Six and Eya promote apoptosis through direct
transcriptional activation of the proapoptotic BH3-only
gene egl-1 in Caenorhabditis elegans
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Citation
Hirose, T., B. D. Galvin, and H. R. Horvitz. “Six and Eya promote
apoptosis through direct transcriptional activation of the
proapoptotic BH3-only gene egl-1 in Caenorhabditis elegans.”
Proceedings of the National Academy of Sciences 107, no. 35
(August 31, 2010): 15479-15484.
As Published
http://dx.doi.org/10.1073/pnas.1010023107
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National Academy of Sciences (U.S.)
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Final published version
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Wed May 25 19:02:30 EDT 2016
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http://hdl.handle.net/1721.1/84507
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Detailed Terms
Six and Eya promote apoptosis through direct
transcriptional activation of the proapoptotic
BH3-only gene egl-1 in Caenorhabditis elegans
Takashi Hirose, Brendan D. Galvin, and H. Robert Horvitz1
Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
Contributed by H. Robert Horvitz, July 14, 2010 (sent for review April 30, 2010)
cell death
| sine oculis | eyes absent | transcription
A
poptosis, also referred to as programmed cell death, plays
fundamental roles in animal development and tissue homeostasis (1). The misregulation of apoptosis is associated with
many human disorders, including cancer and neurodegenerative
and autoimmune diseases (2). Determining how particular cells
are specified to live or die is critical to understanding both normal
animal development and human diseases associated with the
misregulation of apoptotic cell death.
During the development of the Caenorhabditis elegans hermaphrodite, 131 somatic cells undergo programmed cell death (3,
4). Genetic studies of programmed cell death in C. elegans have
defined an evolutionarily conserved pathway that executes this process (5). This pathway consists of four genes, egl-1 [egg-laying defective (egl)], ced-9 [cell-death abnormal (ced)], ced-4, and ced-3, all
of which are conserved from C. elegans to mammals. Although much
is understood about the pathway responsible for the execution of
programmed cell death in both C. elegans and other animals, less is
known about the mechanisms that control how specific cells decide
whether to survive or die by programmed cell death. In C. elegans,
most of the genes identified to control cell-death specification encode transcription factors, some of which are known to directly regulate the transcription of cell-death genes (6–11). For example, the
Snail family transcription factor CES-1 [cell-death specification
(ces)] can directly repress expression of the BH3-only proapoptotic
gene egl-1 and prevent the deaths of the NSM sister cells (6, 7). This
regulatory mechanism is conserved in mammals and has been implicated in human cancer: acute lymphoblastic leukemia results
from an overexpression of the CES-1 homolog Slug, which directly
represses expression of the BH3-only proapoptotic gene Puma, preventing apoptosis of hematopoietic progenitor cells (12, 13).
By contrast, the C. elegans Bar homeodomain transcription
factor CEH-30 [C. elegans homeobox (ceh)] protects specific
neurons from undergoing programmed cell death by acting inwww.pnas.org/cgi/doi/10.1073/pnas.1010023107
dependently of egl-1 (14); ceh-30 also regulates egl-1 transcription (15). The antiapoptotic function of ceh-30 likely explains the
previously observed loss of sensory hair cells of the inner ear and
progressive hearing loss of mice deficient in the function of the
ceh-30 homolog Barhl1 (16).
The identification and characterization of additional factors
that regulate cell type-specific apoptosis in C. elegans promise to
reveal both conserved molecular mechanisms that control celldeath specification and how misregulated apoptosis can cause
human disease. Here, we show that the C. elegans Six family
homeodomain protein CEH-34 and the Eyes absent ortholog
EYA-1 directly activate expression of the proapoptotic BH3-only
gene egl-1 in the M4 sister cell to promote programmed cell death.
We suggest that, in mammals, Six family members and Eya control
apoptosis by directly regulating the transcription of BH3-only
genes and mutations in Six and Eya genes perturb normal apoptosis by misregulating the transcription of BH3-only genes.
Results
Mutants Defective in the Programmed Cell Death of the M4 Sister
Cell. The C. elegans M4 motor neuron is located on the dorsal side
of the anterior bulb of the pharynx and regulates pharyngeal
muscle contraction during feeding (17, 18). The mother of the M4
neuron divides to generate an anterior daughter that survives and
becomes the M4 neuron and a posterior daughter, the M4 sister
cell, which dies by programmed cell death during embryogenesis
(Fig. 1A) (3). To identify genes that control the specification of
the M4 sister-cell death, we performed a genetic screen for
mutants with a surviving M4 sister cell. We used a Pceh-28::4xNLS::
gfp reporter transgene that is specifically expressed in the M4
neuron in wild-type animals and in both the M4 neuron and the
surviving M4 sister cell in ced-3 mutants defective in programmed
cell death (Fig. 1 A and B) (19). Thus, this reporter can be used to
detect M4 sister-cell survival.
We screened the F2 progeny of hermaphrodites mutagenized
with ethyl methanesulfonate (EMS) and, from a total of 72,000
haploid genomes screened, isolated 59 mutants with a surviving
M4 sister cell, which was located variably as in ced-3 mutants (Fig.
1B and Fig. S1). From these 59 mutants, we identified 38 alleles of
ced-3, 13 alleles of ced-4, 3 alleles of egl-1, and 5 mutants that
seemed not to carry alleles of any known cell-death genes. Here,
we focus on two mutants that affected the deaths of both the M4
sister cell and the pharyngeal I1 sister cells, n4820 and n4796.
In n4820 mutants, 80% of the M4 sister cells (n = 120) and 10%
of the I1 sister cells (n = 240) survived (Fig. 1 B and C and Table
S1). In n4796 mutants, 38% of the M4 sister cells (n = 140) and
Author contributions: T.H., B.D.G., and H.R.H. designed research; T.H. and B.D.G. performed research; T.H. and B.D.G. contributed new reagents/analytic tools; T.H., B.D.G.,
and H.R.H. analyzed data; and T.H., B.D.G., and H.R.H. wrote the paper.
