The function of E2F6 in the Polycomb complex
by
Maria F. Courel
Licenciada, Biology
Universidad de Buenos Aires, 1999
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
SEPTEMBER 2005
MASSACHUSETTS INS
©2005 Massachusetts Institute of Technology
All rights reserved
OF TECHNOOGy
LJiB 27 20E
LIBRAIESJ
Signature of Author:
Department of Biology
August 5, 2005
Certified by:
Jacqueline A. Lees
Professor of Biology
Thesis Supervisor
Accepted by:
1--
9
Stephen B. Bell
Professor of Biology
Chairman, Graduate Committee
E
To Juan, Nicolds and all the Burronitosto come
The function of E2F6 in the Polycomb complex
by
Maria F. Courel
Submitted to Department of Biology
on March 31, 2005 in partial fulfillment of the requirements
for the Degree of Doctor of Philosophy in Biology
ABSTRACT
The E2F family of transcription factors are known cell cycle regulators that function at
the G1/S transition. Unlike other E2Fs, E2F6 does not activate transcription and is not regulated
by pocket protein binding. Instead, this protein appears to repress transcription through the
recruitment of the Polycomb Group (PcG) complex. This complex is responsible for the
maintenance of Hox gene expression patterns during development and thus ensures the correct
anterior-posterior segmentation of the embryo. Genetic ablation of PcG proteins leads to
posterior transformations of the axial skeleton as well as other developmental abnormalities such
as hematopoietic, cerebellar and smooth muscle defects. The PcG complex has been implicated
in cell cycle control since several of its members, including the oncoprotein Bmi 1, appear to
repress the transcription of p 1 6INK4 A and pI 9 ARF. In order to determine the biological function of
E2F6, we have generated and characterized E2f6'- mice and mouse embryonic fibroblasts
(MEFs). The mutant mice are viable and survive into adulthood with similar lifespan as their
littermate controls. Furthermore, the E2f6 null MEFs are indistinguishable from wild-type MEFs
in asynchronous proliferation, cell cycle re-entry from quiescence, senescence and E2F target
genes expression levels. These findings suggest that E2F6 does not play a major role in cell cycle
control or that its function can be compensated by the action of other factors. In fact,
preliminary results from combined loss of E2f6 and Bmil suggest that E2F6 may take part in the
Bmi 1-mediated control of the cell cycle. Furthermore, we found that the loss of E2F6 results in
posterior axial skeleton transformations that are reminiscent of the Bmil-deficient mice defects.
The study of the E2f6;Bmil compound mutant mice revealed a dosage-dependent synergism
between E2F6 and Bmi 1. These results indicate that E2F6 participates in segmentation during
murine development. As a whole, our work has provided proof that E2F6 is a bonafide
Polycomb Group protein and, at the same time, has opened the field to a number of interesting
questions.
Thesis supervisor: Jacqueline A. Lees
Title: Professor of Biology
3
ACKNOWLEDGEMENTS
I would like to thank my advisor, Jackie Lees, for her support, encouragement and
training through the years as well as her constant enthusiasm about my project. I am also
grateful to my committee members, Tyler Jacks, Frank Gertler, David Housman and Nick Dyson
for their helpful advice and suggestions.
I would also like to thank the entire Lees Lab for their help and for many hours shared
together. In particular, Jeff Trimarchi, Frances Connor and Paul Danielian have helped me at
different stages of my project with reagents, suggestions and discussions. Mark Garcia has
assisted me with the maintenance of the mouse colony and Laurie Friesenhahn has kindly agreed
to continue some of my work. Tiziana Parisi has become a source of friendship, encouragement
and honest advice since she joined our lab in recent years. I am especially indebted to Eunice
Lee, who has been a great colleague and sincere friend. She has always been willing to share her
expertise and ideas as well as lend a sympathetic ear and I cannot imagine my graduate studies
without her unwavering support and encouragement.
Many thanks are due to my friends and family far and close. My friends all over the
world have followed every detail of my PhD and my friends in Boston have made these past six
years much more fun. My family, by blood and by law, has always encouraged me without
hesitation and has relished and suffered with each of my triumphs and tribulations.
Finally, I would like to thank the two men in my life, Juan and Nicolas, for being my
ultimate source of strength. Their laughter and companionship have carried me through all
difficult times and have brighten up my accomplishments. This work, as well as my life, would
not be the same without them and it is dedicated to them.
4
TABLE OF CONTENTS
Abstract
3
Acknowledgements -----------------------------------------------------
4
Chapter One Introduction
------------------------------------------------Part I: The E2F family of transcription factors
-----------------------------1. Discovery and characterization of the E2F activity
-----------------------------------------------2. The role of E2F in cell cycle control
----------------------------------3. Members of the E2F family
a. DPI and DP2
b. E2F1 ....c. E2F2 and E2F3
d. E2F4 and E2F5
---------------------------------e. E2F6, E2F7 and E2F8
----------------------------------Part 1I: The Polycomb Group complex
1. The homeotic genes and their role in embryo segmentation ---------------------------------2. Identification of the Polycomb Group complex
----------------------------3. The composition of PcG complexes
-----------------------------genes
target
Group
4. The Polycomb
--------------------5. The role of PcG complexes in cellular memory
--------------------6. The role of PcG complexes in cell cycle control
References
Chapter Two E2f6 deletion leads to posterior transformations of the axial skeleton
7
8
8
11
14
18
18
22
25
27
32
36
38
40
41
42
48
-------- 74
Abstract
75
Introduction
Results
Generation of E2f6 mutant mice
E2F6 is dispensable for mouse viability and survival
76
81
81
83
E2f6 null mice display posterior transformations -------------E2F6 is not required for cell proliferation and senescence -
86
88
---------
Discussion
------------------------------Viability and survival of E2f6' mice
Proliferation and senescence of E2f6- MEFs ---------------------------Posterior transformations in E2f6' mice
-------------------------------------------Experimental procedures
92
92
94
95
96
Acknowledgements -----------------------------
98
References
98
Chapter Three E2F6 and Bmi 1 synergize in axial patterning in a gene dosage-dependent
manner
Abstract
-.--
Introduction
Results
-
--
-.-
......-........-
-
--.-
-
103
104
105
110
5
-----------------------Viability and survival of compound mutants
Neurological abnormalities of compound mutants
--------------------transformations
of
E2f6-'-;Bmil'
mice
Axial skeletal
Cell cycle properties of double mutant MEFs
-----------------------Discussion
--------------------Viability and lifespan of compound mutant mice
Neurological abnormalities of E2f6'-;Bmi'1 mice
--------------------Posterior transformations of the E2f6;Bmil double mutant mice
----------Proliferation and senescence of E2f6--;BmiL-' MEFs
------------------Experimental procedures
------------------------------------------Acknowledgements
----------------------------------------------References
Chapter 4 Conclusions
--------------------------------------------------------------------------------------------E2F6 in axial patterning
in
viability
and
survival
E2F6
--------------------------------E2F6 in the control of cell proliferation
Mode of action of E2F6
----..-...-.-..References ---
110
112
115
117
123
124
125
125
126
128
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137
140
143
147
150
6
ChapterOne
Introduction
Part I: The E2F family of transcription factors
The E2F family of transcription factors has long been known to control the
expression of genes that are critical for cell cycle entry and progression such as Cyclin A,
Cyclin E, cdc2, Cdc25A, E2fl, Rb, DHFR, thymidylate synthase, thymidine kinase,
PCNA, ORC1, MCMs and cdc6 (DeGregori, 2002; Dyson, 1998; Helin, 1998; Nevins,
1998; Trimarchi and Lees, 2002). In recent years, however, microarray analysis in
combination with chromatin immunoprecipitation revealed additional E2F-responsive
genes that have roles beyond the GI/S transition such as DNA repair and recombination,
apoptosis, mitosis, differentiation and development (Cam and Dynlacht, 2003;
DeGregori, 2002; Muller et al., 2001; Stevaux and Dyson, 2002).
The E2F transcription factors can activate or repress their target genes in different
contexts (Ferreira et al., 2001; Harbour and Dean, 2000b; Helin, 1998; Muller and Helin,
2000; Zhang and Dean, 2001). The activation of E2F target genes is believed to occur
via chromatin modifications and the recruitment of the transcriptional machinery to
promoters. Two models have been proposed for the E2F-mediated repression of
transcription. In the first model, called "inhibition of activation", pocket protein binding
to the transactivation domain of E2F blocks its ability to stimulate the expression of
target genes. In the second model, called "active repression", E2Fs actively repress the
expression of target genes through recruitment of chromatin-modifying complexes.
1. Discovery and characterization of E2F activity
Studies on the transcriptional activation of the adenovirus early promoters by
Nevins and colleagues led to the discovery of the E2F transcription factors. The
8
adenovirus ElA (early region lA) transforming protein activates the transcription of
several viral and cellular promoters. Initially, Nevins and co-workers detected a cellular
factor that was bound to the early E2 promoter in infected HeLa cells (Kovesdi et al.,
1986a). This factor, called the E2 promoter binding factor (E2F), was later purified from
extracts of not only infected cells but also of uninfected cells albeit at much lower
concentrations (Kovesdi et al., 1986b). Consistent with this finding, EtA induces the E2
promoter binding activity after infection even in the presence of inhibitors of protein
synthesis, demonstrating that the induction of E2F activity is post-translational (Reichel
et al., 1988). In fact, it was demonstrated that E2F is found in complex with other
cellular factors in uninfected cells and, upon infection, ElA dissociates these complexes,
releasing the free E2F activity which can induce expression from the E2 promoter
(Bagchi et al., 1990).
