 1995; Herman and
Hedgecock, 1990). The authors interpreted those experiments as indicating a hyp7 site
of action. hyp7 is a large hypodermal synctitial cell, and nuclei are contributed to hyp7
from many branches of the C. elegans lineage. Conversely, mosaic analysis of the
class B synMuv gene lin-36 was interpreted as indicating a site of action in the Pn.p
cells themselves. However, the results for each of these mosaic experiments are also
consistent with a model in which multiple tissues contribute to the synMuv phenotype.
The site of action of the class B synMuv gene lin-35 has been studied using the
heterologous promoters dpy-7p, which drives expression in the hypodermis, and lin-31p,
which drives expression in the Pn.p cells (Myers and Greenwald, 2005). lin-35 cDNA
expression driven by dpy-7p, but not by lin-31p, rescues the synMuv phenotype of
lin-35, suggesting that lin-35 normally acts in hyp7 (Myers and Greenwald, 2005). More
recently, the class B synMuv gene hpl-2 was reported to act in both hyp7 and the Pn.p
201
cells based on studies using the dpy-7p and lin-31p promoters (Schott et al., 2009). To
determine the site of action of the class A synMuv genes, we used dpy-7p and lin-31p to
drive expression of lin-15A and lin-56. Here we report our findings concerning the
specificities of the dpy-7p and lin-31p promoters and the site of action of the class A
synMuv genes lin-15A and lin-56.
202
Materials and Methods
Strains and genetics
C. elegans strains were cultured using standard methods on OP50 bacteria
(Brenner, 1974). All animals were grown at 20˚C. The wild-type strain used was N2.
The following mutations were used in this study:
LGII: lin-56(n2728) (Thomas et al., 2003)
LGIII: lin-36(n766) (Ferguson et al., 1987)
LGIV: let-60(n1876) (Beitel et al., 1990)
LGX: lin-15A(n767) (Ferguson et al., 1987)
The balancer nT1[qIs51] IV:V (Siegfried et al., 2004) was used.
Plasmid construction
The vectors containing lin-31p and dpy-7p (kindly provided by T. Myers and I.
Greenwald) were converted into Gateway destination vectors by ligating a Gateway
cassette (Invitrogen) into an XmaI site for dpy-7p and a NotI site for lin-31p. Gateway
recombination (Invitrogen) was then used to insert full-length lin-15A and lin-56 cDNAs
downstream of lin-31p and dpy-7p. The dpy-7p::let-60 and lin-31p::let-60 constructs
were created by cloning a full-length let-60 cDNA into the pDONR201 vector (Invitrogen)
and then using Gateway recombination to insert the let-60 cDNA into the dpy-7p and
lin-31p destination vectors.
Transgenic animals
Transgenic animals were made essentially as previously described (Mello et al.,
1991). dpy-7p plasmids were injected at 10 ng/µl, lin-31p plasmids were injected at 100
ng/µl, and myo-3::gfp was used at 50 ng/µl as the coinjection marker.
203
Results
Activity of the dpy-7 and lin-31 promoters
To determine in which cells lin-15A and lin-56 act, we used the promoters of
dpy-7 (dpy-7p) and lin-31 (lin-31p) to perform tissue-specific rescue experiments.
dpy-7p::yfp has been reported to be expressed in the hypodermal syncytium hyp7 but
not in the Pn.p cells, whereas lin-31p::cfp has been reported to be expressed in the
Pn.p cells but not in hyp7 (Myers and Greenwald, 2005). The use of these promoters
led to the proposal that the class B synMuv gene lin-35 has a hyp7 site of action (Myers
and Greenwald, 2005). We used the Gateway system (Invitrogen) to make constructs
for our experiments. To determine if the approximately 100 base pair recombination
sites added by the use of the Gateway system affected the rescuing activity of the dpy-7
and lin-31 promoters, we made dpy-7p::lin-35 and lin-31p::lin-35 constructs using the
Gateway system. Similar to the non-Gateway versions previously studied (Myers and
Greenwald, 2005), we found that our dpy-7p::lin-35 construct made using the Gateway
system could rescue the lin-35(lf) synMuv phenotype, but our lin-31p::lin-35 Gateway
construct could not (data not shown).
To confirm the reported specificities of dpy-7p and lin-31p, we attempted to
rescue the vulvaless (Vul) phenotype caused by a loss-of-function mutation in let-60
Ras. Approximately half of let-60(n1876) homozygotes derived from heterozygous
mothers are inviable, while the other half are viable and Vul; all progeny of let-60(n1876)
homozygotes die as L1 larvae (Beitel et al., 1990). Mosaic experiments have
established that several genes in the EGF/Ras pathway of vulval development act in the
Pn.p cells and suggest the same is the case for let-60 (Koga and Ohshima, 1995;
Lackner et al., 1994; Miller et al., 1996; Simske and Kim, 1995; Yochem et al., 1997).
Previous experiments using the lin-31p and dpy-7p promoters to express a gain-offunction let-60 allele suggested that lin-31p but not dpy-7p drives expression in the Pn.p
cells, as lin-31p::let-60(gf) but not dpy-7p::let-60(gf) produced a multivulva (Muv)
phenotype (Myers and Greenwald, 2005). However, we found that expression of the
wild-type let-60 allele driven by either dpy-7p or lin-31p was sufficient to rescue the Vul
204
phenotype of let-60(n1876) mutants (Table 1). This result indicates that dpy-7p can
drive expression in both hyp7 and the Pn.p cells. By contrast, Myers and Greenwald
(2005) reported that a dpy-7p::yfp construct is visibly expressed in hyp7 but not in the
Pn.p cells or in their parent cells, the P cells. These findings can be reconciled if the
dpy-7 promoter used both by us and by Myers and Greenwald (2005) drives a low level
of Pn.p expression, sufficient for rescue in our experiments but below the threshold for
visible detection in their experiments. Consistent with this hypothesis, a larger fragment
of the dpy-7 promoter drives expression in the P cells (Gilleard et al., 1997).
lin-15A and lin-56 might function in the Pn.p cells, hyp7, or both
We tested if expression of lin-15A and lin-56 cDNAs under the control of either
dpy-7p or lin-31p could rescue the synMuv phenotype of the null alleles lin-15A(n767)
and lin-56(n2728), respectively, in a lin-36(n766) class B synMuv mutant background.