The authors declare no conflict of interest.
1
To whom correspondence should be addressed. E-mail: horvitz@mit.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1010023107/-/DCSupplemental.
PNAS | August 31, 2010 | vol. 107 | no. 35 | 15479–15484
DEVELOPMENTAL
BIOLOGY
The decision of a cell to undergo programmed cell death is tightly
regulated during animal development and tissue homeostasis.
Here, we show that the Caenorhabditis elegans Six family homeodomain protein C. elegans homeobox (CEH-34) and the Eyes absent
ortholog EYA-1 promote the programmed cell death of a specific
pharyngeal neuron, the sister of the M4 motor neuron. Loss of
either ceh-34 or eya-1 function causes survival of the M4 sister cell,
which normally undergoes programmed cell death. CEH-34 physically interacts with the conserved EYA domain of EYA-1 in vitro. We
identify an egl-1 5′ cis-regulatory element that controls the programmed cell death of the M4 sister cell and show that CEH-34
binds directly to this site. Expression of the proapoptotic gene
egl-1 in the M4 sister cell requires ceh-34 and eya-1 function. We
conclude that an evolutionarily conserved complex that includes
CEH-34 and EYA-1 directly activates egl-1 expression through a 5′
cis-regulatory element to promote the programmed cell death of
the M4 sister cell. We suggest that the regulation of apoptosis by
Six and Eya family members is conserved in mammals and involved
in human diseases caused by mutations in Six and Eya.
A
A
B
B
C
C
Fig. 1. Mutants defective in M4 sister-cell death. (A) A schematic representation of the M4 lineage in the wild-type and ced-3 mutants is shown.
The M4 sister cell undergoes programmed cell death in the wild-type and
survives in ced-3 animals. (B) Merged epifluorescence and Nomarski images
of the pharynx in the wild-type, ced-3(n717), n4820, and n4796 animals
carrying the Pceh-28::4xNLS::gfp transgene are shown. Arrow, the M4 neuron;
arrowhead, the surviving M4 sister cell. (Scale bar, 20 μm.) (C) The penetrances of M4 sister survival in the indicated strains are shown.
47% of the I1 sister cells (n = 240) survived (Fig. 1 B and C and
Table S1). By contrast, the n4820 and n4796 mutations did not
affect the programmed cell deaths of the NSM sister cells or the
VC homologs of the ventral nerve cord (Table S1). Thus, n4820
and n4796 affect M4 sister-cell and I1 sister-cell deaths but do not
affect all programmed cell deaths.
n4820 Is a Noncoding Allele of the Proapoptotic BH3-Only Gene
egl-1. The mutation n4820 complemented ced-3(n717) and ced-4
(n1162) but failed to complement egl-1(n1084 n3082) for M4 sistercell survival (Table S2). We mapped n4820 to chromosome V, the
same chromosome as egl-1. Whereas the egl-1(n4820) mutation
caused survival of the M4 sister cell and the I1 sister cells but not
of other cells fated to die (Table S1), an egl-1 null allele causes
survival of essentially all somatic cells that die by programmed cell
death (Table S1) (20). These data indicate that egl-1(n4820) is an
atypical egl-1 allele that specifically affects the programmed cell
deaths of the M4 sister cell and the I1 sister cells.
We determined the sequence of the egl-1 locus of egl-1(n4820)
mutants and found two mutations. One is a C-to-T mutation located 4.9 kb upstream of the egl-1 start codon, and the other is
a G-to-C mutation located 4.2 kb upstream of the egl-1 start codon
(Fig. 2A). To determine which mutation caused survival of the M4
sister cell, we performed rescue experiments for the phenotype of
M4 sister survival of egl-1(n4820) mutants by injecting an egl-1
genomic fragment containing 6.5 kb upstream of the egl-1 start
codon, the egl-1 coding region, and 2.2 kb downstream of the egl-1
stop codon. Four of five transgenic lines that contained a wildtype egl-1 genomic fragment and three of six transgenic lines that
contained an egl-1 genomic fragment carrying the G-to-C mutation were rescued for the phenotype of M4 sister survival (i.e., the
M4 sister cells died in the rescued lines) (Fig. 2B and Fig. S2).
Zero of five transgenic lines that contained an egl-1 genomic
fragment carrying either the C-to-T mutation or both the G-to-C
and C-to-T mutations were rescued for the phenotype of M4 sister
survival (Fig. 2B and Fig. S2). Furthermore, we isolated a 268-bp
genomic deletion, n5347Δ, lacking the region of the C-to-T mutation but not the region of the G-to-C mutation (Fig. 2A). In
n5347Δ mutants, 90% of the M4 sister cells (n = 118) and 39% of
the I1 sister cells (n = 240) survived; n5347Δ did not affect the
15480 | www.pnas.org/cgi/doi/10.1073/pnas.1010023107
Fig. 2. n4820 is an egl-1 noncoding allele. (A) The egl-1(n4820) isolate
contained two mutations in the egl-1 5′ upstream noncoding region: C-to-T
(n4820) and G-to-C (n5334). The extent of the n5347Δ deletion is shown. The
exons of egl-1 are shown as black boxes. (B) The indicated egl-1 genomic
fragments were injected into egl-1(n4820 n5334) animals to test for rescue
of the phenotype of M4 sister-cell survival. The results of these rescue
experiments are indicated. (C) DNA sequence surrounding the site altered by
the n4820 mutation is shown for C. elegans and related species. Nucleotides
identical to those in C. elegans are indicated by bold, and the nucleotide
mutated in n4820 animals is highlighted in red.
deaths of the NSM sister cells (0% NSM sister survival; n = 240)
or the VC homologs (0.0 extra VC homologs/animal; n = 120).