The transcriptional activation of viral and cellular promoters by ElA was
dependent on binding of E2F to specific DNA sequences in these promoters (5'TTTCCCGC-3') (Trimarchi and Lees, 2002). DNase footprinting was performed to
identify the precise binding site of E2F, revealing two distinct regions of binding at
positions -33 to -49 and -53 to -71 of the E2 promoter (Kovesdi et al., 1987; Yee et al.,
1987). Yee et al (1987) demonstrated that a purified E2F factor could induce
transcription from a promoter containing two E2F-binding sites.
Simultaneously, La Thange and Rigby (1987) characterized a sequence-specific
transcription factor, DRTF1 (differentiation regulated transcription factor 1), the activity
of which is down-regulated during F9 embryonal carcinoma stem cells differentiation (La
Thangue and Rigby, 1987). Soon it was clear that E2F and DRTF1 are the same factor.
9
E2F/DRTF1 was found in several protein complexes, one of which also contained
the Retinoblastoma protein (pRB), a known EtA binding protein (Bandara and La
Thangue, 1991; Chellappan et al., 1991; Kaelin et al., 1991; Partridge and La Thangue,
1991; Stevaux and Dyson, 2002; Whyte et al., 1988; Whyte et al., 1989). pRB had been
previously identified as the tumor suppressor that when mutated leads to the development
of several human tumors such as retinoblastoma, osteosarcoma, small cell lung
carcinoma, breast and prostate cancer (Weinberg, 1992). In fact, pRB mutation or loss is
detected in at least one third of human tumors. The finding that EtA causes the
dissociation of pRB from E2F (Bagchi et al., 1990; Bagchi et al., 1991; Bandara and La
Thangue, 1991; Chellappan et al., 1991), suggested that EtA's transforming capacity was
a result of the release of E2F and that the pRB tumor suppressor functions in opposition
by sequestering E2F. Interestingly, the pRB region that is required for ElA and E2F
binding is a common site for naturally occurring inactivating mutations of the Rb gene,
indicating that the tumors that arise from Rb mutation may be a consequence of E2F
deregulation (Hu et al., 1990).
Several studies demonstrated that E2F regulates the expression of many cellular
proteins that are necessary for cell cycle progression and S phase entry. A few examples
are MYC (Hiebert et al., 1989; Thalmeier et al., 1989), DHFR (Blake and Azizkhan,
1989), thymidine kinase (Dou et al., 1992) and cdc2 (Dalton, 1992). Instead, pRB
specifically inhibits the transcription of these genes and this repression is overridden by
EtA (Zamanian and La Thangue, 1992). Taken together, these data supported a model in
which free E2F promotes cell cycle progression and pRB negatively controls cell
proliferation by sequestering E2F. However, it was later shown that the pRB-E2F
10
complex could also actively inhibit the transcription of target genes when bound to their
promoter (Hamel et al., 1992; Weintraub et al., 1992).
2. The pRB/E2F pathway and cell cycle control
In order for a cell to divide and produce two identical daughter cells, it must first
faithfully replicate its DNA and then segregate the replicated chromosomes into two
separate cells. In addition, most cells double their mass and duplicate their cytoplasmic
organelles in each cell cycle. To ensure that these events have been successfully carried
out before dividing into two daughter cells, the cell is required to pass through a number
of checkpoints that block cell cycle progression when the requirements for the next
downstream process are not met. The major checkpoints known to date are a - the GI/S
checkpoint (also called restriction point) which prevents entry into S phase in the
absence of growth signals or when the cell has not reached a critical mass, b - the G2
checkpoint which ensures that the DNA is fully replicated, the environment is favorable
and the cell is big enough before allowing the cell to enter mitosis, and c - the Metaphase
checkpoint which blocks cell division when the replicated chromosomes are not properly
aligned on the spindles.
The restriction point separates the G 1 phase into two functionally different distinct
parts. Progression through GI before reaching the restriction point requires continuous
stimulation by mitogenic signals and a high rate of protein synthesis. Once the cell
passes through the restriction point, it becomes independent of mitogenic and inhibitory
signals and is committed to S phase entry (Sherr, 1996; Zetterberg et al., 1995).
The role of E2F in the control of GI/S transition by stimulating the expression of
11
important cell cycle regulators, as well as components of the nucleotide biosynthesis and
DNA replication machineries, has been well established (Dyson, 1998; Nevins, 1998;
Trimarchi and Lees, 2002). The indication that E2F is a major downstream target of pRB
(Bagchi et al., 1991; Bandara and La Thangue, 1991; Chellappan et al., 1991; Chittenden
et al., 1991) has led to intense studies that identified a pathway in which E2F is regulated
through the cell cycle by its interactions with pRB and other pocket proteins, p107 and
p130 (Stiegler et al., 1998). Positive regulators, such as cyclin-dependent kinases (cdks),
and negative regulators, such as the cdk inhibitors (CKIs), converge on this pathway and,
in turn, regulate the activity of pRB and related proteins.
It has been clearly demonstrated that the pRB/E2F pathway mainly governs
progression through the GI restriction point. During quiescence (GO) and early G1,
hypophosphorylated pRB binds to and negatively regulates E2F complexes, thereby
blocking entry into S phase (Kato et al, 1993). Upon mitogenic stimulation, D-type
cyclins (DI, D2 and D3) are induced and subsequently assembled into complexes with
their catalytic partners, CDK4 and CDK6 (Kato et al., 1994). The resulting cyclin Ddependent kinases phosphorylate pRB late in G1, causing the release of free E2Fs, which
in turn activates the transcription of cell cycle regulators and ensures S phase entry
(DeGregori et al., 1995). Specific inhibitors of these kinases (the INK4 proteins,
p21WAF1/CIP1, p27KIP1 and p57KIP2) can block cyclin D-dependent kinase activity
and cause G1 arrest in response to several signals (Peter, 1997).
These events initiate a positive feedback loop that leads to progressive rounds of
pRB phosphorylation and cell cycle progression. Upon E2F release, it transactivates the
cyclin E gene (DeGregori et al., 1995; Ohtani et al., 1995) causing a rise in cyclin
12
E/CDK2 activity, which in turn hyperphosphorylates pRB. In addition, E2F1 is also an
E2F target gene (Hsiao et al., 1994; Johnson et al., 1994b). Subsequently, the
inactivation of pRB becomes mitogen-independent and can no longer be blocked by the
cdk inhibitors. Once the cell enters S phase, cyclin E/CDK2 is inactivated by CDK2mediated phosphorylation of cyclin E and it is subsequently degraded by ubiquitindependent proteolysis (Clurman et al., 1996; Won and Reed, 1996).
Cyclin A- and cyclin B-dependent kinases are responsible for maintaining pRB
hyperphosphorylation until mitosis. Cyclin A transcription is also regulated by E2F
(DeGregori et al., 1995) and the cyclin A/CDK2 complex will begin to accumulate
during S phase. This ensures that the E2F-mediated transcription is terminated at the end
of S phase since cyclin A-CDK2 binds to the E2F complex and phosphorylates it thereby
blocking DNA binding (Xu et al., 1994).
The importance of the pRB/E2F pathway in the restriction checkpoint is
underscored by the findings that virtually all human tumors contain mutations that disrupt
this pathway (Bartek et al., 1996; Sherr, 1996). The most common mutation found in
sporadic human cancers is loss of function mutations of the Rb gene. However,
inactivation of the p16INK4 A cyclin kinase inhibitor and amplification or translocation of
the cyclin D gene are also naturally occurring mutations in several sporadic human
tumors. These mutations are generally mutually exclusive since inactivation of a single
step in this pathway is sufficient to achieve deregulation of E2F activity.
13
3. Members of the mammalian E2F family
To date, there are ten genes that encode components of the mammalian E2F
transcriptional activity (Figure 1). These genes can be classified into two subfamilies, the
E2F subfamily (E2f] through E2J8) and the DP subfamily (Dpi and Dp2). The
endogenous E2F activity is achieved by heterodimerization of one E2F subunit and one
DP subunit (Bandara et al., 1993; Huber et al., 1993), generating a basic helix-loop-helix
transcription factor that recognizes the consensus DNA sequence 5'-TTTCGCGC-3'.
The DP proteins can associate with E2F1 through E2F6 interchangeably and this
association is necessary for DNA binding. The functional specificity, however, is
conferred by the E2F subunit.