The synMuv double mutant strains lin-36(n766); lin-15A(n767) and lin-56(n2728); lin36(n766) both exhibit a Muv defect with 99.6% penetrance (Tables 2 and 3). We
established eight lines for the dpy-7p::lin-15A construct and six lines for the
lin-31p::lin-15A construct in a lin-36(n766); lin-15A(n767) background. Similarly, we
established six lines for the dpy-7p::lin-56 construct and five lines for the lin-31p::lin-56
construct in a lin-56(n2728); lin-36(n766) background. All eight dpy-7p::lin-15A lines
exhibited modest rescue of the lin-36(n766); lin-15A(n767) synMuv phenotype, with on
average a 72% penetrant Muv defect (Table 2). The lin-31p::lin-15A lines also exhibited
rescue with a 93% penetrant Muv defect (Table 2), but to a lesser extent (p-value <
0.0001) than the dpy-7p::lin-15A lines. Similarly, both the dpy-7p::lin-56 and
lin-31p::lin-56 constructs were able to rescue the synMuv phenotype of lin-56(n2728);
lin-36(n766) mutants, although the dpy-7p::lin-56 construct conferred stronger rescue
with a 9% penetrant Muv defect compared to the 42% penetrant Muv defect with
lin-31p::lin-56 (Table 3).
205
Discussion
Mosaic analyses of different synMuv genes have been interpreted as suggesting
a site of action in either hyp7 or the Pn.p cells (Hedgecock and Herman, 1995; Herman
and Hedgecock, 1990; Thomas and Horvitz, 1999). More recently, studies using tissuespecific expression with the heterologous promoters dpy-7p and lin-31p have been
interpreted as supporting a hyp7 site of action for the class B gene lin-35 (Myers and
Greenwald, 2005). We found that localized expression of lin-15A or lin-56 under the
control of either dpy-7p or lin-31p can partially rescue loss of lin-15A or lin-56,
respectively. One possible explanation for the rescue with either lin-31p or dpy-7p is
that lin-15A and lin-56 have a single site of action in a tissue that expresses both lin-31
and dpy-7, although it is unclear what tissue this could be. The only known overlap in
their expression is the Pn.p cells, as determined by published reporter expression
patterns (Myers and Greenwald, 2005) and our functional assays. However, mosaic
analysis clearly indicates that lin-15AB does not act solely in the Pn.p cells (Herman
and Hedgecock, 1990). The explanation we prefer is that lin-15A and lin-56 act in
multiple tissues, including both hyp7 and the Pn.p cells. Consistent with such a model,
LIN-15A and LIN-56 are expressed ubiquitously in all cells of the animal (Chapter Five).
Mosaic analyses have been interpreted as suggesting a Pn.p site of action for lin-36
and a hyp7 site of action for lin-15AB and lin-37 (Hedgecock and Herman, 1995;
Herman and Hedgecock, 1990; Thomas et al., 1990). However, the mosaic analyses of
lin-15 and lin-37 actually produced very different results: loss of lin-15 in many different
tissues causes a mutant phenotype, while loss of lin-37 in most tissues does not cause
a mutant phenotype (Hedgecock and Herman, 1995; Herman and Hedgecock, 1990).
Furthermore, none of these mosaic experiments is inconsistent with a site of action in
multiple tissues, including both hyp7 and the Pn.p cells and possibly other cells. There
might be a threshold of lin-15A and lin-56 activity required to prevent a synMuv
phenotype, and expression in either hyp7 or the Pn.p cells provides sufficient activity to
reach that threshold.
lin-31p-driven expression of lin-35 is not sufficient to rescue loss of lin-35
function, but lin-31p-driven expression of lin-15A or lin-56 can partially rescue loss of
206
lin-15A or lin-56, respectively. Therefore, the expression requirements for these
synMuv genes must differ in some way. Because lin-3 EGF is globally ectopically
expressed at a high level only in class AB synMuv double mutants (Chapter Two), it is
unlikely that the class A synMuv genes lin-15A and lin-56 function in different cells than
the class B synMuv gene lin-35, although it is conceivable they could function at
different times in the same cells. More likely, both sets of synMuv genes act in the
same cell(s), but the function of the class A synMuv genes lin-15A and lin-56 require a
lower level of expression than the class B synMuv gene lin-35. That several class B
synMuv genes, including lin-35, are partially haplo-insufficient, while no such haploinsufficiency has been observed for the class A synMuv genes, is consistent with this
possibility (E. C. Andersen, A. M. Saffer, and H. R. Horvitz, unpublished results).
lin-3 EGF is the only relevant target of the class A synMuv genes in vulval
development, and in class AB synMuv double mutants lin-3 is ectopically expressed in
all or nearly all cells (Chapter Two). Therefore, the site of action where the class A and
B synMuv genes repress ectopic lin-3 EGF expression is the entire animal. lin-3 EGF is
capable of acting at a distance to control vulval cell fates (Katz et al., 1995; Thomas et
al., 1990). The contribution of each cell to the synMuv phenotype should primarily
reflect a combination of the size of that cell and its proximity to the Pn.p cells. The site of
action for the synMuv phenotype might also reflect which cells express the appropriate
protease activity to cleave the membrane-bound LIN-3 precursor and release the
soluble EGF domain of LIN-3. Given their locations, hyp7 and the Pn.p cells are likely
to be major sites of action of the synMuv genes, but other cells probably contribute as
well. This is analogous to human tumors, in which growth factors produced by both the
tumor and the surrounding microenvironment can promote cancerous growth (Hanahan
and Weinberg, 2000).
Acknowledgments
We thank Takashi Hirose for helpful comments about this chapter.
207
Table 1: Expression of let-60 cDNA under the control of either dpy-7p or lin-31p
rescues the vulvaless phenotype of let-60(n1876)
genotype a
% vulvalessb (n)c
wild-type
let-60(n1876)
let-60(n1876) Ex[lin-31p::let-60] line 1
let-60(n1876) Ex[lin-31p::let-60] line 2
let-60(n1876) Ex[lin-31p::let-60] total
let-60(n1876) Ex[dpy-7p::let-60] line 1
let-60(n1876) Ex[dpy-7p::let-60] line 2
let-60(n1876) Ex[dpy-7p::let-60] line 3
let-60(n1876) Ex[dpy-7p::let-60] line 4
let-60(n1876) Ex[dpy-7p::let-60] total
0% (many)
100% (20)
31.8% (22)
58.8% (17)
43.6% (39)d
50.0% (12)
72.7% (11)
12.3% (57)
9.1% (22)
22.5% (102)d
a
all let-60(n1876) animals descended from let-60(n1876)/nT1[qIs51] mothers.
b
% Vulvaless, percentage of animals which lacked a vulva as determined by the
internal hatching of their progeny.
c
n, number of animals examined.
d
P-value <0.0001 compared to let-60(n1876) by Fisherʼs Exact Test.