Together, these results establish that the C-to-T mutation was
responsible for the survival of the M4 sister cell and the I1 sister
cells in egl-1(n4820) mutants. To distinguish the n4820 C-to-T
mutation, which causes survival of the M4 sister cell and the I1
sister cells, from the G-to-C mutation, we designated the G-to-C
mutation n5334 (Fig. 2A). The DNA sequence immediately
surrounding the n4820 mutation is highly conserved in other
nematode species (Fig. 2C). Based on these results, we suggest
that the n4820 region defines a binding site for a transcriptional
activator that promotes egl-1 expression in the M4 sister cell and
the I1 sister cells and that this binding site is defective in n4820
and n5347Δ mutants.
n4796 Is an Allele of ceh-34, a Six Family Homeodomain Gene. We
mapped n4796 to a 150-kb interval of chromosome V almost
completely covered by six cosmids (Fig. S3). We performed rescue
experiments for the phenotype of M4 sister survival of n4796
animals by injecting cosmids in this interval. The cosmid C10G8
rescued the phenotype of M4 sister survival of n4796 animals (Fig.
S3). C10G8 encodes eight predicted genes, including ceh-34. A
genomic fragment that contains only ceh-34 rescued the phenotype
of M4 sister survival of n4796 animals (Fig. S3). n4796 animals
have a G-to-A mutation in the splice acceptor site of the second
exon of ceh-34 (Fig. 3A). RNAi of ceh-34 caused survival of the M4
sister cell (38% of M4 sisters survived; n = 123). These results
indicate that n4796 is a reduction-of-function allele of ceh-34.
ceh-34 encodes a 256 amino acid protein that is similar to Six
family homeodomain proteins, which typically function as transcription factors (21). Six family homeodomain proteins contain
a conserved Six domain that mediates protein–protein interactions and a homeodomain that binds DNA (21) (Fig. 3B). Both
domains are evolutionarily conserved from C. elegans to humans
(Fig. S4). The CEH-34 protein is 48% and 63% identical to the
human protein SIX1 within the Six domain and the homeodomain, respectively.
We tested whether the functions of C. elegans CEH-34 and human SIX are evolutionarily conserved. We showed that expression of a ceh-34 cDNA under the control of the ceh-34 promoter in
a ceh-34(n4796) mutant rescued the defect in M4 sister-cell death
(Table S3). We then substituted a human SIX1 cDNA for the
ceh-34 cDNA in this construct and found that expression of SIX1
also rescued the defect in M4 sister-cell death of ceh-34(n4796)
Hirose et al.
sisters survived; n = 120) and the I1 sister cells (19% of I1 sisters
survived; n = 240) but did not affect the NSM sister cells or the VC
homologs (Fig. 4A and Table S1). Null mutations in vab-3 and
dac-1, the C. elegans orthologs of eyeless and dachshund, respectively, did not cause survival of the M4 sister cell (Fig. 4A).
These results indicate that the C. elegans eyes absent ortholog eya-1
is required for the deaths of the M4 sister cell and the I1 sister cells
and suggest that eya-1 might function with the Six family homeodomain gene ceh-34 to promote programmed cell death.
A
B
C
D
E
F
Fig. 3. n4796 is an allele of the Six family homeodomain gene ceh-34. (A)
Genomic organization of the ceh-34 locus. The location and nature of the
n4796 mutation are indicated. The red lowercase letter indicates the splice
acceptor site of the n4796 mutation in the first intron, and the uppercase
letters indicate DNA sequences in the second exon. The extent of the tm591Δ
deletion is indicated. (B) Representation of the domain structure of the CEH34 protein. The yellow box indicates the Six domain (Six), and the orange
box indicates the homeodomain (HD). (C–F) Epifluorescence images showing
expression of the ceh-34::gfp transgene. (C) The early embryo, (D) 1.5-fold
stage embryo, (E) first-stage (L1) larva, and (F) the pharynx of an L1 larva at
higher magnification. Cells expressing the ceh-34::gfp transgene are identified in F. BWM, body-wall muscle. (Scale bars: C, D, and F, 10 μm; E, 50 μm.)
mutants (Table S3). We conclude that the functions of C. elegans
ceh-34 and human SIX are evolutionarily conserved.
EYA-1 Forms an Evolutionarily Conserved Complex with the Six Family
Homeodomain Protein CEH-34. The Six family homeodomain pro-
teins of Drosophila and mammals physically interact with the
conserved C-terminal Eya domains of Eya proteins (25, 26). Because mutations of ceh-34 and eya-1 caused the same phenotype
of M4 sister-cell and I1 sister-cell survival, we examined whether
CEH-34 physically interacts with EYA-1. We fused GST to the Cterminal Eya domain of EYA-1 (GST-EYA-1C) and found that
this protein specifically bound CEH-34 (Fig. 4B). CEH-34 and
EYA-1 also have been shown by others to both act to specify
coelomocyte cell fate and interact physically (22).
If CEH-34 physically interacts and functions with EYA-1 in vivo,
the two proteins must be expressed in at least some of the same
cells. Indeed, the expression pattern of an eya-1::gfp transgene
capable of rescuing the phenotype of eya-1 mutants is similar to
that of our ceh-34::gfp transgene; eya-1 is predominantly expressed
in pharyngeal cells (27). We observed expression of both the
A
B
To determine the expression pattern of ceh-34, we constructed
a translational ceh-34::gfp reporter transgene and showed that it
can rescue the phenotype of M4 sister survival of ceh-34(n4796)
mutants. Expression of this ceh-34::gfp transgene began during
embryogenesis (Fig. 3 C and D). CEH-34::GFP was localized to
the nuclei of expressing cells (Fig. 3 C–F), consistent with its likely
function as a transcription factor. During embryonic morphogenesis and larval development and throughout adulthood, expression of the ceh-34::gfp transgene was seen predominantly in
pharyngeal cells (Fig. 3 D and E). A similar expression pattern for
a ceh-34::gfp transgene has been independently observed (22). We
observed that our ceh-34::gfp transgene was expressed in all
pharyngeal neurons (M4, I1, MI, I3, M3, NSM, MC, I2, I4, I5, I6,
M1, M2, and M5), some pharyngeal muscle cells (pm1 and pm2)
and pharyngeal epithelial cells (e1 and e3), and some body wall
muscles around the anterior pharynx (Fig. 3F).