The E2F subfamily can be subdivided into three groups based on their structure,
transcriptional ability, expression patterns, pocket protein binding and functional
properties. The first group, comprised of E2F1, E2F2 and E2F3a, are strong
transcriptional activators, are expressed in a cell cycle-dependent manner and interact
exclusively with pRB (Helin et al., 1992; Lees et al., 1993). Overexpression of these
proteins is sufficient not only to induce quiescent cells to enter the cell cycle (Johnson et
al., 1993; Lukas et al., 1996; Qin et al., 1994), but also to override cell cycle arrest in
response to TGF- , p16INK4A, p21CIP1 and p27KIP1 (DeGregori et al., 1995; Mann
and Jones, 1996; Schwarz et al., 1995). Furthermore, in primary cells, overexpression of
E2F1, E2F2 or E2F3a leads to transformation (Johnson et al., 1994a; Shan and Lee, 1994;
Singh et al., 1994; Xu et al., 1995) whereas microinjection of antibodies against E2F3a
14
TAD
CA
DBD
DIM MB
PP
E2F1
E2F2
-9erna uom
E2F3a
*
E2F3b
*111w]
E2F4
E2F5
E
L
919u9"1
E2F6
E2F7
_______________________alternative
E2F8
_______________________splicing
DPI
DP2
U
alternative
"I'll'
""liz
splicing
Figure 1. The mammalian E2F family of transcription factors.
The conserved domains of the E2F and DP proteins are indicated in the diagram. CA,
Cyclin A binding domain. DBD, DNA binding domain. DIM, dimerization domain.
MB, Marked box. TAD, transactivation domain. PP, Pocket protein binding domain.
causes cell cycle arrest. Finally, E2f3-deficient mouse embryonic fibroblasts (MEFs)
display a delay in cell cycle re-entry after serum arrest (Humbert et al., 2000b) and MEFs
lacking E2F1, E2F2 and E2F3 are unable to proliferate (Wu et al., 2001). These findings
led to the belief that E2F1, E2F2 and E2F3a, called the activating E2Fs, are involved in
the induction of cellular proliferation.
The second group, composed of E2F4 and E2F5, are weak transcriptional
activators (Muller et al., 1997; Verona et al., 1997), are expressed constitutively
throughout the cell cycle (Ikeda et al., 1996; Moberg et al., 1996) and interact with all
members of the pocket proteins (Beijersbergen et al., 1994; Dyson et al., 1993; Hijmans
et al., 1995; Vairo et al., 1995). Another difference with the activator E2Fs is that they
are also regulated by their different subcellular localization during the cell cycle (Gaubatz
et al., 2001; Magae et al., 1996; Muller et al., 1997; Verona et al., 1997). These E2Fs
seem to function as repressors by recruiting pocket proteins and chromatin modifying
enzymes to E2F target genes (Iavarone and Massague, 1999). Although E2F3b has not
been characterized extensively yet, initial studies have led to its classification with the
repressive E2Fs. First, the patterns of expression of E2F3b are reminiscent of E2F4 and
E2F5 (He et al., 2000; Leone et al., 2000). Second, E2F3b has been recently described to
act as a transcriptional repressor of the p19ARF gene (Aslanian et al., 2004). Based on
these observations, this group of proteins has been called the repressive E2Fs and are
believed to play a role in exit from the cell cycle and terminal differentiation.
The third group of E2Fs, comprised by E2F6, E2F7 and E2F8, are pocket proteinindependent repressors of transcription whose biological functions remain to be
elucidated (Cartwright et al., 1998; de Bruin et al., 2003; Gaubatz et al., 1998; Logan et
16
al., 2004; Maiti et al., 2005; Morkel et al., 1997; Trimarchi et al., 1998). However, this is
a more heterogeneous group. Although all three proteins appear to be expressed in a cell
cycle-dependent manner, there are differences in the timing of peak expression. E2F6
reaches its highest level during mid-Gi whereas E2F7 and E2F8 peak during S phase
(Dahme et al., 2002; de Bruin et al., 2003; Maiti et al., 2005). Furthermore, the structural
analysis of these proteins also indicates that E2F7 and E2F8 are more closely related
(Figure 1). E2F6 possesses conserved DNA binding, heterodimerization and marked box
domains and lacks the pocket protein binding and transactivation domains (Cartwright et
al., 1998; Gaubatz et al., 1998; Morkel et al., 1997; Trimarchi et al., 1998). In contrast,
E2F7 and E2F8 possess two DNA binding domains and lack the rest of the E2F
conserved domains (de Bruin et al., 2003; Logan et al., 2004; Maiti et al., 2005).
Despite the mounting evidence that the activating and repressive E2Fs have
opposing functions in the regulation of gene expression, it is unclear if each E2Fresponsive gene is regulated by all the different E2F complexes. Recently, it has been
suggested that E2F target specificity may not be due to differential DNA binding but
rather to the differential ability of E2F to interact with additional transcription factors
(Gaubatz et al., 1998; Giangrande et al., 2003; Giangrande et al., 2004).
Both E2F and pRB are evolutionarily conserved with orthologs found in
Drosophilamelanogaster(Du et al., 1996; Dynlacht et al., 1994a; Ohtani and Nevins,
1994; Sawado et al., 1998; Stevaux et al., 2002), Caenorhabditiselegans (Ceol and
Horvitz, 2001; Page et al., 2001), Xenopus laevis (Stevaux and Dyson, 2002) and several
plant species (Albani et al., 2000; Magyar et al., 2000; Sekine et al., 1999; Suzuki and
Hemmati-Brivanlou, 2000). Drosophilahas two E2Fs (dE2F1 and dE2F2), one DP
17
(dDP) and two pRBs (RBF1 and RBF2). Xenopus laevis possesses two E2Fs (EFLl and
EFL2), one DP (DPL1) and one RB (LIN35).
a. DPI and DP2
DPI (dimerization partner 1 or DRTF1-binding protein) was isolated based on its
ability to bind DRTF1/E2F (Girling et al., 1993). Subsequent cloning revealed a DNA
binding domain that resembles that of E2F1 and recognizes the same E2F consensus
DNA binding site (Girling et al., 1993; Helin et al., 1993). In fact, DPI and E2F1
heterodimerize. This association results in stronger DNA binding and cooperative
activation of an E2F-responsive promoter and is required for stable interaction with pRB
(Helin et al., 1993; Krek et al., 1993). Several groups used different methods to clone the
DPI homolog, DP2 (Ormondroyd et al., 1995; Rogers et al., 1996; Wu et al., 1995;
Zhang and Chellappan, 1995). Both DPI and DP2 can heterodimerize with most E2F
family members in vivo.
Mice deficient for Dpi die by embryonic day 12.5 due to failure of
extraembryonic lineages to proliferate (Kohn et al., 2004). To study the role of DPI in
the embryo proper, chimeras were generated and the resulting embryos survive until midto late gestation suggesting that DPI is dispensable for embryonic development (Kohn et
al., 2004).
b. E2F1
The E2f] gene was simultaneously cloned by three different groups through
cDNA expression library screening for pRB binding proteins (Helin et al., 1992; Kaelin
18
et al., 1992; Shan et al., 1992). The E2F1 protein was found to interact exclusively with
the pocket domain of pRB in vitro and in vivo and this interaction was competed by viral
proteins known to disrupt the pRB-E2F association such as ElA. This protein copurified with the E2F activity and could bind to E2F recognition sites causing a 10-fold
activation of the adenovirus E2 promoter. Moreover, the E2F1 protein was found to be
expressed in a cell cycle-dependent manner with highest levels during GI/S.
Sequence analysis of the E2f] gene revealed regions that allowed DNA binding in
a sequence-specific manner, DP dimerization, transactivation and pocket protein binding.
The DP dimerization domain is composed of a hydrophobic heptad repeat (a putative
leucine zipper) and a marked box motif, which has also been implicated in DNA bending
(Cress and Nevins, 1996). Furthermore, E2FL contains a cyclin A binding domain in the
amino terminal region (Devoto et al., 1992; Krek et al., 1994; Mudryj et al., 1991;
Pagano et al., 1992a; Xu et al., 1994). This domain allows specific binding to cyclin
A/CDK2 and cyclin A/CDC2 complexes resulting in phosphorylation of the DP subunit
and loss of E2F DNA binding and transactivation activities during S phase (Dynlacht et
al., 1994b; Krek et al., 1995; Xu et al., 1994). There is also evidence that E2F1 is
phosphorylated by cyclin A/CDK complexes resulting in its targeting for degradation
(Adams and Kaelin, 1996; Peeper et al., 1995). Lastly, E2F1 possesses a nuclear
localization signal and is, therefore, found exclusively in the nucleus (Muller et al., 1997;
Verona et al., 1997).
Overexpression studies have shown that E2F1 can override the cell cycle arrest
induced by serum deprivation or by growth arrest signals such as TGF-3 or cdk inhibitors
(DeGregori et al., 1995; Johnson et al., 1993; Kowalik et al., 1995; Mann and Jones,
19
1996; Schwarz et al., 1995; Shan and Lee, 1994) as well as transform primary fibroblasts
(Johnson et al., 1994a; Shan and Lee, 1994; Singh et al., 1994; Xu et al., 1995). Finally,
inhibition of E2F1 activity is necessary for differentiation of several lineages as ectopic
expression of E2F1 can inhibit the differentiation of C2C12 myocytes (Wang et al.,
1995), epidermal keratinocytes (Paramio et al., 2000), squamous keratinocytes (Wong et
al., 2003) and chondrocytes (Wong et al., 2003). In agreement with this, E2F1
overexpression induces DNA synthesis in post-mitotic cerebellar neurons (Suda et al.,
1994). However, E2F1 seems to promote differentiation of adipocytes through induction
of PPAR-y (Fajas et al., 2002), suggesting that the role of E2F1 in terminal differentiation
might be tissue-specific.