208
Table 2: Expression of lin-15A under the control of either dpy-7p or lin-31p
rescues the synMuv phenotype
Genotype
% Muva (n)b
lin-36(n766); lin-15A(n767)
lin-36(n766); lin-15A(n767) Ex[lin-31::lin-15A] line 1
lin-36(n766); lin-15A(n767) Ex[lin-31::lin-15A] line 2
lin-36(n766); lin-15A(n767) Ex[lin-31::lin-15A] line 3
lin-36(n766); lin-15A(n767) Ex[lin-31::lin-15A] line 4
lin-36(n766); lin-15A(n767) Ex[lin-31::lin-15A] line 5
lin-36(n766); lin-15A(n767) Ex[lin-31::lin-15A] line 6
lin-36(n766); lin-15A(n767) Ex[lin-31::lin-15A] total
lin-36(n766); lin-15A(n767) Ex[dpy-7::lin-15A] line 1
lin-36(n766); lin-15A(n767) Ex[dpy-7::lin-15A] line 2
lin-36(n766); lin-15A(n767) Ex[dpy-7::lin-15A] line 3
lin-36(n766); lin-15A(n767) Ex[dpy-7::lin-15A] line 4
lin-36(n766); lin-15A(n767) Ex[dpy-7::lin-15A] line 5
lin-36(n766); lin-15A(n767) Ex[dpy-7::lin-15A] line 6
lin-36(n766); lin-15A(n767) Ex[dpy-7::lin-15A] line 7
lin-36(n766); lin-15A(n767) Ex[dpy-7::lin-15A] line 8
lin-36(n766); lin-15A(n767) Ex[dpy-7::lin-15A] total
99.6% (251)
88.4% (96)
88.1% (102)
100% (73)
94.5% (91)
99.0% (104)
94.2% (104)
93.1% (569)c
75.9% (54)
91.8% (73)
47.6% (82)
60.5% (38)
87.8% (74)
79.6% (54)
94.5% (55)
100% (18)
71.6% (448)c
a
% Muv, percentage of animals that were Muv.
b
n, number of animals examined.
c
P-value <0.0001 compared to lin-36(n766); lin-15A(n767) by Fisherʼs Exact Test.
209
Table 3: Expression of lin-56 under the control of either dpy-7p or lin-31p
rescues the synMuv phenotype
Genotype
% Muva (n)b
lin-56(n2728); lin-36(n766)
lin-56(n2728); lin-36(n766) Ex[lin-31::lin-56] line 1
lin-56(n2728); lin-36(n766) Ex[lin-31::lin-56] line 2
lin-56(n2728); lin-36(n766) Ex[lin-31::lin-56] line 3
lin-56(n2728); lin-36(n766) Ex[lin-31::lin-56] line 4
lin-56(n2728); lin-36(n766) Ex[lin-31::lin-56] line 5
lin-56(n2728); lin-36(n766) Ex[lin-31::lin-56] total
lin-56(n2728); lin-36(n766) Ex[dpy-7:lin-56] line 1
lin-56(n2728); lin-36(n766) Ex[dpy-7:lin-56] line 2
lin-56(n2728); lin-36(n766) Ex[dpy-7:lin-56] line 3
lin-56(n2728); lin-36(n766) Ex[dpy-7:lin-56] line 4
lin-56(n2728); lin-36(n766) Ex[dpy-7:lin-56] line 5
lin-56(n2728); lin-36(n766) Ex[dpy-7:lin-56] line 6
lin-56(n2728); lin-36(n766) Ex[dpy-7:lin-56] total
99.6% (234)
29.1% (86)
61.9% (97)
35.7% (42)
16.7% (30)
44.7% (38)
41.6% (293)c
18.3% (60)
13.0% (88)
8.9% (79)
5.7% (88)
5.8% (69)
3.9% (102)
8.6% (486)c
a
% Muv, percentage of animals that were Muv.
b
n, number of animals examined.
c
P-value <0.0001 compared to lin-56(n2728); lin-36(n766) by Fisherʼs Exact Test.
210
Appendix Three
Progress towards identifying proteins that bind to the lin-3 promoter
Adam M. Saffer and H. Robert Horvitz
Appendix Three: Progress towards identifying proteins that bind to the lin-3
promoter
211
Introduction
Vulval development in C. elegans is regulated by an epidermal growth factor
(EGF)/Ras pathway. There are six equivalent Pn.p cells in C. elegans, and in wild-type
animals three of the six Pn.p cells are induced to adopt vulval cell fates by the LIN-3
EGF ligand (Hill and Sternberg, 1992). The other three cells adopt non-vulval cell fates.
Loss-of-function mutations in lin-3 cause a vulvaless phenotype in which none of the six
cells adopts vulval cell fates, while overexpression of lin-3 results in a Muv phenotype in
which all six cells adopt a vulval cell fate (Hill and Sternberg, 1992).
Expression of lin-3 is precisely regulated both spatially and temporally. Some
tissues, such as the pharynx, express lin-3 throughout development (Hwang and
Sternberg, 2004). Other cells, such as some of the descendants of the Pn.p cells that
form the vulva, express lin-3 at specific stages of development (Chang et al., 1999).
Expression of lin-3 in the anchor cell is responsible for the induction of vulval
development, and ablation of the anchor cell results in a vulvaless phenotype (Hill and
Sternberg, 1992; Kimble, 1981). Outside of the few cells and tissues in which it is
normally expressed, lin-3 is very tightly repressed; this repression is disrupted in
synMuv mutants (Chapter Two).
Because lin-3 is expressed in distinct tissues at different times, there are likely to
be multiple cis regulatory elements required for the normally precise expression pattern
of lin-3. The lin-3(e1417) mutation causes a vulvaless phenotype but not other defects
known to result from strong loss-of-function mutations in lin-3 (Liu et al., 1999). The lin3(e1417) mutation affects an element located in a lin-3 intron, and this conserved 59 bp
element is both necessary and sufficient to drive expression in the anchor cell (Hwang
and Sternberg, 2004). Electrophoretic mobility shifts assays (EMSA) indicate that NHR25 and HLH-2 can bind to this element in vitro (Hwang and Sternberg, 2004). RNAi of
hlh-2 at the L2 and L3 larval stages when vulval cell fates are specified causes a loss of
lin-3 expression in the anchor cell, but other roles of nhr-25 in development have
confounded attempts to determine its role in regulating lin-3 expression in vivo (Hwang
and Sternberg, 2004).
212
In Chapter Two, we describe the identification and characterization of the
mutation lin-3(n4441), which dominantly causes a class A synMuv phenotype.
lin-3(n4441) disrupts a cis-acting regulatory element in the lin-3 promoter required for
the class A synMuv genes to tightly repress lin-3 and prevent leaky ectopic expression.
lin-3(n4441) is a point mutation, and the extent of the class A synMuv repressive
element in the lin-3 promoter is not known. Additionally, it is not known what proteins
bind to the lin-3 promoter at the site of the lin-3(n4441) mutation. The protein(s) that
bind to this site could be one or more class A synMuv proteins, non-synMuv proteins
that are regulated by the class A synMuv proteins, or a combination of synMuv and nonsynMuv proteins. Here we report preliminary experiments to investigate the extent of
the class A synMuv repressive element in the lin-3 promoter and identify proteins that
bind to this element.