Eyes Absent Ortholog EYA-1 Is Required for M4 Sister- and I1 SisterCell Death. Six family homeodomain genes were originally identi-
fied in studies of Drosophila eye development (23). The Drosophila Six family homeodomain gene sine oculis is required for
proper eye specification and functions in a conserved transcriptional network that includes the paired-box (Pax) homeodomain
gene eyeless (ey) and the transcriptional cofactors eyes absent (eya)
and dachshund (dac) (24). We tested whether the C. elegans
orthologs of these three genes promote M4 sister-cell death. We
found that a deletion allele of eya-1, the C. elegans eyes absent
ortholog, caused survival of both the M4 sister cell (49% of M4
Hirose et al.
C
D
E
F
G
H
Fig. 4. (A) The penetrances of M4 sister-cell survival in the indicated mutants
are shown. Pax6, paired-like homeodomain gene; Eya, mammalian eyes absent
homolog; Dach, mammalian dachshund homolog. (B) CEH-34 binds the Cterminal domain of EYA-1 (EYA-1C) in vitro; 5% of input proteins and bound
proteins were subjected to electrophoresis and visualized using autoradiography. The positions of CEH-34 and luciferase are indicated by arrows. (C–H)
ceh-34::gfp and eya-1::gfp transgenes are expressed in the M4 sister cells in
ced-3 mutants. (C and F) Expression of the ceh-34::gfp and eya-1::gfp transgenes, respectively. (D and G) Expression of the M4 neuron and the M4 sistercell marker Pceh-28::4xNLS::mCherry. (E and H) Merged images. Arrows indicate
the M4 neurons, and arrowheads indicate the M4 sister cells. (Scale bar, 10 μm.)
PNAS | August 31, 2010 | vol. 107 | no. 35 | 15481
DEVELOPMENTAL
BIOLOGY
CEH-34 Is Expressed Predominantly in the Nuclei of Pharyngeal Cells.
ceh-34::gfp transgene and an eya-1::gfp transgene in the M4 sister
cell in ced-3(n717) mutants (Fig. 4 C–H). These results suggest that
CEH-34 and EYA-1 form an evolutionarily conserved complex in
the M4 sister cell to promote programmed cell death.
CEH-34 Binds to the egl-1 5′ cis-Regulatory Element Defined by egl-1
(n4820). Because egl-1(n4820) and ceh-34(n4796) both specifically
affected the programmed cell deaths of the M4 sister cell and the
I1 sister cells, we examined whether egl-1 and ceh-34 might
functionally interact to regulate these programmed cell deaths.
Interestingly, the DNA sequence surrounding the egl-1(n4820)
mutation is similar to the consensus-binding sequence of Sine
Oculis (SO), the Drosophila homolog of CEH-34 (Fig. 5A) (28).
To test if CEH-34 can bind directly to this presumptive egl-1 cisregulatory element, we performed gel-shift assays. We observed
that CEH-34 indeed binds to this sequence (Fig. 5B).
To test the specificity of this interaction, we performed competition experiments with unlabeled competitors. Both the wildtype competitor and competitors with mutations outside the SO
consensus-binding sequence (wild type, I, and V in Fig. 5A)
competed with the labeled wild-type probe for CEH-34 binding
(Fig. 5C). By contrast, competitors with mutations in the SO consensus-binding sequence (II, III, IV, and scrambled in Fig. 5A) did
not efficiently compete with the labeled wild-type probe for CEH34 binding (Fig. 5C). These results indicate that CEH-34 binds to
the SO consensus-binding sequence of an egl-1 cis-regulatory element surrounding the egl-1(n4820) mutation.
Because the egl-1(n4820) mutation is located within the SO
consensus-binding sequence, we examined whether the egl-1
(n4820) mutation affects CEH-34 binding. Whereas wild-type
competitor efficiently competed for CEH-34 binding, a competitor
containing the egl-1(n4820) mutation did not compete for CEH-34
binding as efficiently as the wild-type competitor (Fig. 5D). These
results indicate that the egl-1(n4820) mutation reduces CEH-34
binding to this egl-1 cis-regulatory element.
egl-1 Expression in the M4 Sister Cell Requires ceh-34 and eya-1
Function. Given that CEH-34 directly binds to the egl-1 cis-regula-
tory element in vitro and that CEH-34 forms a complex with EYA-1,
A
C
we examined if ceh-34 or eya-1 mutations alter egl-1 expression in
the M4 sister cell. In ced-3 mutants, a Pegl-1::4xNLS::gfp reporter
transgene that contains the egl-1 cis-regulatory element required for
CEH-34 binding was expressed in the M4 sister cell (100% of animals; n = 119) but not in the M4 neuron (0% of animals; n = 119),
strongly suggesting that egl-1 expression regulates the death of
the M4 sister cell (Fig. 6 A, B, C, and G). We observed that in 55%
of ced-3; ceh-34(n4796) animals (n = 97) and 64% of eya-1
(ok654Δ); ced-3 animals (n = 89), the M4 sister cell did not express the Pegl-1::4xNLS::gfp transgene (Fig. 6 D–G). These results
establish that ceh-34 and eya-1 are necessary for normal egl-1
expression in the M4 sister cell and suggest that ceh-34 and eya-1
activate egl-1 expression to promote M4 sister-cell death.