E2F1 has also been implicated in the induction of apoptosis through p53dependent and independent mechanisms as a result of overexpression or in response to
DNA damage (DeGregori et al., 1997; Hiebert et al., 1995; Hsieh et al., 1997; Kowalik et
al., 1995; Lissy et al., 2000; Phillips et al., 1999; Qin et al., 1994; Shan and Lee, 1994).
Similarly, in flies, ectopic expression of dE2F1 in the imaginal discs also leads to
apoptosis (Asano et al., 1996; Du et al., 1996). It is thought that the E2F induced
apoptosis is blocked during normal cell cycle progression by pRB and the Ras-P13 kinase
signaling pathway (Hallstrom and Nevins, 2003; Harbour and Dean, 2000a; Lipinski and
Jacks, 1999).
E2F1 is believed to trigger p53-mediated apoptosis partly through direct induction
of
p
1 9
ARF,
a known E2F target gene, which in turn inhibits the MDM2-mediated
degradation of p53 (Bates et al., 1998). However, both through in vivo and in vitro
experiments, p1 9 ARF was shown to be dispensable for the E2F1-mediated p53-dependent
20
apoptosis (Lindstrom and Wiman, 2003; Tolbert et al., 2002; Tsai et al., 2002),
suggesting that there must be alternative mechanisms. One possibility is that E2F1
regulates p53 in a transcriptional independent manner (Hsieh et al., 2002; Nip et al.,
2001; Phillips et al., 1999; Stanelle et al., 2003). For example, E2F1 could induce p53
stabilization by directly binding to it (Hsieh et al., 2002) or by inducing its
phosphorylation (Rogoff et al., 2002).
Several mechanisms have been proposed for the p53-independent apoptosis
triggered by E2F1. First, p73 has been shown to be transactivated by E2F1 and required
for the E2F1-mediated apoptosis in cells lacking p53 (Irwin et al., 2000; Lissy et al.,
2000; Stiewe and Putzer, 2000). Second, E2F1 can inhibit anti-apoptotic signals such as
NF-kappa B (Phillips et al., 1999) and Mcl-i (Croxton et al., 2002; Elliott et al., 2001).
Finally, recent studies have shown that E2FL can also induce propaptotic genes such as
Apaf-1 (Moroni et al., 2001), caspases (Nahle et al., 2002), BH3-only proteins (Nahle et
al., 2002) and SIVA (Nahle et al., 2002).
Lastly, a clear link between E2F1 and DNA damage control has also been
established. Upon DNA damage, E2F1 protein levels rise, probably due to
phosphorylation and stabilization of the protein (Lin et al., 2001; Stevens et al., 2003),
leading to either apoptosis (Huang et al., 1997; Lin et al., 2001; Stevens and La Thangue,
2003) or cell cycle arrest through inhibition of E2F1 functions by TopBP1 (Liu et al.,
2003). Furthermore, E2F1 associates with the Mre 1 recombination and repair complex
(Maser et al., 2001).
Mutant mice for E2fl have been generated and have supported the in vitro
findings demonstrating a role for E2F1 in cell proliferation and apoptosis (Yamasaki et
21
al., 1996). These mice are viable and fertile but exhibit testicular atrophy, exocrine
dysplasia, impaired pancreatic growth and increased tumorigenesis as well as increased T
cell numbers and splenomegaly (Fajas et al., 2004; Lissy et al., 2000; Yamasaki et al.,
1996). This suggests that E2F1 functions both as a tumor suppressor, probably through
its capacity to induce apoptosis, and as an oncogene, through its ability to stimulate cell
proliferation. Consistent with this notion, Pierce et al (Pierce et al., 1998a; Pierce et al.,
1998b; Pierce et al., 1999; Pierce et al., 1998c) have shown that the overexpression of
E2F1 in tissue-specific transgenic mice both predisposes the mice to a variety of
spontaneous tumors and prevents the formation of skin tumors following a carcinogenesis
protocol. Loss of E2f] in the Rb heterozygous background reduces the frequency of
pituitary and thyroid tumors demonstrating that E2F1 deregulation is an important factor
in the pRB-related tumorigenesis (Yamasaki et al., 1998). However, E2fT' MEFs
display normal proliferative properties suggesting that the loss of E2F1 is compensated
by the activities of the other activating E2Fs (Humbert et al., 2000b).
c. E2F2 and E2F3
Electrophoretic mobility shift assays suggested that E2F1 could not account for
all the E2F DNA binding activity. Therefore, a cDNA library was screened at lowstringency hybridization with an E2F1 probe and two clones that encoded proteins with
high degree of sequence conservation to E2F1 were isolated (Ivey-Hoyle et al., 1993;
Lees et al., 1993). The conservation was observed throughout the protein including the
cyclin A binding, nuclear localization signal, DNA binding, dimerization, transactivation
and pRB binding domains. These E2F-like proteins, called E2F2 and E2F3, are very
22
similar to E2F1 not only in structure but also in function and regulation. They are
expressed in a cell cycle-dependent manner, are found exclusively in the nucleus and
bind cyclin A/CDK complexes (Devoto et al., 1992; Leone et al., 2000; Mudryj et al.,
1991; Muller et al., 1997; Pagano et al., 1992b; Verona et al., 1997). E2F2 and E2F3 can
bind wild-type but not mutant E2F recognition sites and activate the transcription of E2Fresponsive genes. They also bind exclusively to pRB and are strong transcriptional
activators. In fact, they behave in a similar manner to E2F1 in overexpression assays by
inducing cell cycle progression and transformation of primary fibroblasts (DeGregori et
al., 1997; Lukas et al., 1996; Xu et al., 1995).
In contradiction to initial studies (DeGregori et al., 1997; Kowalik et al., 1998;
Leone et al., 2001; Lissy et al., 2000), recent findings show that, like E2F1, E2F2 and
E2F3 are also capable of inducing apoptosis although this is still in debate in the field
(Vigo et al., 1999; Ziebold et al., 2001).
Recently, it was shown that the E2f3 locus encodes for two different proteins: the
original E2F3, now called E2F3a, and the novel E2F3b (He et al., 2000; Leone et al.,
2000). An intronic promoter within the first intron of the E2f3 locus allows expression of
E2F3b from an alternative exon 1. The result is a protein that differs from E2F3a in that
the 122 residues of the N-terminus are replaced by 6 distinct amino acids while the rest of
the protein remains identical. Thus, E2F3b contains all the conserved domains described
for E2F3. Two pieces of data suggest that E2F3b possesses distinct functional
characteristics from E2F2 and E2F3a and is therefore classified with the repressive E2Fs.
First, E2F3b is expressed throughout the cell cycle with peak levels in GO when it is
associated with pRB. Second, E2F3b has recently been shown to participate in the
23
transcriptional repression of the p19ARF gene (Aslanian et al., 2004). However, unlike
E2F4 and E2F5, E2F3b seems to associate primarily with pRB during quiescence (Leone
et al., 2000).
Despite the characterization of E2F2 as an activator E2F, homozygous loss of the
E2J2 gene in mice provides evidence for its role in transcriptional repression of genes
necessary for cell proliferation in T lymphocytes. This leads to an accumulation of
autoreactive effector/memory T lymphocytes that are responsible for the late-onset
autoimmune disease in E2J2 mutant mice (Murga et al., 2001). Furthermore, compound
mutants for E2f] and E2J2 are highly predisposed to tumor formation and exhibit some
signs of autoimmunity (Zhu et al., 2001). These results suggest that E2F2 may also
function to negatively regulate cellular proliferation at least in certain tissues.
Mice deficient for E2F3 were generated and they have demonstrated a role for
E2F3 in normal embryonic development as well as in normal cell proliferation in
response to mitogenic signals (Humbert et al., 2000b). It is important to note that these
mice lack both E2F3a and E2F3b making it difficult to assess the contribution of each of
these proteins. Only 25% of expected numbers of E2f3 mutant mice are born and MEFs
lacking E2F3 are not able to induce the expression of several E2F-responsive genes in
response to mitogens. The E2f3-deficient adult mice develop congestive heart failure but
in contrast to E2F1, E2f3-deficiency does not lead to increased tumor formation.
Interestingly, loss of E2F3 in the Rb heterozygous background suppresses the pituitary
tumors like E2F1 but enhances the thyroid carcinomas metastasis (Ziebold et al., 2003).
Almost all the defects observed in mice lacking either E2F1 or E2F3 were exacerbated in
the compound mutant (Cloud et al., 2002). Thus, it appears that E2F1 and E2F3 have
24
distinct roles as well as overlapping functions. The overlapping role of all the activating
E2Fs in cell cycle entry is underscored by the cell proliferation block observed in MEFs
deficient for all three activating E2Fs (Wu et al., 2001).
d. E2F4 and E2F5
The discovery of p107 and p130 suggested that E2F1, E2F2 and E2F3, which only
bind pRB, could not account for all of the cellular E2F activity (Cobrinik et al., 1993;
Shirodkar et al., 1992). This led several laboratories to embark on a search for the
missing E2F(s) and, as a result, E2F4 and E2F5 were cloned (Beijersbergen et al., 1994;
Ginsberg et al., 1994; Hijmans et al., 1995; Sardet et al., 1995). These proteins have
significant homology to E2F1 (approximately 50%) but much higher homology to each
other (78% similarity). They are truncated at the N-terminus and therefore lack the
cyclin A binding domain as well as the nuclear localization signal (Beijersbergen et al.,
1994; Ginsberg et al., 1994; Hijmans et al., 1995; Leone et al., 2000; Sardet et al., 1995).