213
Materials and Methods
Strains and genetics
C. elegans strains were cultured using standard methods on OP50 bacteria
(Brenner, 1974). All animals were grown at 20˚C except where noted otherwise. The
wild-type strain used was N2.
The following mutations were used in this study:
LGII: lin-8(n2731) and lin-56(n2728) (Thomas et al., 2003) and lin-38(n751) (Ferguson
and Horvitz, 1989)
LGIV: F13E9.13(n5408) (this study)
LGX: lin-15A(n767), lin-15B(n744), and lin-15AB(n765) (Ferguson and Horvitz, 1989)
Transgenic animals
The lin-3 locus was amplified from genomic DNA with Platinum Taq DNA
Polymerase High Fidelity (Invitrogen) using the primers
5ʼ-GTAACGCCAGGGTTTTCCCAGTCACGACGCACCCAAATGTCCTTGAACCAAC-3ʼ and
5ʼ-GCGGATAACAATTTCACACAGGAAACAGCAGAAGGACACCCGTAAGTTCATTG-3ʼ, and the resulting PCR
product was cloned into the vector pRS426 (New England Biolabs) by yeast-mediated
ligation (Oldenburg et al., 1997). The lack of PCR-induced mutations was confirmed by
determining the sequence of the entire insert. The lin-3(n4441) mutation was added to
this plasmid with the QuikChange system (Stratagene) and confirmed by DNA
sequence determination. Transgenic animals were made essentially as previously
described (Mello et al., 1991). lin-3 plasmids were injected at various concentrations
along with 50 ng/µl myo-3::GFP plasmid and 100 ng/µl 1 kb Plus DNA ladder
(Invitrogen).
Nuclear extracts
C. elegans was grown in liquid S-medium supplemented with 1X AntibioticAntimycotic (Invitrogen) and HB101 bacteria. Animals were harvested by centrifugation.
214
To isolate embryos, we resuspended the animals in a solution of 1.5N NaOH and 12%
NaOCl with vigorous mixing for 5-10 minutes. Embryos or mixed stage animals were
resuspended in 1X phosphate buffered saline (PBS) solution, flash frozen in liquid
nitrogen, and stored at -80˚ C. Nuclear extracts were prepared essentially as previously
described (Hope, 1999). The nuclear preparation buffer was supplemented with
Complete EDTA-free protease inhibitor cocktail tablets (Roche). Briefly, animals were
homogenized using a Wheaton stainless steel tissue grinder. Nuclei were pelleted by
centrifugation at 4000g, and the cytoplasmic extract was removed. Unwanted debris
was then separated from the nuclei by centrifugation at 100g. Finally, proteins were
extracted from nuclei by incubation in a high salt buffer. Protein concentrations of the
extracts were determined using the Coomassie Plus Protein Assay (Pierce).
Electrophoretic mobility shift assays (EMSA) with C. elegans nuclear extracts
Complementary oligonucleotides (Integrated DNA Technologies), with or without
a 5ʼ biotin molecule, were annealed to produce double-stranded probes for the EMSA
experiments.
The following oligonucleotides were used for EMSA and affinity purification experiments:
AMS245:
5ʼ-Biotin-CTCGTAATCAGTGGTGACTCGAATTTTGAAGATGTTGCGACCGTGCTCCATTGGACTCTCC-3ʼ
AMS246:
5ʼ-GGAGAGTCCAATGGAGCACGGTCGCAACATCTTCAAAATTCGAGTCACCACTGATTACGAG-3ʼ
AMS251:
5ʼ-CTCGTAATCAGTGGTGACTCGAATTTTGAAGATGTTGCGACCGTGCTCCATTGGACTCTCC-3ʼ
AMS252:
5ʼ-CTCGTAATCAGTGGTGACTCGAATTTTGAAAATGTTGCGACCGTGCTCCATTGGACTCTCC-3ʼ
AMS253:
5ʼ-GGAGAGTCCAATGGAGCACGGTCGCAACATTTTCAAAATTCGAGTCACCACTGATTACGAG-3ʼ
AMS254:
5ʼ-GGTATACTGAGGTATTTTAGGGTCACTTGGCGATTTGCTCCGTGACCCATGCCATCACCAT-3ʼ
AMS255:
5ʼ-ATGGTGATGGCATGGGTCACGGAGCAAATCGCCAAGTGACCCTAAAATACCTCAGTATACC-3ʼ
AMS290: 5ʼ-Biotin-CTCGTAATCAGTGGTGACTCGAATTTTGAAAATGTTGCGACCGTGCTCCATTGGACTCTCC-3ʼ
Wild-type labeled probe for EMSA was made by annealing AMS245 and
AMS246. Unlabeled wild-type competitor probe was made by annealing AMS246 and
AMS251. Unlabeled lin-3(n4441) mutant competitor probe was made by annealing
215
AMS252 and AMS253. Unlabeled scrambled probe was made by annealing AMS254
and AMS255.
Nuclear extracts were applied to Micro Bio-Spin 6 Chromotography columns (BioRad) equilibrated with distilled water to remove the nuclear extraction buffer. Each 20 µl
EMSA reaction contained 20 fmol biotin-labeled double-stranded oligonucleotide probe,
various concentrations of unlabeled double-stranded competitor probe, 1 µg
Poly(deoxyinosinic-deoxycytidylic) acid (Sigma), 4.5 µg bovine serum albumin, 20 mM
HEPES pH 7.6, 10 µM ZnSO4, 12% glycerol, 1 mM EDTA, 100 mM KCl, 10 mM MgCl2,
and 1 mM DTT. Reactions were analyzed by electrophoresis on non-denaturing 0.5X
TBE gels with 8% polyacrylamide. After electrophoresis, binding reactions were
transferred to BrightStar-Plus nylon membrane (Ambion) and UV-crosslinked using the
Autocrosslink function of a UV Stratalinker 1800. Biotin-labeled probes were detected
using the Chemiluminescent Nucleic Acid Detection Module (Pierce).
EMSA with in vitro transcribed and translated proteins
The gateway recombination system (Invitrogen) was used to clone a cDNA
corresponding to each class A synMuv gene into the pDest14 vector (Invitrogen), which
contains a T7 RNA polymerase binding site. For mcd-1, a cDNA corresponding to the
long isoform of mcd-1 was used (Chapter Four). For all other class A synMuv genes a
cDNA corresponding to the full-length known transcript was used (Clark et al., 1994;
Davison et al., 2005; Huang et al., 1994; Chapters Four and Five). pDest14::cDNA
constructs were used as templates for in vitro transcription and translation reactions
with the TnT Quick Coupled Transcription/Translation system (Promega). 35S -labeled
methionine was added to each reaction, and the success of each reaction was
determined by denaturing gel electrophoresis followed by detection of the 35S using a
phosphor screen.