Discussion
ceh-34 and eya-1 Promote M4 Sister-Cell Death by Directly Activating
Transcription of the Proapoptotic Gene egl-1. From a genetic screen
for mutants defective in the death of the M4 sister cell and
a subsequent candidate-gene approach, we identified two genes
that specify the cell-death fate of the M4 sister cell: the Six family
homeodomain gene ceh-34 and the eyes absent ortholog eya-1.
Based on our findings, we propose a model for the regulation of
M4 sister-cell death (Fig. 6H). In wild-type animals, a complex
consisting of the CEH-34 and EYA-1 proteins binds to the egl-1
5′ cis-regulatory element and activates egl-1 expression in the M4
sister cell, causing the M4 sister cell to die. In ceh-34 or eya-1
mutants, a functional CEH-34-EYA-1 complex is not formed,
and egl-1 is not expressed, causing the M4 sister cell to survive. In
egl-1(n4820) mutants, the CEH-34-EYA-1 complex cannot bind
to the egl-1 5′ cis-regulatory element, and egl-1 is not expressed,
causing the M4 sister cell to survive. The same transcriptional
regulatory mechanism likely, at least in part, promotes I1 sistercell death, because ceh-34(n4796), eya-1(ok654Δ), and egl-1(n4820)
also caused survival of the I1 sister cells.
We propose that CEH-34 and EYA-1 function cell autonomously in the M4 sister cell to activate egl-1 expression and promote programmed cell death. Consistent with this hypothesis, we
observed that ceh-34 and eya-1 were expressed in the M4 sister
cell. Whereas ceh-34 and eya-1 were expressed in both the M4
B
D
Fig. 5. CEH-34 binds to the egl-1 5′ cis-regulatory element defined by egl-1(n4820). (A) Sine Oculis (SO) consensus-binding sequence and the DNA sequences
of oligonucleotide probes used in the gel-shift assays are shown. Mutated bases are shown in red lowercase letters. Y indicates C or T, S indicates G or C, and N
indicates A, C, G, or T. (B) The indicated amount of reticulocyte lysate expressing CEH-34 was mixed with the wild-type radioactive probe. The shifted band
and free probe are indicated by arrowheads. (C) The indicated unlabeled competitors were preincubated in 200-fold molar excess to the radiolabeled probe.
The shifted band and free probe are indicated by arrowheads. (D) The indicated unlabeled competitors were preincubated in 0- to 200-fold molar excess to
the radiolabeled probe. The shifted band and free probe are indicated by arrowheads.
15482 | www.pnas.org/cgi/doi/10.1073/pnas.1010023107
Hirose et al.
A
B
C
lation of the programmed cell deaths of the M4 sister cell and I1
sister cells.
Functions of ceh-34 and eya-1 in Regulating Programmed Cell Death
Are Evolutionarily Conserved. CEH-34 family Six proteins and
E
F
G
H
Fig. 6. egl-1 expression in the M4 sister cell requires ceh-34 and eya-1. (A–F)
The Pegl-1::4xNLS::gfp and Pceh-28::4xNLS::mCherry transgenes were coexpressed in ced-3(n717) animals (A–C) and ced-3(n717); ceh-34(n4796) animals
(D–F). (A and D) Expression of the Pegl-1::4xNLS::gfp transgene. (B and E)
Expression of the Pceh-28::4xNLS::mCherry transgene in the M4 neuron and
M4 sister cell. (C and F) Merged images. Arrows indicate the M4 neuron;
arrowheads indicate the M4 sister cell. Both cells are circled. (Scale bar,
5 μm.) (G) Percent of animals lacking expression of the Pegl-1::4xNLS::gfp
transgene in the M4 sister cell is indicated for ced-3(n717), ced-3(n717); ceh34(n4796), and eya-1(ok654Δ); ced-3(n717) animals. (H) A model for the
control of M4 sister-cell death by CEH-34 and EYA-1 is shown for the wildtype, ceh-34, eya-1, or egl-1(n4820) mutants.
neuron and M4 sister cell, egl-1 was expressed only in the M4 sister
cell destined to die but not in the M4 neuron destined to survive.
This observation suggests that ceh-34 and eya-1 are necessary but
not sufficient to activate egl-1 expression in the M4 sister cell.
Other factors that activate egl-1 expression in the M4 sister cell or
that repress egl-1 expression in the M4 neuron likely also interact
with CEH-34 and EYA-1 in causing the M4 sister cell to die or the
M4 neuron to survive (Fig. 6H).
ceh-34 and eya-1 Function More Broadly in Pharyngeal Development.
Although ceh-34 and eya-1 regulate the programmed cell deaths
of the M4 sister cell and the I1 sister cells, these genes also
function in other aspects of development. ceh-34(tm591Δ) deletion mutants arrest as early larvae and exhibit abnormal pharyngeal morphology with decreased expression of the ceh-28
reporter transgene that we used as a reporter for the M4 neuron
and M4 sister cell. eya-1 deletion mutants exhibit various defects,
including abnormal pharyngeal morphology (27). Both eya-1 (27)
and ceh-34 (our results) are predominantly expressed in pharyngeal cells. These observations suggest that ceh-34 and eya-1 are
involved in aspects of pharyngeal development besides the reguHirose et al.
EYA-1 family Eya proteins function in a diversity of organisms in
a variety of developmental processes, including apoptosis, cell
survival, and cell differentiation (24). We observed that human
SIX1 can rescue the defect in M4 sister-cell death of ceh-34
(n4796) mutants. This result suggests that the mechanisms of Six
family protein function in regulation of apoptosis are evolutionarily conserved between C. elegans and humans.