Instead, E2F4 contains two leucine/isoleucine-rich nuclear export signals, which trigger
the export of E2F4 from the nucleus when it is not bound to pocket proteins (Gaubatz et
al., 2001). Both proteins require DP for efficient DNA binding to promoters containing
E2F sites. E2F4 associates with all members of the pocket proteins whereas E2F5
preferentially associates with p130. Although E2F4 and E2F5 lack the cyclin A binding
domain, they are phosphorylated by cyclin/CDK complexes through p107- and p130mediated interactions (Devoto et al., 1992; Faha et al., 1992; Ginsberg et al., 1994; Lees
et al., 1992; Shirodkar et al., 1992). The expression patterns of E2F4 and E2F5 are very
different than those of the activating E2Fs as they are constitutively expressed throughout
25
the cell cycle with peak levels in mid-G 1 phase before the activating E2Fs are detectable.
E2F4 and E2F5 are much less effective than the activating E2Fs in releasing cells
from quiescence when overexpressed (DeGregori et al., 1997; Lukas et al., 1996).
Consistently, overexpression of the repressive E2Fs cannot overcome a p16INK4A-induced
cell cycle arrest (Mann and Jones, 1996). Furthermore, reporter assays using mutants of
the E2F sites within the promoters of E2F-responsive genes revealed an increase in
reporter activity during GO/G1 suggesting that while E2Fs are responsible for activation
of transcription during GI/S, they also negatively regulate the expression of target genes
during GO/G 1 (Dalton, 1992; Lam and Watson, 1993). In fact, E2F4-p130 complexes
were bound to promoters during GO/G 1 and are thought to be the main complex that
represses the transcription of E2F-responsive genes during quiescence and early G1
(Takahashi et al., 2000). This repression appears to be achieved by the recruitment of
HDAC1 and mSin3B corepressor complexes (Rayman et al., 2002). Taken together,
these data suggest that E2F4 and E2F5 function to repress E2F target genes during GO.
The generation of E2f4 and E2f5 mutant mice uncovered specific functions for
these proteins in development. Mice lacking E2F5 displayed increased numbers of
cerebrospinal fluid-producing epithelial cells in the choroids plexus and as a result
developed hydrocephalia (Lindeman et al., 1998). In contrast, loss of E2F4 lead to
cranoiofacial defects, hematopoietic abnormalities due to defects in maturation and a
reduction in the thickness of the gut epithelium (Humbert et al., 2000a; Rempel et al.,
2000). The analysis of E2f4;E2f5 compound mutant revealed that the repressive E2Fs
also perform overlapping functions in development since loss of both proteins results in
neonatal lethality (Gaubatz et al., 2000). Surprisingly, mutation of the repressive E2f4 in
26
the Rb heterozygous background suppresses the development of pituitary and thyroid
tumors as well as the inappropriate gene expression and proliferation (Lee et al., 2002).
The tumor suppression appears to occur through release of p107 and p130 molecules,
which in the absence of pRb, can bind and inhibit the deregulated activating E2Fs.
While it is clear that the repressive E2Fs play a role in terminal differentiation,
depending on the context the regulation can be positive or negative. For example, loss of
E2F4 leads to spontaneous adipogenesis in a pocket protein-independent manner
(Landsberg et al., 2003) but E2F4 overexpression promotes the differentiation of the P12
neuronal precursors and keratinocytes (Paramio et al., 2000; Persengiev et al., 1999).
In contrast to the obvious role of the repressive E2Fs in development and
differentiation, studies of the mutant MEFs have shown that E2F4 and E2F5 are
dispensable for normal cell cycle progression (Gaubatz et al., 2000; Humbert et al.,
2000a; Lindeman et al., 1998). However, E2f4;E2f5 double mutant MEFs failed to arrest
in GI in response to p 1 6INK 4 A, indicating that they are necessary for pocket proteinmediated arrest (Gaubatz et al, 2000).
e. E2F6, E2F7 and E2F8
E2F6, E2F7 and E2F8, the most recently identified E2Fs, are repressors of
transcription that act independently of pocket proteins. However, this is not a cohesive
group since there are significant differences between these proteins. E2F7 and E2F8,
share unique structural features that distinguish them from all other E2F members (de
Bruin et al., 2003; Logan et al., 2004; Maiti et al., 2005). These proteins possess two
DNA binding domains and lack the heterodimerization, marked box, pocket protein
27
binding and transactivation domains. Modeling studies suggest that, in order to bind
DNA, the two DNA binding domains must interact probably mimicking the E2F/DP
heterodimer structure. This organization resembles the E2F-like proteins from
Arabidopsis thaliana(Vlieghe et al., 2005). Thus, E2F7 and E2F8 associate with E2F
consensus recognition sites in a DP-independent manner.
Both proteins are expressed in a cell cycle dependent manner with peak levels
during S phase, at least in part, through E2F regulation. Furthermore, they display
similar patterns of expression in adult tissue with highest expression in skin and testes.
At least E2F7 has been shown to associate with a subset of E2F-responsive promoters
during S phase and overexpression of E2F7 or E2F8 delays the proliferation of MEFs.
However, their biological functions remain to be determined.
In contrast, E2F6 is much more conserved to the rest of the E2F family. This
protein was identified by several groups based on the sequence conservation of the E2F
DNA binding domain (Cartwright et al., 1998; Gaubatz et al., 1998; Morkel et al., 1997;
Trimarchi et al., 1998). However, once the full-length sequence was obtained, it was
clear that the homology extended also to the dimerization domain, composed of the
leucine zipper and the marked box, as well as to the nuclear localization signal (Figure 1).
Yet, E2F6 is truncated at the C-terminus and therefore lacks the pocket protein binding
and transactivation domains. Consistent with this structure, it has been shown that E2F6
is a nuclear protein that binds DP but not pocket proteins and that the E2F6/DP
heterodimer is able to associate with DNA at E2F binding sites. It appears that the
E2F6/DP complex has a preference for a 5'-TTTCCCGC-3' E2F recognition site instead
of the E2F consensus site from the E2 promoter (5'-TTTCGCGC-3'). In addition, E2F6
28
overexpression fails to induce transcription of a reporter gene. Instead, it seems to
repress E2F-dependent transcription in a dominant negative manner and this repression is
dependent on an intact marked box domain. E2F6 levels are low during GO and start to
rise during the GO/Gi transition with maximum expression in mid-Gi (Dahme et al.,
2002).
Initial studies showed that overexpression of E2F6 delays exit from S phase
resulting in an accumulation of cells in S phase (Cartwright et al., 1998) whereas E2F6
microinjection into serum-arrested cells prevents S phase entry upon serum re-addition
(Gaubatz et al., 1998). In agreement with the latter experiment, E2F6 has been shown to
bind and repress the GI/S-, but not the G2/M-regulated, E2F target genes during S phase
(Giangrande et al., 2004). The implication is that E2F6 would repress the GI/S target
genes at the end of S phase while allowing E2F-mediated transcription of G2/M target
genes. In this model, E2F6 is proposed to stimulate S phase progression. Yet another
contradiction comes from the finding that E2F6 preferentially occupies certain E2F target
promoters in GO cells (Ogawa et al., 2002). This discrepancy could be due to differences
in cell types since Giangrande et al. (2004) used T98G cells whereas Ogawa et al. (2002)
used BJ-1 cells Significantly, Ogawa et al. (2002) did not detect E2F4 and p130 bound
to promoters during GO in opposition to a report that in T98G cells, E2F4 and p130
occupy target promoters in GO and early G 1 phases (Takahashi et al., 2000).
In an attempt to understand the biological significance of E2F6, a yeast twohybrid screen has identified RYBP (Ring 1 and YY 1 binding protein) as an E2F6
interactor (Trimarchi et al., 2001). RYBP is a member of the mammalian Polycomb
Group (PcG) complex (which will be discussed in more detail in the second part of this
29
introduction). In fact, E2F6 associates with a number of the mammalian PcG proteins
including RingIA, MEL-18, Mphl and Bmi-1, as determined by co-immunoprecipitation
assays. At least the interaction with RYBP seems to be through direct contacts with the
marked box domain, which is responsible for the E2F6-mediated repression. More
recently, another E2F6-containing complex has been purified from cultured cells and
mass spectrometry analysis has identified Mga and Max, chromatin modifiers such as
histone methyltransferases, heterochromatin protein 1 gamma (HPly) and several PcG
proteins such as RING1, RING2, MBLR, h-I(3)mbt-like protein and YAF2 (Ogawa et al.,
2002). Finally, another yeast two-hybrid screen identified another PcG protein, EPC1, as
an E2F6 interactor (Attwooll et al., 2005). The complex containing E2F6 and EPC1 also
contains DPI, the PcG protein EZH2 and Sin3B. Interestingly, EZH2 is required for
cellular proliferation, suggesting a role for E2F6 in the regulation of cell cycle
progression. Although there is controversy over the identity of the PcG proteins that
interact with E2F6, it is most likely that the E2F6-mediated repression is achieved by
recruitment of repressive complexes such as the PcG complexes.