Oligonucleotides AMS245 and AMS251 were annealed and then labeled with 32P
using gamma-32P ATP and T4 polynucleotide kinase. Binding reactions contained 2 µl
of each transcription and translation reaction and 20 fmol radiolabeled oligonucleotide
216
probe. All other conditions were identical to the EMSA experiments performed with
nuclear extracts.
Affinity purification of lin-3p binding proteins
Wild-type double-stranded oligonucleotides were made by annealing AMS245
and AMS246. lin-3(n4441) mutant double-stranded oligonucleotides were made by
annealing AMS253 and AMS290. Probes were bound to Dynabeads M-280 streptavidin
magnetic beads (Invitrogen) according to the manufacturerʼs directions. Nuclear
extracts were placed in a binding buffer equivalent to the conditions used for the gels
shifts, with 1 µg Poly(deoxyinosinic-deoxycytidylic) acid, 4.5 µg bovine serum albumin,
20 mM HEPES pH 7.6, 10 µM ZnSO4, 12% glycerol, 1 mM EDTA, 100 mM KCl, 10 mM
MgCl2, and 1 mM DTT. The 1.25 mL binding reaction was incubated for 30 minutes
with 500 pmol of lin-3(n4441) oligonucleotides bound to 2.5 µg streptavidin beads. The
binding reaction was then added to a second tube with an additional 500 pmol of lin3(n4441) oligonucleotides bound to 2.5 µg streptavidin beads. Finally, the binding
reaction was added to a tube with 200 pmol of wild-type oligonucleotides bound to 1 µg
streptavidin beads. Each tube of oligonucleotide-bound beads was then washed four or
five times with 1 mL of wash buffer consisting of 20 mM HEPES pH 7.6, 10 µM ZnSO4,
12% glycerol, 1 mM EDTA, 100 mM KCl, 10 mM MgCl2, and 1 mM DTT. Proteins were
eluted from the oligonucleotide DNA fragments with an elution buffer consisting of 20
mM HEPES pH 7.6, 10 µM ZnSO4, 12% glycerol, 1 mM EDTA, 1 M KCl, 10 mM MgCl2,
and 1 mM DTT. 1 µl of the unbound supernatant and all of each eluate were then
analyzed by gel electrophoresis on a 10% denaturing polyacrylamide gel. The gel was
stained with SYPRO Ruby protein gel stain (Invitrogen) and imaged on a Typhoon 9400
imager. Mass spectrometry was performed by the Proteomics Core Facility in the Koch
Institute for Integrative Cancer Research at MIT.
217
Results
The effect of the lin-3(4441) mutation on vulval development is not replicated with
a repetitive extrachromosomal array
lin-3(n4441) is a G-to-A transition at nucleotide 30904 of cosmid F36H1 and
dominantly causes a class A synMuv phenotype (Chapter Two). Alignments of the
region 5ʼ to lin-3, including the site mutated in lin-3(n4441) animals, between sequences
from C. elegans and the related nematodes Caenorhabditis briggsae and
Caenorhabditis remanei revealed no substantial conservation in the region surrounding
the lin-3(n4441) site. For biochemical experiments to identify proteins that bind to the
element affected by the lin-3(n4441) mutation, it would be useful to know the extent of
this element. Therefore, we attempted to design a system to study the effect of lin-3
promoter mutations on vulval development.
If the class A synMuv phenotype caused by the lin-3(n4441) mutation was
replicated by transgenic animals carrying an extrachromosomal array consisting of the
lin-3 locus with the n4441 mutation, then it would be possible to use such a system to
evaluate the effects of other lin-3 promoter mutations. However, when a plasmid
carrying the wild-type lin-3 genomic locus is injected at concentrations of 30 ng/µl or
more, a highly penetrant Muv defect results (Hill and Sternberg, 1992; our data not
shown). We reasoned that injecting the lin-3 locus at lower concentrations might cause
either no mutant phenotype or a low penetrance Muv defect that could be modified. The
lin-3 locus caused no noticeable defect when injected into wild-type animals at a
concentration of 0.1 ng/µl, and less than a 1% penetrant Muv defect when injected at 1
ng/µl (data not shown). When the lin-3 locus was injected into wild-type animals at a
concentration of 10 ng/µl, a weak Muv defect with 6% penetrance resulted (Table 1).
The lin-3 plasmid caused a slightly stronger Muv defect when injected into lin-15B(n744)
class B synMuv mutants than when injected into wild-type animals (Table 1). We
expected the lin-3(n4441) plasmid to confer a similar penetrance Muv defect to the wildlin-3(+) plasmid when injected into wild-type animals. Conversely, in a class B synMuv
mutant we expected the lin-3(n4441) plasmid to cause a much stronger Muv phenotype
218
than the lin-3(+) plasmid. However, we did not observe any differences between the
lin-3(+) and lin-3(n4441) constructs in these experiments, as both plasmids caused a
similar penetrance Muv defect in lin-15B(n744) and wild-type backgrounds (Table 1).
Therefore, the effects of the lin-3(n4441) mutation on lin-3 expression were not
recreated by this system. The method we used to create these transgenic animals
results in multicopy, repetitive extrachromosomal arrays (Mello et al., 1991; Stinchcomb
et al., 1985), which might be regulated differently than endogenous loci.
EMSA experiments detect a sequence-specific, lin-3-promoter-binding protein
The simplest mechanism by which the lin-3(n4441) mutation could cause a class
A synMuv phenotype is by disrupting a binding site for a protein that is required for the
repression of lin-3. The protein(s) that binds to this site could be one or more of the
class A synMuv proteins, and/or one or more non-synMuv proteins regulated by the
class A synMuv proteins. To investigate how the class A synMuv proteins repress lin-3
mRNA expression, we attempted to identify what proteins bind to this site. There are no
known consensus transcription factor binding sites that include the site of the
lin-3(n4441) mutation according to the Transfac database of known transcription binding
sites (http://www.gene-regulation.com). To identify proteins that bind to this site we
performed EMSA experiments using a 60 bp double-stranded oligonucleotide probe
centered around the site of the lin-3(n4441) mutation. EMSA experiments were
performed with nuclear extracts from C. elegans to allow the identification of unknown
binding proteins and proteins that bind only as part of a complex. Nuclear extracts were
prepared from large-scale liquid cultures of C. elegans embryos. The vulval cell-fate
decision occurs at the late L2 to early L3 stage, but synMuv proteins are expressed
throughout development, including embryogenesis (Ceol and Horvitz, 2001, 2004;
Davison et al., 2005; Harrison et al., 2006; Harrison et al., 2007a), and synMuv
mutations affect cell-fate decisions at multiple points in development (Jiang and
Sternberg, 1998). Therefore, any binding activity consisting of or dependent on synMuv
proteins is likely to be present at all stages of development including embryos, and
embryos have previously been used successfully for biochemical studies of synMuv
219
proteins (Harrison et al., 2006). Nuclear extracts were incubated for 30 minutes at room
temperature with labeled probe, and the mixtures were then assayed by gel
electrophoresis on a native non-denaturing polyacrylimide gel (Figure 1A). There were
multiple shifted bands, all of which were effectively competed by an excess of unlabeled
probe. The shifted bands were unaffected by an excess of unlabeled scrambled probe
that contained the same base composition of the wild-type probe in a randomized order,
indicating that the bands reflected sequence-specific DNA-binding activity and not
general DNA-binding activities. By contrast, when a probe carrying the lin-3(n4441)
mutation was used as the unlabeled competitor, all of the bands except one were
effectively competed. This observation suggests that the remaining band represents a
protein or protein complex that binds to sequences in the lin-3 promoter but not the
lin-3(n4441) mutant promoter. This specific binding activity was successfully competed
by a wide range of concentrations of unlabeled wild-type competitor. The specific
binding activity was not competed by lin-3(n4441) mutant competitor, and the binding
activity was actually increased by the addition of unlabeled lin-3(n4441) competitor
(Figure 1B). Such an increase could be caused by a titration of other DNA-binding
proteins that interfere with the specific binding activity. Concentrations of lin-3(n4441)
competitor up to a 900-fold excess over the labeled probe did not lower the specific
binding activity (data not shown). The specific binding activity was also present in
nuclear extracts made from mixed stage cultures of C. elegans. No additional shifted
bands representing proteins that specifically bind to wild-type but not lin-3(n4441)
mutant probe were identified when various concentrations of MgCl2 (ranging from 0 mM
to 60 mM) and KCl (ranging from 0 mM to 400 mM) were used for the buffer (data not
shown).