Eya function in apoptosis is also likely conserved from C. elegans to mammals. Just as eya-1 promotes M4 sister-cell death in
C. elegans, the overexpression of mammalian Eya proteins promotes apoptosis of murine myeloid cells (29). By contrast, during
embryonic development, C. elegans eya-1 mutants have excessive
programmed cell deaths (27), suggesting a protective function of
eya-1. Such a protective function of eya-1 might also be evolutionarily conserved, because loss of Eya function causes increased
apoptosis in Drosophila (30), zebrafish (31), and mice (32). Eya
proteins act as protein phosphatases (33) as well as transcriptional
cofactors, and the phosphatase activity of Eya proteins on histone
variant H2AX is required to protect mammalian kidney embryonic cells from apoptosis after DNA damage (34). These observations indicate that Eya proteins can function in either
promoting or inhibiting apoptosis, depending on cellular context,
and more generally, that the function of Eya in regulating apoptosis is evolutionarily conserved from C. elegans to mammals.
Human Diseases Caused by Mutations in Six Family Homeodomain
Genes or eyes absent Homologs Likely Result in Part from Misregulation
of Transcription of BH3-Only Genes and Apoptosis. Six family home-
odomain genes and eyes absent genes have been implicated in
mammalian apoptosis and various human diseases, including
cancer (35, 36), cardiomyopathy (37), and developmental disorders (38–40). Mutations in SIX3 were identified in human
patients with holoprosencephaly (HPE), a severe malformation of
the forebrain characterized by an incomplete segregation of the
cerebral hemispheres (38). In mice, a reduction of Six3 function
can cause an HPE-like phenotype as well as decreased apoptosis
along the ventral midline of the telencephalon and in optic stalks
and increased apoptosis in the lateral dorsal telencephalon of
embryos of a phenotypically normal heterozygous Sonic hedgehog
mutant (41). Mutations in SIX1 and EYA1 were identified in
human patients with branchio-oto-renal (BOR) syndrome, which
is characterized by craniofacial abnormalities, hearing loss, and
kidney defects (39, 40). Six1- or Eya1-deficient mice showed increased apoptosis in the inner ear and kidney, likely causing
a lack of inner ear structure and failure of kidney development
(32, 42, 43). In these mice, increased or decreased apoptosis is
seen in tissues in which abnormalities also are observed in human
HPE or BOR patients. These observations suggest that the misregulation of apoptosis caused by a mutation in Six or Eya is likely
involved in these diseases. Because Six and Eya likely function in
transcriptional regulation, transcriptional misregulation of target
genes likely causes altered apoptosis in mice with a mutation in
Six or Eya. Based on our findings, we suggest that mammalian Six
and Eya proteins directly regulate transcription of BH3-only
genes, that altered apoptosis in mammals carrying Six or Eya
mutations is caused by misregulation of transcription of BH3-only
genes, and that, in humans, such altered apoptosis is at least in
part responsible for the HPE and BOR syndromes.
Materials and Methods
See SI Materials and Methods for a full description.
PNAS | August 31, 2010 | vol. 107 | no. 35 | 15483
DEVELOPMENTAL
BIOLOGY
D
ACKNOWLEDGMENTS. We thank Brian Harfe (University of Florida College
of Medicine, Gainesville, FL) and Andrew Fire (Stanford University School
of Medicine, Stanford, CA) for providing pBH46.53, Asako Sugimoto (RIKEN
Center for Developmental Biology, Kobe, Japan) for eya-1 strains and the
eya-1::gfp construct, Yuichi Iino (University of Tokyo, Tokyo) for pENTR-gcy-
10, Barbara Conradt (Dartmouth Medical School, Hanover, NH) for bcIs24,
Karen Oegema (University of California at San Diego, La Jolla, CA) for mCherry,
Yuji Kohara (National Institute of Genetics, Mishima, Japan) for ceh-34 and
eya-1 cDNAs, Andrew Fire for gfp vectors, and members of the H.R.H. laboratory for technical support and discussions. We also thank Nirav Amin (Cornell
University) and Jun Liu (Cornell University, Ithaca, NY) for sharing information
about ceh-34 and eya-1, the Caenorhabditis Genetic Center, which is funded
by the National Institutes of Health, National Center for Research Resources,
for strains, the Sanger Institute (Hinxton, UK) for cosmids, and Shohei Mitani at
the National BioResource project (Tokyo Women’s Medical University, Tokyo)
for the ceh-34(tm591) strains. This work was supported by the Howard Hughes
Medical Institute. T.H. was supported in part by the Ministry of Education,
Science, Technology, Sports, and Culture of Japan. H.R.H is an Investigator
of the Howard Hughes Medical Institute and the David H. Koch Professor of
Biology at Massachusetts Institute of Technology.
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Strains and Genetics. C. elegans strains were cultured and maintained on
NGM plates at 20 °C as described (44). The Bristol strain N2 was the standard
wild-type strain, and the Hawaiian strain CB4856 was used for SNP mapping.
In Vitro GST Fusion Protein-Binding Experiments. In vitro GST fusion proteinbinding experiments were performed essentially as described (25).
Gel Mobility Shift Assays. Gel mobility shift experiments were performed
essentially as described (28).
15484 | www.pnas.org/cgi/doi/10.1073/pnas.1010023107
Hirose et al.