A suggested alternative mechanism for the E2F6-mediated repression is
methylation of histone H3 at lysine 9 through the recruitment of a euchromatic-specific
histone methyltransferase to DNA (Ogawa et al., 2002). This methylated lysine 9 is
known to recruit the heterochromatic protein 1 (HP1) resulting in formation of a
repressive heterochromatic structure. In contrast to this model, E2F6-regulated
promoters do not seem to contain histone H3 methylated lysine 9 (Oberley et al., 2003).
Instead, E2F1 is recruited to the target promoters in the absence of E2F6 indicating that
30
E2F6 is able to repress transcription simply by preventing the activating E2Fs from
binding to DNA.
To identify E2F6 target genes, Oberley et al. (2003) have used chromatin
immunoprecipitation followed by CpG island microarrays in cancer cells. The results
from this screen were confirmed as they were up-regulated when E2F6 was knockeddown using RNA interference. Many of these genes encoded proteins that are involved
in tumor suppression and maintenance of chromatin structure such as brcal, ctip, art27,
hplca and rbap48 (Oberley et al., 2003).
Despite in vitro evidence that suggest roles for E2F6 in cell cycle control as well
as in mouse development, the biological significance of E2F6 remains unknown. E2f6
mutant mice may significantly broaden our understanding of E2F6 and its function.
31
Part II: The Polycomb group complex
The Polycomb group (PcG) proteins were first identified in Drosophilaas
repressors of the homeotic (Hox) genes that are responsible for the segmentation
patterning of the embryo. In early fly development, the expression patterns of the Hox
genes are established by the segmentation genes. About four hours into embryogenesis,
the transcriptional state of Hox genes is no longer controlled by these genes but by the
trithorax group (TrxG) and the PcG complexes. These complexes maintain the active or
inactive state of Hox genes transcription. Specifically, the TrxG complex maintains the
active state of those Hox genes that were initially on, whereas the PcG group maintains
the inactive state of those genes that were initially off.
Many mammalian PcG proteins have been identified based on homology to
members of the DrosophilaPcG genes or by yeast two-hybrid screens (See Table 1). In
mammals, there are many more PcG proteins suggesting a higher level of complexity and
redundancy than in the fly. However, both in mammals and flies, there are at least two
distinct polycomb complexes, commonly known as PRC1 and PRC2 (Table 1). The
PRC2 complex functions early in development and initiates polycomb-mediated
repression. The PRC1 complex functions later in development maintains the repression
at later stages of development. However, there is mounting evidence suggesting that
there may be many different "early" and "late" PcG complexes with different protein
compositions.
32
1. The homeotic genes and their role in segmentation
Segmentation was defined by Bateson in 1894 as a repetition of pattern elements
along the major anterior-posterior (A/P) axis of the body. He also described homeosis as
the phenomenon in which one segment is transformed toward the identity of another
(Bateson, 1894). The process of segmentation is best understood in the fruitfly,
Drosophilamelanogaster. The basic segmentation of Drosophila is established through
the subdivision of the embryo body into increasingly specified body regions by the
sequential action of a hierarchy of regulatory genes (reviewed in (Akam, 1987).
Initially, the egg polarity is specified by maternally active coordinate genes; then,
zygotically active segmentation genes determine the patterning of the embryo (NussleinVolhard et al., 1980; Nusslein-Volhard and Wieschaus, 1980). There are three groups of
segmentation genes: the gap, pair-rule and segment polarity genes. Mutations in these
genes result, respectively, in gaps in the array of segments, deletion of alternate segments
and defects in the sequence of pattern elements within segments. Finally, the homeotic
(Hox) genes determine the unique identity of equivalent segmental units (Anderson et al.,
1985; Anderson and Nusslein-Volhard, 1984; Lewis, 1978; Nusslein-Volhard et al.,
1985; Nusslein-Volhard et al., 1980; Nusslein-Volhard and Wieschaus, 1980; Wieschaus
et al., 1984) and their mutation usually leads to homeotic transformations in which one
segment is changed into another. However, it is important to note that the Hox genes have
also been implicated in the processes of morphogenesis and organogenesis (Hombria and
Lovegrove, 2003) as well as in cell cycle control (Del Bene and Wittbrodt, 2005)
During development, Hox genes are expressed in specific zones on the A/P axis
with very sharp anterior boundaries and less well-defined posterior boundaries. This
33
expression pattern is established in the syncytial blastoderm about two hours into
embryogenesis and is maintained throughout the course of development. The gap and
pair-rule proteins are responsible for specifying the initial Hox genes expression
boundaries. Specifically, gap proteins are repressors whereas pair-rule proteins are
activators of transcription and the correct Hox gene expression results from the
coordinated actions of these two classes of transcription factors (Muller and Bienz, 1992;
Qian et al., 1993; Shimell et al., 1994; Zhang and Bienz, 1992).
The first two homeotic mutants isolated were bithorax and antennapedia. The
bithorax mutant has a transformation of the halteres into an extra pair of wings (Lewis,
1978; Lewis, 1998) whereas the antennapediamutant transforms the antenna on the head
of the fly into an extra pair of thoracic legs (Kaufman et al., 1990; Plaza et al., 2001).
Since then, massive genetic screens designed to identify lethal mutations that alter the
pattern of the larval cuticle led to the discovery of many more of these regulatory genes
in Drosophila(Nusslein-Volhard et al., 1985; Nusslein-Volhard and Wieschaus, 1980).
Subsequently, multiple Hox genes were found in all bilateral organisms. Mutational
studies in mice revealed homeotic transformations of the axial skeleton confirming that
vertebrate Hox genes control the anterior-posterior patterning in a similar manner to that
of their invertebrate counterparts. Loss of function mutations of Hox genes in both flies
and mice lead to anterior transformations whereas gain of function mutations result in
posterior ones. This ability of a more posterior Hox gene to impose its function on more
anterior genes is called posterior prevalence.
Each of the Hox gene products contains a 60-amino acid homeodomain (HD) that
is conserved across the species (reviewed by (Scott et al., 1989). The structure of the HD
34
has been determined by nuclear magnetic resonance and crystallographic studies. These
domains are related to the helix-turn-helix motif of prokaryotic DNA-binding proteins.
The Hox proteins are monomeric transcription factors that positively or negatively
regulate the expression of multiple target genes. In vitro experiments have shown that
they recognize the DNA sequences 5'-TAAT-3' (Laughon, 1991). Since this consensus
sequence is very short, modulators and cofactors are thought to assist Hox proteins in
assembling specific activation and repression complexes on the regulatory elements of
Hox target genes (Kornberg, 1993; Mann and Chan, 1996). Although many Hox target
genes have been described, only a few have been shown to be directly regulated by the
Hox genes (Biggin and McGinnis, 1997; Liang and Biggin, 1998). It has been proposed
that the Hox proteins function by controlling the expression of other genes called
"realizators" that are directly involved in controlling cell proliferation, survival, shape
changes and rearrangements (Garcia-Bellido, 1975). We now know that in many cases,
the downstream targets of Hox proteins are actually networks that control the expression
of the realizators.
Interestingly, the Hox genes are arranged in clusters where the position and order
of orthologs are conserved in different species and are collinear with the order of the
anterior boundaries of expression and the timing of activation during development
(Zakany and Duboule, 1999a; Zakany and Duboule, 1999b). While Drosophila contains
only one cluster of Hox genes, mammals have four Hox genes clusters (HOXA, HOXB,
HOXC and HOXD).
35
2. Identification of the Polycomb Group complex
Mutations in the Hox genes lead to transformation of one body segment into
another along the A/P axis. Similar phenotypes were observed in mutants of genes that
lie outside of the Hox gene clusters. Initially the extra sex combs (esc) mutant was
described by Slifer in 1940 and later, Lewis found the polycomb (Pc) mutant in 1947.
Both of these mutations lead to additional sex combs on the second and third pairs of legs
in males, instead of only on the first leg, where they usually belong. To date, 18 such
genes have been identified and the group has been termed the Polycomb Group (PcG)
(Paro, 1990; Ringrose and Paro, 2004). The posterior transformations observed in the
PcG mutants are due to inappropriate derepression of Hox gene expression, equivalent to
a gain-of-function mutation in Hox genes (Kennison, 1995; Kennison, 2004). Years later,
the Trithorax Group (TrxG) was identified as regulators of Hox genes that function in
opposition to the PcG proteins (reviewed by (Ringrose and Paro, 2004). Consistently,
mutations in the 17 genes identified so far suppress the extra sex comb phenotype.
For most of the DrosophilaPcG genes, multiple mammalian counterparts have
been isolated (See Table 1) and they also appear to play an important role in the
maintenance of Hox gene repression (Satijn and Otte, 1999; van Lohuizen, 1999). While
there seems to be a higher degree of complexity in mammals, the proteins are fairly well
conserved. In fact, many mammalian proteins are able to partially rescue the phenotypes
caused by mutations on their Drosophilaorthologs. These include the mouse Pc
ortholog, M33 (Muller, 1995), the mouse polyhomeotic ortholog and the human
pleiohomeotic ortholog, YY 1 (Jooss et al., 1995).