To determine if the specific binding activity is either a class A synMuv protein or
dependent on a class A synMuv protein, we repeated the EMSA experiments with
nuclear extracts made from class A synMuv mutants. The lin-8(n2731) nonsense
mutation and the lin-15A(n767) small deletion are both likely null alleles (Clark et al.,
1994; Davison et al., 2005; Huang et al., 1994). The lin-56(n2728) mutation is a large
deletion that removes the entire lin-56 coding region (Chapter Five). We used the
220
lin-38(n751) missense mutation, because lin-38 null mutations cause larval lethality
(Chapter Four). Nuclear extracts made from each of those four class A synMuv mutants
possessed the specific binding activity, and the size of the shifted band did not appear
to be altered (Figure 1C). Therefore, the specific binding activity is unlikely to be a class
A synMuv protein, nor is it likely to be a complex that includes a class A synMuv protein.
Furthermore, the expression and DNA-binding activity of the protein(s) that make up the
specific binding activity do not require class A synMuv activity. However, because we
used a missense allele of lin-38 and did not test mcd-1 in this assay, it is possible that
the binding activity consists of or requires either MCD-1 or LIN-38. If an antibody that
recognized some component of the specific binding activity was added to the binding
reaction, then the size of the complex and hence its migration in gel electrophoresis
experiments could be altered. No shift in the migration of the specific binding activity
was observed when antibodies that recognize LIN-8, LIN-15A, or LIN-56 were added to
the binding reaction (data not shown).
Affinity purification of lin-3p binding proteins
To determine what proteins specifically bind to the lin-3 promoter but not the
lin-3(n4441) mutant promoter, we attempted to purify these proteins on the basis of their
sequence-specific DNA binding activity. In EMSA competition assays, the specific
binding activity had no affinity for a lin-3(n4441) mutant probe (Figure 1B). Therefore,
we reasoned that unwanted DNA-binding proteins would be removed by incubating
nuclear extracts with lin-3(n4441) mutant oligonucleotides, and the desired DNA-binding
activity could then be affinity purified by incubating the cleared extracts with wild-type
oligonucleotides (Figure 2A). Nuclear extract from mixed stage liquid cultures of wildtype animals was used to obtain the maximal amount of protein. Nuclear extract
containing approximately 1.2 mg of protein was incubated with lin-3(n4441) mutant
oligonucleotides bound to magnetic beads in the same buffer conditions used for the
EMSA experiments. Unbound supernatant was then incubated with a second aliquot of
lin-3(n4441) mutant oligonucleotides and subsequently with beads bound to wild-type
oligonucleotides. After washing the beads several times, we eluted potential sequence-
221
specific binding proteins using a high salt buffer. The concentration of salt used was
several times higher than the concentration require to disrupt all binding activities in the
EMSA experiments (data not shown). When the purified candidate sequence-specific
binding proteins were assayed using denaturing gel electrophoresis, most of the bands
were also present in eluates from the lin-3(n4441) mutant oligonucleotides and in the
unbound supernatant (Figure 2B). Therefore, these bands likely represent highly
expressed nuclear proteins that were not sufficiently removed by incubation with the
lin-3(n4441) mutant oligonucleotides. If this experiment is repeated, it would be good to
increase the amount of the lin-3(n4441) oligonucleotides. Additionally, a single band of
approximately 35 kD was identified that bound specifically to the wild-type lin-3 probe
and did not appear to correspond to a highly expressed nuclear protein (Figure 2B).
This band was excised from the gel and analyzed by mass spectrometry. One peptide
was identified that corresponded to the histone H1.1 protein HIS-24. However, only a
single peptide corresponding to HIS-24 was isolated and the predicted size of HIS-24 of
21.3 kD is much smaller than the band we observed (although such an apparent size
difference could be a consequence of post-translational protein modifications). Eight
peptides were identified that correspond to the F13E9.13 protein. The predicted size of
the F13E9.13 protein is 30 kD. However, the structure of the F13E9.13 gene has not
been experimentally determined, so the actual size of the protein could be either more
or less than 30 kD.
We tested if F13E9.13 is involved in vulval development by inactivating F13E9.13
by feeding RNAi in wild-type animals, the class A synMuv mutant lin-15A(n767), the
class B synMuv mutant lin-15B(n744), and the class AB synMuv mutant lin-15AB(n765)
backgrounds. F13E9.13 RNAi did not cause a Muv phenotype in any background, nor
did it cause any noticeable suppression of the lin-15AB(n765) synMuv phenotype. The
F13E9.13(n5408∆) deletion, which removes the entire F13E9.13 coding region, did not
cause lethality or any obvious abnormalities when homozygous. Homozygous
F13E9.13(n5408∆) did not either cause a synMuv phenotype or suppress the synMuv
phenotype of lin-15AB(n765) (Table 2).