Supporting Information
Hirose et al. 10.1073/pnas.1010023107
SI Materials and Methods
We used the following mutations and integrants, which were
described by Riddle et al. (1) unless otherwise noted. LGI: eya-1
(ok654Δ, tm759Δ), nIs177[Pceh-28::4xNLS::gfp; lin-15AB(+)] (this
study), nIs226[ceh-34::gfp; lin-15AB(+)] (this study); LGII: nIs343
[Pegl-1::4xNLS::gfp; lin-15AB(+)] (this study), nIs352[eya-1::gfp; rol6(su1006)] (this study); LGIII: ced-4(n1162), ced-9(n1950), dac-1
(gk211Δ, gk213Δ), nIs176[Pceh-28::4xNLS::gfp; lin-15AB(+)] (this
study); LGIV: ced-3(n717), nIs175[Pceh-28::4xNLS::gfp; lin-15AB
(+)] (this study); LGV: ceh-34(n4796, tm591Δ) (this study), dpy-11
(e224), egl-1(n1084 n3082, n4820 n5334, n5347Δ) (2) (this study);
LGX: bcIs24 [Ptph-1::gfp; lin-15AB(+)] (3), lin-15(n765ts), nIs106
[Plin-11::gfp; lin-15AB(+)] (4), nIs283[Pgcy-10::4xNLS::gfp; lin-15AB
(+)] (this study), nIs349[Pceh-28::4xNLS::mCherry; lin-15AB(+)]
(this study), vab-3(e648, ju468) (5); extrachromosomal arrays:
tjEx21[eay-1(+); rol-6(su1006); sur-5::gfp] (6), tjEx25[eya-1::gfp; rol6(su1006)] (6). Information about the tm (provided by S. Mitani,
Tokyo Women’s Medical University, Tokyo), gk, and ok alleles can
be found at www.wormbase.org.
Genetic screens were performed using ethyl methanesulfonate
(EMS) mutagenesis as described (7). SNP mapping was performed to map ceh-34(n4796) and egl-1(n4820) essentially as
described (8).
Phenotypic Analysis and Microscopy. Animals were examined for
gross developmental defects using a dissecting microscope and
a compound microscope equipped with Nomarski optics. Programmed cell deaths were scored using gfp reporters and a compound microscope equipped with fluorescence optics. M4 sistercell death was scored using nIs175, nIs176, or nIs177 at the L1
stage, I1 sister-cell death was scored using nIs283 at the L4 stage,
NSM sister-cell death was scored using bcIs24 at the L1 stage (3),
and the deaths of the VC homologs were scored using nIs106 at
the L4 stage (4). To score expression of the Pegl-1::4xNLS::gfp
reporter transgene in the M4 sister cell, gravid animals carrying
nIs343 and nIs349 were put on NGM plates. After 24 h, L1 larvae
were scored for expression of the Pegl-1::4xNLS::gfp transgene
using a compound microscope equipped with fluorescence optics.
Molecular Biology and Transgenes. The Pceh-28::4xNLS::gfp and
Pceh-28::4xNLS::mCherry transgenes contained 2.4 kbp of 5′ promoter of ceh-28 in pPD122.56 carrying either gfp or mCherry.
The Pgcy-10::4xNLS::gfp transgene contained 1.8 kbp of 5′ promoter of gcy-10 in pPD122.56. The ceh-34–rescuing transgene
contained 4.0 kbp of 5′ promoter, the coding region, and 0.5 kbp
3′ of the stop codon in pGEM-T Easy vector. The ceh-34::gfp
transgene contains 3.8 kbp of 5′ promoter and the coding region
in pPD95.77. The egl-1–rescuing transgene contained 6.5 kbp of
5′ promoter, the coding region, and 2.2 kbp 3′ of the egl-1 stop
codon. The QuikChange II XL Site-Directed Mutagenesis Kit
(Stratagene) was used to make egl-1 constructs containing one or
both of the point mutations n4820 or n5334. The Pegl-1::4xNLS::
gfp transgene contained 6.5 kbp of the 5′ promoter of egl-1 in
pPD122.56. For in vitro transcription and translation, ceh-34
cDNA fragments containing their entire coding regions were
cloned in pGEM-T Easy. For a GST-EYA-1C fusion protein, the
C-terminal region of EYA-1 corresponding to amino acids 220–
503 was cloned in pGEX-4T-3.
In Vitro GST Fusion Protein-Binding Experiments. GST-EYA-1C fusion proteins were expressed in Escherichia coli strain BL21 and were
purified using glutathione Sepharose 4B (Amersham Biosciences).
Full-length [35S]methionine-labeled CEH-34 and luciferase were
expressed using a reticulocyte lysate in vitro transcription and translation system (Promega). [35S]methionine-labeled CEH-34 and luciferase were incubated with GST-EYA-1C bound to glutathione
Sepharose 4B for 2 h at 4 °C in binding buffer containing 20 mM
Hepes (pH 7.7), 150 mM NaCl, 0.1% Nonidet P-40, 2.5 mM MgCl2,
0.1 mM EDTA, 1 mM DTT, and 10% glycerol. The beads were
washed three times with binding buffer, and the bound proteins were
eluted by boiling in SDS/PAGE sample buffer for 5 min. The samples
were subjected to 6–18% gradient Tris·HCl polyacrylamide gel
electrophoresis (BioRad) followed by autoradiography.
Germ-line Transformation. Germ-line transformation experiments
were performed as described (9). gfp or mCherry transgenes were
injected at 50–100 μg/mL into lin-15(n765ts) or ced-3(n717); lin15(n765ts) animals with 50 μg/mL of pL15EK as a coinjection
marker (10). For ceh-34 rescue experiments, cosmids or genomic
constructs were injected at 10 μg/mL into ceh-34(n4796) animals
with 50 μg/mL of Plin-44::gfp as a coinjection marker (11). For egl-1
(n4820 n5334) rescue experiments, the egl-1 transgenes were injected at 2 μg/mL into egl-1(n4820 n5334) animals with 50 μg/mL
Plin-44::gfp as a coinjection marker (11).