36
Mutant mice for many PcG proteins display axial skeleton transformations and
deregulation of Hox gene expression like the Drosophilamutants. Unlike the fly genes,
the mammalian PcG genes are expressed differentially in different tissues and cell types
(Alkema et al., 1997b; Lessard et al., 1998; Pearce et al., 1992; Raaphorst et al., 2001;
Raaphorst et al., 2000; Sewalt et al., 1998; van Lohuizen et al., 1998). This probably
explains the mutant-specific phenotypes, which include cerebellar defects in the Bmildeficient mice, male-to-female sex reversal in M33 mutant mice, smooth muscle atrophy
in Mel18 mutant mice and neural crest defects in Mph-deficient mice (Akasaka et al.,
1996; Akasaka et al., 1997; Core et al., 1997; Katoh-Fukui et al., 1998; Raaphorst et al.,
2001; Takihara et al., 1997; Tokimasa et al., 2001).
However, although the transformations in mutant mice are highly reminiscent of
those found in flies, they are relatively mild compared to the drastic malformations
observed in the Drosophilamutants. This may reflect a higher level of functional
redundancy between the mammalian PcG proteins. The idea of redundancy in Hox gene
repression was first suggested by Jirgens (1985) based on the phenotypes of double and
triple mutants flies for the Polycomb Group genes. If the PcG proteins formed one
functional complex, it would be expected that loss of one protein in the complex should
completely abolish the function of the complex. Therefore the loss of additional proteins
should not make a difference (Peterson and Herskowitz, 1992). However, in all cases
examined, double and triple mutants for Polycomb Group genes have more severe
phenotypes than the single mutants (Cheng et al., 1994; Moazed and O'Farrell, 1992;
Peterson and Herskowitz, 1992). Subsequently, the analysis of double mutants in mice
have also revealed a high degree of redundancy (Akasaka et al., 2001; Bel et al., 1998).
37
3. The composition of PcG complexes
The PcG proteins act in large multiprotein complexes that modify chromatin to
maintain the repressed states of their target genes (Alkema et al., 1997a; Franke et al.,
1992; Gunster et al., 1997). At least two distinct multiprotein Polycomb Group
complexes have been purified (Table 1) (Lund and van Lohuizen, 2004). First, the
Polycomb Repressive Complex 2 (PRC2) is a 600 kDa complex that was purified from
Drosophilaembryos and Hela cells and is involved in the initiation of silencing. It
contains ESC, Enhancer of zeste (E(z)), Suppressor of zeste 12 (Su(z)12) and a small
number of additional proteins depending on the purification protocol used (Table 1).
These include histone deacetylases and histone methylases (Cao et al., 2002; Czermin et
al., 2002; Kuzmichev et al., 2004; Muller et al., 2002; Tie et al., 2001; van der Vlag and
Otte, 1999). Consistent with their role in early development, deletion of PRC2 genes in
mice result in early embryonic lethality during gastrulation (O'Carroll et al., 2001;
Schumacher et al., 1998). Second, the Polycomb Repressive Complex 1 (PRC1) is a 1-2
MDa complex that was purified from DrosophilaSchneider cells and human Hela cells
and is implicated in the stable maintenance of gene repression. The core components of
this complex are Polycomb, Polyhomeotic, Posterior Sex Combs and RING (Table 1)
(Francis and Kingston, 2001; Lavigne et al., 2004). Mutant mice for most PRC1 genes
survive until birth due to partial functional redundancy (Akasaka et al., 2001; Core et al.,
1997; Takihara et al., 1997; van der Lugt et al., 1994b). An exception is RnJ2/Ringlbdeficiency, which leads to an early lethal phenotype similar to that of PRC2-deficient
mice (Voncken et al., 2003). Although studies demonstrate that PRC1 functions later in
38
Drosophila
proteins
Human proteins
Mouse proteins
EED
EZH1
Eed
Ezhl/Enx2
EZH2
Ezh2/Enxl
PRC2/Initiation complex
Esc
E(z)
Su(z)12
PRC1/Maintenance complex
Pc
SUZ12
CBX2/HPCl
CBX4/HPC2
Cbx2/M33
Cbx4/Mpc2
CBX8/HPC3
Ph
EDRl/HPHl
EDR2/HPH2
EDR3/HPH3
RINGl/RNF1/RINGlA
RNF2/RINGlB
BMIl
RNFI lO/ZFP144
ZNF134
YYl
Ringi/Ringla
Rnf2/Ring lb
Bmil
Rnf lI0/Zfp144/Mel18
Znf 134/Mblr
Yyl
Scm
SCML1
SCML2
Scmhl
Scmh2
PcI
PHFl
Psc
Pho
Edrl/Mphl/Rae28
Edr2/Mph2
Pho-like
Table 1. Polycomb Group Complexes in flies, humans and mice. Adapted from
(Valk-Lingbeek et al., 2004).
development than PRC2, the mode of action of these complexes is currently under debate
and will be discussed later in this chapter.
Importantly, there is strong evidence that the function and composition of these
core complexes in vivo is modulated in different tissues (Otte and Kwaks, 2003) and at
different target genes (Rastelli et al., 1993; Strutt and Paro, 1997). Furthermore, many
other proteins have been reported to associate with PcG proteins (Mulholland et al., 2003;
Sewalt et al., 2002; Trimarchi et al., 2001; van der Vlag and Otte, 1999) suggesting that
39
an enormous functional diversity can be achieved by multiple combinations of core and
auxiliary factors.
4. The Polycomb Group target genes
In Drosophila,the PcG proteins have been found to bind to more than 100 sites in
polytenic chromosomes from larval salivary gland (Rastelli et al., 1993; Zink and Paro,
1989). The binding specificity has been proposed to be achieved through the cisregulatory PcG response elements (PREs) that enable both the PcG and TrxG proteins to
bind and maintain the status of transcriptional activity over many cell generations. The
PREs are large sequences (100-300 bp) and tend to be very divergent. PREs act in
groups of two or more elements that are modulated by tissue-specific enhancers and
boundary elements (Barges et al., 2000; Mihaly et al., 1997; Pirrotta et al., 1995; Simon
et al., 1993). Interestingly, only five proteins have been shown to have a sequencespecific DNA-binding activity (GAGA, PSQ, Zeste, PHO and PHO-like) (Simon et al.,
1993). In mammals, much less is known about the PcG proteins binding to DNA.
However, although no PREs have been identified, some targets are known (Hanson et al.,
1999). For example, the deregulation of Hoxc4, Hoxc5, Hoxc6, Hoxc8 and Hoxc9 in the
Bmil-'- mice indicate that these are controlled by the Bmil-containing PcG complex (van
der Lugt et al., 1996).
In addition to the Hox genes, it appears that the PcG and TrxG proteins regulate
the expression of a variety of genes such as the segmentation genes (Kassis, 1994;
Maurange and Paro, 2002; McKeon et al., 1994; Pelegri and Lehmann, 1994) and genes
involved in patterning of the imaginal discs which will later give rise to the adult
40
structures (Ringrose et al., 2003). The mammalian PcG proteins have also been shown to
regulate genes implicated in cellular proliferation and tumorigenesis such as p16INK4 A and
p19ARF (Jacobs and van Lohuizen, 2002).
5. The role of PcG complexes in cellular memory
The specialized state of cells is maintained throughout many rounds of cell
division that are necessary for body growth during Drosphila development. In fact,
imaginal discs that are transplanted into a different larvae form the appropriate structures
and appendages for which they were initially specialized. This maintenance of cellular
memory is a dynamic process that is accomplished by the concerted functions of the PcG
and TrxG proteins in the regulation of their many target genes.
First, these proteins must find the target gene while the initial determining
transcription factors (i.e., segmentation genes) are still present. Indeed, PC and TRX
have been found to bind the Bithorax Complex (BX-C) well before the segmentation
genes disappear (Orlando et al., 1998). Furthermore, recent data indicates that the
segmentation gene hunchback recruits the PcG complex to repressed target genes through
the dMi2 deacetylase complex (Kehle et al., 1998; Zhang and Bieker, 1998).
Second, the PcG and TrxG proteins must assess the activity of the promoter.
Transgenic studies suggest that PREs act as silencers by default (Sengupta et al., 2004)
and that the TrxG proteins directly oppose this repression (Klymenko and Muller, 2004).
This suggests that the active state of a promoter may lead to the recruitment of TrxG
proteins through an unknown mechanism.
41
Third, the PcG and TrxG complexes must preserve the repressed or active state of
(Kuzmichev et al., 2002)transcription of their target genes, respectively. It has been
shown that PcG proteins do not exclude transcription factors from silenced promoters as
it was previously thought (Saurin et al., 2001). Several alternative mechanisms of
repression were proposed including the sumoylation and subsequent activation of
transcriptional corepressors (Kagey et al., 2003), modulation of the polymerase activity
(Dellino et al., 2004; Wang et al., 2004), histone methylation (Lachner et al., 2003)) and
deacetylation (Czermin et al., 2002; Huang et al., 2004; Kuzmichev et al., 2002; Poux et
al., 2001; Tie et al., 2001; van der Vlag and Otte, 1999). In contrast, the activation
mechanism seems to involve histone methylation (Beisel et al., 2002; Strutt et al., 1997)
and acetylation (Cavalli and Paro, 1999).
Finally, after each cell division, the same transcriptional state must be maintained.