222
EMSA experiments to test direct interactions between the lin-3 promoter and
class A synMuv proteins
We performed additional EMSA experiments to directly test if the class A synMuv
proteins can bind the lin-3 promoter in vitro. Class A synMuv proteins were produced by
in vitro transcription and translation using a reticulocyte lysate system. To ensure that
the proteins were full-length, we assayed them using denaturing gel electrophoresis. All
five class A synMuv proteins were successfully translated, although all five appeared
slightly larger on the gel than their predicted molecular weights (Figure 3A). We had
previously observed a similar phenomenon of class A synMuv proteins appearing larger
than predicted using denaturing gel electrophoresis with recombinant class A synMuv
proteins expressed in E. coli. EMSA was performed identically to the previous
experiments with nuclear extracts, except that the oligonucleotide probe was labeled
with 32P instead of biotin, as the biotin detection system reacted with some component
of the in vitro transcription and translation system to produce an extremely high
background when the biotin-labeled probes were detected following electrophoresis.
The only shifted bands observed with class A synMuv proteins were also present when
in vitro transcribed and translated luciferase protein was tested, indicating that none of
the class A synMuv proteins binds to the lin-3 promoter fragment in this assay (Figure
3B).
223
Discussion
We have found that in the context of a repetitive extrachromosomal array, the
lin-3(n4441) mutation does not affect expression of lin-3. One possible explanation is
that the mutation we have identified at nucleotide 30904 of cosmic F36H1 is not the
mutation that causes the class A synMuv phenotype of lin-3(n4441). However, our
mapping experiments show that this possibility is very unlikely (Chapter Two).
Alternatively, the extrachromosomal array may not faithfully recapitulate normal lin-3
regulation. Supporting this possibility, Cui et al. (2006) reported that they saw no
difference in the expression of lin-3 reporter constructs in wild-type and synMuv mutant
background. There might be sequences upstream or downstream of lin-3 that are
required for synMuv repression of lin-3 but are not included in our construct. The
construct we used contains approximately 1.4 kb of sequences upstream of the start of
the lin-3 coding region, including most of the upstream gene, and approximately 800 bp
of sequences 3ʼ to lin-3. However, it is possible for cist regulatory elements to be much
further away, so we cannot rule out the possibility that synMuv regulation of lin-3
requires distant cist regulatory elements. Another explanation for our failure to observe
any effects of lin-3(n4441) in this assay is that the context of the extrachromosomal
array alters normal synMuv regulation. Arrays made by germline injection typically
contain hundreds of copies of the injected DNA. This high copy number might override
repression by the class A synMuv proteins. Additionally, extrachromosomal arrays are
highly repetitive, with many copies of the same sequence recombined together (Mello et
al., 1991). Expression from these repetitive sequences is regulated differently than that
from normal chromosomal genes. For example, most extrachromosomal arrays are
silenced in the germline (Kelly et al., 1997). Furthermore, expression from repetitive
extrachromosomal arrays is affected by mutations in many synMuv genes (Hsieh et al.,
1999). Recently, a technique for integrating single copy DNA into defined sites in the C.
elegans genome has been developed (Frokjaer-Jensen et al., 2008). Future transgene
studies of the lin-3 locus should use single copy insertions to avoid the myriad of
concerns with using repetitive extrachromosomal arrays.
224
Our observations implicate the protein F13E9.13 as a candidate lin-3 regulatory
protein, based on its ability to interact either directly or indirectly with a wild-type
fragment of the lin-3 promoter but not the corresponding lin-3(n4441) mutant fragment.
We have had difficulty amplifying F13E9.13 cDNA fragments by RT-PCR, and no cDNA
for F13E9.13 has been identified by Yuji Koharaʼs large-scale cDNA identification effort
(http://www.wormbase.org), suggesting that F13E9.13 is not expressed at a high level.
Those considerations make it much less likely that we identified F13E9.13 because it is
a contaminating highly expressed nuclear protein. F13E9.13 has no known DNAbinding domains but could have been purified as part of a complex that includes a DNAbinding protein that specifically interacts with the lin-3 promoter. Alternatively, it is
possible that F13E9.13 is a DNA-binding protein and possesses a DNA-binding domain
distinct from previously characterized DNA-binding domains. F13E9.13 contains a BtpA
domain, but the molecular function of the BtpA domain is not known. btpA was
identified in the cyanobacterium Synechocystis 6803, and btpA mutants have impaired
accumulation of the proteins that form the core of the photosystem I complex
(Bartsevich and Pakrasi, 1997). Fractionation experiments indicate that BtpA is a
peripheral membrane protein associated with thylakoid membranes (Zak et al., 1999).
However, proteins with BtpA domains are present in organisms that do not carry out
photosynthesis, including C. elegans, flies, frogs, and fish, so this domain must have
other functions (Pfam 24.0, http://pfam.sanger.ac.uk/).
It remains to be determined if F13E9.13 has a role in vulval development. RNAi
or a deletion of F13E9.13 did not cause a class A or B synMuv phenotype or suppress
the synMuv phenotype. Additionally, neither RNAi nor a deletion of F13E9.13 caused
lethality, a phenotype that would obscure a role in vulval development. Either chromatin
immunoprecipitation (ChIP) or EMSA experiments with F13E9.13 protein could
determine if the F13E9.13 protein binds to the lin-3 promoter or is otherwise localized
there.
A major outstanding question is if any of the class A synMuv proteins are present
at the lin-3 promoter, either by directly binding DNA in a sequence-specific fashion or as
part of a complex with another DNA-binding protein. We did not detect any binding of in
225
vitro transcribed and translated class A synMuv proteins to a 60 bp fragment of the lin-3
promoter in EMSA experiments. However, there are numerous reasons why an
interaction might not be seen in such an assay. The DNA fragment could be too short
and missing sequences essential for the interaction. The proteins created by in vitro
transcription and translation might be missing essential modifications required for
function. Essential protein cofactors might be missing or buffer conditions might not be
correct to allow for the interaction. Many of these same caveats apply to EMSA with
nuclear extracts. Additionally, we lack viable null mutations in lin-38 and mcd-1
(Chapter Four), so it is not possible to determine if the specific binding activity that we
identified contains or is dependent on LIN-38 or MCD-1. In short, it is still possible that
one or more of the class A synMuv proteins bind to the lin-3 promoter at the site of the
lin-3(n4441) mutation. We believe that the best way to test this hypothesis is to perform
ChIP experiments. ChIP requires specific antibodies, and antibodies have been raised
against only some of the class A synMuv proteins. Antibodies that recognize LIN-8 and
LIN-56 are functional for both western blots and whole-mount staining, while antibodies
that recognize LIN-15A are functional for whole-mount staining but not for western blots
(Davison, 2003; Davison et al., 2005; E. Davison and H.R.H, personal communication).
Furthermore, the existing class A synMuv antibodies might not be functional for
immunoprecipitation. The simplest way to circumvent these problems is to fuse each
class A synMuv gene to an epitope tag. For example, we have made a GFP-tagged
version of lin-15A that can rescue the class A synMuv phenotype caused by loss of
lin-15A function. Rescuing plasmids exist for all of the class A synMuv genes except
mcd-1, and a similar approach could be taken to produce epitope-tagged versions of the
other class A synMuv proteins. Established anti-GFP antibodies and ChIP protocols
could then be used to definitively test if the class A synMuv proteins are present at the
lin-3 promoter. If the class A synMuv proteins are present at the lin-3 promoter then
additional experiments can determine how the class A synMuv proteins directly repress
lin-3 expression. If the class A synMuv proteins are not present at the lin-3 promoter
then other approaches could identify proteins that do interact with the class A synMuv
element in the lin-3 promoter (see Chapter Six).