Gel Mobility Shift Assays. A 30-bp double-stranded radioactive
probe was generated by annealing two partially overlapping
26-bp oligonucleotides and filling in a 5′ overhang using α-32P-CTP
and Klenow polymerase; unincorporated α-32P-CTP was removed
using a gel-filtration column packed with Sephadex G-25 (Amersham Biosciences). Full-length CEH-34 protein was expressed by
in vitro transcription and translation using a reticulocyte lysate
(Promega); 0.5–2 μL reticulocyte lysate was incubated with 10,000
cpm 32P-labeled probe in 20 mM hepes (pH 7.9), 100 mM KCl,
1 mM DTT, 0.3% BSA, 0.01% Nonidet P-40, and 20% glycerol
for 1 h at room temperature. The reactions were analyzed by
electrophoresis using nondenatured 6% polyacrylamide gels in
0.5% TBE buffer followed by autoradiography. For competition
experiments, CEH-34 protein was preincubated with unlabeled
competitors for 20 min at room temperature followed by incubation with 32P-radioactive probes.
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programmed cell death and interacts with the Bcl-2-like protein CED-9. Cell 93:
519–529.
3. Thellmann M, Hatzold J, Conradt B (2003) The Snail-like CES-1 protein of C. elegans
can block the expression of the BH3-only cell-death activator gene egl-1 by
antagonizing the function of bHLH proteins. Development 130:4057–4071.
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ortholog EYA-1 is required for tissue differentiation and plays partially redundant
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9. Mello CC, Kramer JM, Stinchcomb D, Ambros V (1991) Efficient gene transfer in C.
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gene lin-44, which controls the polarity of certain asymmetric cell divisions, encodes
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Fig. S1. The merged epifluorescence of the Pceh-28::4xNLS::gfp transgene and Nomarski images are presented. Genotypes are (A) ced-3(n717), (B) egl-1(n4820),
and (C) ceh-34(n4796). The surviving M4 sister cell is similarly located at the ventral side of the posterior bulb in these mutants. The arrows indicate the M4
neuron, and the arrowheads indicate the surviving M4 sister cell in the posterior bulb. (Scale bar, 20 μm.)
Fig. S2. An egl-1(n4820) C-to-T mutation is responsible for survival of the M4 sister cell. The transgenes containing the egl-1 genomic fragment of the wild
type egl-1(+), egl-1(n5334), egl-1(n4820), and egl-1(n4820 n5334) were used to transform egl-1(n4820 n5334) animals, and the animals carrying transgenes
were assayed for the phenotype of M4 sister-cell survival; 60 animals were scored for each strain.
Fig. S3. SNP mapping of ceh-34(n4796); n4796 was mapped between nucleotides 4549 on C52A10 (map position −2.4) and 38015 on F08F3 (map position −1.9)
on chromosome V. Cosmid C10G8, highlighted in red, rescued the phenotype of M4 sister-cell survival of ceh-34(n4796) mutants. A fragment of C10G8 containing
only ceh-34 rescued the phenotype of M4 sister-cell survival. The number of rescued lines is indicated.
Hirose et al. www.pnas.org/cgi/content/short/1010023107
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Fig. S4. Alignment of C. elegans CEH-34 protein sequence with that of Drosophila sine oculis (SO) and human SIX1. Accession numbers: NP_504419 for CEH-34,
NP_476733 for SO, and CAA62974 for human SIX1. The CLUSTAL W algorithm was used to align the amino acids. Identical amino acids and similar amino acids
are indicated with inverted boxes and gray boxes, respectively. A yellow underline indicates the extent of the Six domain, and the orange underline indicates
the extent of the homeodomain.
Table S1. egl-1(n4820), ceh-34(n4796), and eya-1(ok654Δ) promote survival of the M4 sister cell and I1 sister cells but not of the NSM
sister cells or the VC homologs
Genotype*
Percent M4 sister survival†
Percent I1 sister survival‡
Percent NSM sister survival‡
0
94
97
80
38
49
0
57
52
10
47
19
0
100
100
0
0
1
Wild type
ced-3(n717)
egl-1(n1084 n3082)
egl-1(n4820)
ceh-34(n4796)
eya-1(ok654Δ)¶
Extra VC homologs†
0.0
4.9
5.0
0.0
0.0
0.0
±
±
±
±
±
±
0.0§
0.3
0.1
0.0
0.1§
0.1
*These strains contained integrated gfp reporter transgenes to score survival of the indicated cell type as described in Materials and Methods.
†
One hundred twenty animals were scored.
‡
Two hundred forty cells were scored.
§
These strains contained dpy-11(e224), which does not affect programmed cell death.
¶
eya-1(ok654Δ) animals were the progeny of eya-1(ok654Δ); tjEx21[eay-1(+); rol-6(su1006); sur-5::gfp] animals. eya-1(ok654Δ) animals that lose the eya-1(+)rescuing extrachromosomal array were scored.
Table S2.
n4820 is an allele of egl-1
Genotype
Wild type
egl-1(n1084 n3082)
egl-1(n1084 n3082)/+
egl-1(n4820)
egl-1(n4820)/+
egl-1(n4820)/egl-1(n1084 n3082)
Percent M4 sister survival
n
0
97
0
80
0
71
120
120
24
120
24
24
All strains contained integrated gfp reporter transgenes to score survival
of the M4 sister cell.
Table S3. Human SIX can rescue the defect in M4 sister-cell death of ceh-34(n4796) mutants
Genotype
Wild-type
ceh-34(n4796)
ceh-34(n4796);
ceh-34(n4796);
ceh-34(n4796);
ceh-34(n4796);
Ex[Pceh-34::ceh-34 cDNA] line 1
Ex[Pceh-34::ceh-34 cDNA] line 2
Ex[Pceh-34::SIX1 cDNA] line 1
Ex[Pceh-34::SIX1 cDNA] line 2
Percent M4 sister survival
n
0
38
0
0
8
10
120
140
90
90
100
90
All strains contained integrated gfp reporter transgenes to score survival of the M4 sister cell. For each
transgene, Pceh-34::ceh-34 cDNA and Pceh-34::SIX1 cDNA, two independent transgenic lines, were scored.
Hirose et al. www.pnas.org/cgi/content/short/1010023107
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