226
Acknowledgments
We thank Nicolas Paquin for helpful comments about this chapter.
227
Table 1: lin-3(+) and lin-3(n4441) affect vulval development
similarly when present on a repetitive extrachromosomal array
a
Background
Plasmida
N2
N2
lin-15B(n744)
lin-15B(n744)
lin-3(+)
lin-3(n4441)
lin-3(+)
lin-3(n4441)
% multivulva ± s.d. (n)b
6.0 ± 7.0
8.7 ± 15
34 ± 25
33 ± 26
(10)
(4)
(4)
(5)
lin-3 plasmids were injected at a concentration of 10 ng/µl, along with myo-3::GFP at
50 ng/µl and DNA ladder at 100 ng/µl.
b
Animals were scored as Muv if any ventral ectopic protrusions were observed. %
multivulva shown is the average of between 3 and 10 independent lines. s.d., standard
deviation. n, number of lines scored.
228
Table 2: Deletion of F13E9.13 does not cause or suppress the synMuv
phenotype
% multivulva (n)a
genotype
20ºC
25˚ C
F13E9.13(n5408∆)
0%
(655)
0 % (446)
F13E9.13(n5408∆); lin-15A(n767)
0.1 % (765)
0.4 % (460)
F13E9.13(n5408∆); lin-15B(n744)
0%
(647)
0 % (379)
F13E9.13(n5408∆); lin-15AB(n765)
100 % (279)
N.D.
a
Animals were scored as Muv if any ventral ectopic protrusions were observed.
n, total number of animals scored.
229
Figure 1: Identification of a sequence-specific lin-3p-binding protein
(A) A 60 bp biotin-labeled double-stranded oligonucleotide centered around the site of
the lin-3(n4441) mutation was incubated with wild-type nuclear extracts. Unlabeled
competitor oligonucleotides were added to a 50-fold excess. The n4441 mutant
competitor is identical to the wild-type oligonucleotide except for the lin-3(n4441)
mutation. The scrambled competitor contains the same base composition as the wildtype oligonucleotide, but the bases are randomized into a different order. The band that
specifically binds to the wild-type lin-3 promoter sequence is indicated with an arrow.
(B) EMSA experiments were performed as in (A) with either no unlabeled competitor or
different concentrations of wild-type or mutant competitor oligonucleotides.
(C) EMSA experiments were repeated using nuclear extracts made from wild-type
animals or class A synMuv mutants.
230
n4441 mutant competitor
wild-type competitor
B
C
-
Nuclear extract:
n4441 competitor:
5x
wild-type
-
+
10x
50x
lin-8(n2731)
-
+
m
ut
sc
an
ra
t
m
bl
ed
1
44
n4
w
ild
ne
no
A
50x competitor:
-ty
pe
Figure 1
150x
5x
10x
50x
lin-56(n2728) lin-38(n751)
-
+
-
+
150x
lin-15A(n767)
-
+
Figure 2: Purification of sequence-specific lin-3p binding protein.
(A) Purification scheme. Biotin-labeled double-stranded oligonucleotides were bound to
streptavidin-conjugated magnetic beads. Nuclear extract from mixed-stage wild-type
animals was first incubated with lin-3(n4441) mutant oligonucleotides bound to beads.
Unbound supernatant was then incubated with a second batch of lin-3(n4441) mutant
oligonucleotides. Supernatant that had not bound to any of the lin-3(n4441) mutant
oligonucleotides was then incubated with wild-type oligonucleotides. Finally, each tube
of oligonucleotide-bound beads was washed several times, and a high salt buffer was
used to elute binding proteins.
(B) Denaturing gel electrophoresis analysis of the purification outlined in (A). The band
in lane 7 indicated by an arrow represents a potential sequence-specific binding protein
that binds to lin-3p(+) but not lin-3p(n4441).
(C) The band indicated by an arrow in lane 7 of (B) was excised and analyzed by mass
spectrometry. Eight peptides corresponding to the protein F13E9.13 were identified.
The predicted F13E9.13 protein (http://www.wormbase.org) is shown, and peptides
identified by mass spectrometry are underlined.
232
Figure 2
A
unbound
supernatant
unbound
supernatant
elute from beads
with high salt
Nuclear
extract
lin-3p
binding
protein(s)
n4441 oligos
on beads (#1)
B
1
2
3
4
n4441 oligos
on beads (#2)
5
6
WT oligos
on beads
7
Lane
Contents
1
unbound
supernatant
116 kD
97 kD
2
Final wash from
n4441 oligo (#1)
66 kD
3
Eluate from n4441
oligo (#1)
4
Final wash from
n4441 oligo (#2)
5
Eluate from n4441
oligo (#2)
6
Final wash from WT
oligo
7
Eluate from WT oligo
200 kD
45 kD
31 kD
C
F13E9.13 protein
MVQRVVVSKLLNSSRPLVFGMIHVPALPGTPSNTLPMSAILKK
VRKEADVYFKNGVDGVIVENMHDVPYVKPPASPEIVSSMALA
SDQLVKSRDAHHPAALTGIQILAAANREALAVAYTTGMDFIRA
EGFVYSHVADEGWIDGCAGGLLRYRSSLKAENIAIFTDIKKKH
SAHSVTSDVSIHEMAKDAKFNCADGVIVTGSATGSAASLEEMI
QVMKVQEFPVLIGSGINGKNAREFVKAHGFIVGSDFKIGGEW
KNDLDSGRISKFMKHVNTLKR
Figure 3: EMSA with in vitro transcribed and translated class A synMuv proteins
(A) Each class A synMuv protein was made by in vitro transcription and translation.
35
S-labeled proteins were assayed by denaturing gel electrophoresis.
(B) EMSA using a 32P-labeled 60 bp double-stranded oligonucleotide probe incubated
with in vitro transcribed and translated class A synMuv proteins. The lane labeled “all
class A synMuv proteins” contained an equal mix of each of the five class A synMuv
proteins.
234
al
lc
ss
-1
6
8
la
D
-5
-3
5A
-8
e
A
as
fe
r
-1
C
M
LI
N
LI
N
LI
N
LI
N
ci
n
pr
ot
ei
ns
-1
5
ra
s
LI A
N
-3
8
LI
N
-5
6
M
C
D
-1
LI
N
-8
ci
fe
LI
N
Lu
size (kD)
Lu
ot
ei
B
pr
A
no
e
Figure 3
200
150
100
75
50
37
25
20
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