In both flies and mammals, the PcG proteins dissociate from DNA during mitosis
(Buchenau et al., 1998; Miyagishima et al., 2003; Voncken et al., 1999) whereas it is not
known how the TrxG proteins behave during mitosis yet. Histone methylation is a very
stable modification and therefore could account for the cellular memory after mitosis
(Lachner et al., 2003). However, it has also been suggested that the PREs themselves are
important for remembering the transcriptional state of the genes they regulate (Busturia et
al., 1997; Sengupta et al., 2004).
6. The role of PcG complexes in cell cycle control
Although the PcG complex is well known for its role in segmentation through the
regulation of Hox gene expression, it has become increasingly clear that this group of
42
negative regulators of transcription are also involved in cell cycle control. Most of the
evidence in support of this idea comes from the study of Bmi 1.
Bmil was originally identified in a screen for genes that could cooperate with EliMyc in lymphomagenesis (Haupt et al., 1991; van Lohuizen et al., 1991). Retroviral
insertional mutagenesis was performed with the Moloney murine leukemia virus on E Myc transgenic mice, which were screened for a decrease in the latency period of pre-Bcell lymphoma formation. A frequent site of integration (35-47%) was near the Bmil (Bcell-specific Moloney murine leukemia virus insertion site 1) gene, which resulted in its
overexpression. Bmil is a 324 amino acid protein with high degree of homology to the
DrosophilaPsc and Su(z)2, two members of the PRC1 complex (Brunk et al., 1991; van
Lohuizen et al., 1991). The predicted amino acid sequence reveals several structural
motifs. The N-terminal portion of the protein contains a cysteine-rich zinc finger motif
called the RING finger, which was later found to be required for Bmil's oncogenic
capacity (Cohen et al., 1996). Although it has not been possible to demonstrate that
Bmil binds directly to DNA, it contains a helix-turn-helix-turn-helix-turn (HTHTHT)
motif in the center of the protein, which is a potential DNA-binding domain. Bmi 1 also
has two putative nuclear localization signals, KRRR and KRMK, but only the latter
seems to be required for its nuclear localization (Cohen et al., 1996; Haupt et al., 1991).
Finally, this protein contains a PEST domain, which is rich in proline, glutamic acid,
serine and threonine and is associated with rapid intracellular protein degradation (Rogers
et al., 1986).
Consistent with the retroviral mutagenesis studies, transgenic mice that overexpress
both Bmi 1 and Myc accelerate the onset of both B- and T-cell lymphomas supporting that
43
Bmil is an oncogene (Haupt et al., 1993). Further evidence of Bmil's oncogenic
properties comes from reports that Bmil can transform Ratla cells by itself (Cohen et al.,
1996) and MEFs in cooperation with Ras (Jacobs et al., 1999b). Furthermore, Bmildeficient MEFs have a reduced proliferation rate and senesce prematurely (Jacobs et al.,
1999a). These oncogenic effects of Bmil appear to be mediated by its ability to repress
the tumor suppressors p16INK4 Aand
pI9 ARF,
the products of two alternative reading frames
of the Ink4a/Arf locus. Indeed, Bmil-'- MEFs as well as lymphocytes have increased
expression of both p 1 6 INK 4 Aand
decrease of p 1 6 INKAand
pI 9 ARF
pI 9 ARF.
Conversely, Bmil overexpression results in
protein levels (Itahana et al., 2003; Jacobs et al., 1999a).
Deletion of the Ink4a/Arf locus partially rescues the Bmil mutant phenotypes (Jacobs et
al., 1999a). However, it has not been possible to determine whether the repression of the
Ink4a/Arf locus is direct or not.
In the past couple of years, the role of Bmi 1 in cell cycle control has been
extended to include the regulation of stem cell self-renewal and proliferation of
hematopoietic and neural stem cells (HSCs and NSCs) (Lessard and Sauvageau, 2003;
Leung et al., 2004; Molofsky et al., 2003; Park et al., 2003). The participation of Bmil in
the proliferation of these cells also involves the repression of the Ink4a/Arf locus
suggesting that this is a general function of Bmi 1 in different cell types and tissues.
It has been recently shown that Bmi 1 is required for the self-renewal of peripheral
and central nervous system stem cells (Molofsky et al., 2003). In fact, loss of Bmil leads
to the postnatal depletion of NSCs in the cerebellum. As a result, Bmil mutant mice have
a smaller cerebellum due to severe loss of both the molecular and granular layer neurons
(van der Lugt et al., 1994a). A substantial rescue of the cerebellar size is observed in
44
Bmil;Ink4a/Arf double-deficient mice (Jacobs et al., 1999a). The partial rescue by loss
4
of the Ink4a/Arf locus is in agreement with the upregulation of both p 1 6 INK Aand pI9
ARF
in NSCs lacking Bmil (Molofsky et al., 2003; Park et al., 2003). However, it appears
that the contributions of p16INK4 Aand
pI9 ARF
may be different in different cell types and
tissues (Bruggeman et al., 2005; Molofsky et al., 2005).
Sonic hedgehog (Shh) secreted by Purkinje cells guides cerebellum development
by driving a postnatal wave of proliferation of the cerebellar granule neuronal progenitors
(CGNPs) in the external granular layer (EGL), which subsequently become post-mitotic,
migrate inward and differentiate into cerebellar granule neurons (Goldowitz and Hamre,
1998). Bmil mutant CGNPs have been shown to have an impaired proliferative response
upon Shh stimulation (Leung et al., 2004). Based on these results, it has been proposed
that Shh binds the Patched receptor in the CGNPs leading to inhibition of smoothened
and activation of the Gli transcription factors. The Gli transcription factors induce the
expression of Bmi 1, which in turn leads to the down-regulation of the Ink4a/Arf locus
ensuring cell proliferation can continue (Leung et al., 2004).
Bmil also regulates the proliferative activity of normal hematopoietic stem and
progenitor cells (Lessard and Sauvageau, 2003; Park et al., 2003). Consistent with this
role, the expression of PcG genes during human hematopoietic cell differentiation is
highly regulated (Jacobs and van Lohuizen, 2002); Bmil is specifically expressed in
CD34+ immature progenitor cells, but is absent in differentiated cells (Lessard et al.,
1998). Furthermore, the number of HSCs in postnatal Bmil mutant mice is markedly
reduced with no detectable self-renewal of adult HSCs. As a consequence, mice deficient
for Bmi 1 suffer from various defects in the hematopoietic system such as hypoplasia in
45
spleen and thymus, reduction in overall T cell numbers, defects in B cell development
and an impaired proliferative response of lymphoid precursors to IL-7 (van der Lugt et
al., 1994b). These defects are also partially rescued by Ink4a/Arf loss as indicated by
restored lymphocyte counts (Jacobs et al., 1999b).
In light of the transforming properties of Bmi 1 from in vitro studies as well as its
capacity to cooperate with Myc in lymphomagenesis in vivo, it is not surprising that
Bmi 1 has been found to be overexpressed in several human cancers. These include
mantle cell lymphoma, colorectal carcinoma, liver carcinomas, non-small cell lung
cancer, pediatric brain tumors and medulloblastomas (Bea et al., 2001; Hemmati et al.,
2003; Kim et al., 2004a; Kim et al., 2004b; Leung et al., 2004; Neo et al., 2004;
Vonlanthen et al., 2001). Significantly, the finding that Bmil is involved in stem cell
self-renewal and proliferation raises the question of whether any of these tumors are of
stem cell origin.
In summary, Bmil is involved in the control of proliferation of several cell types, at
least in part, through the control of p16 INK4Aand
pI9 ARF
expression. Importantly, other
PcG proteins have also been implicated in cell cycle control (Gil et al., 2005). In fact,
M33, Mel18 and Cbx7 have been shown to regulate the Ink4a/Arf locus expression (Core
et al., 2004; Gil et al., 2004; Jacobs et al., 1999a). However, the mechanism by which
Bmi 1 (and other PcG proteins) is recruited to the Ink4a/Arf locus promoters remain to be
elucidated. Interestingly, the p19ARF promoter contains E2F binding sites (Bates et al.,
1998) and E2F6 has been found in association with members of the mammalian PcG
complex including Bmil (Trimarchi et al., 2001). These findings raise the possibility that
E2F6 recruits Bmi 1 to the p1 9 ARF promoter for its PcG-mediated repression.
46
E2F6 is a unique member of the E2F family of transcription factors that cannot be
regulated by the pocket proteins. The aim of this study is to understand the biological
significance of E2F6 with respect to its significant homology to the E2F transcription
factors and its potential role in the PcG-mediated transcriptional repression. The study of
mutant mice and MEFs has been critical for defining the role of the individual E2Fs in
cell cycle progression and tumorigenesis. Furthermore, mice and MEFs deficient for
mammalian PcG genes have also been essential in confirming the biological significance
of PcG proteins in development as well as in uncovering novel roles in the control of cell
cycle and tumorigenesis. Thus, to gain insight into the role that E2F6 plays in vivo, E2f6deficient mice were generated and characterized in a wild-type (Chapter 2) or Bmil
mutant (Chapter 3) background.
47
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73
Chapter Two
E2f6 deletion leads to posterior transformations of the axial skeleton
Maria Courel & Jacqueline A. Lees
The author contributed all text and figures. All mouse work and cell culture experiments
were done in the laboratory of Jacqueline A. Lees.
ABSTRACT
E2F6 is a member of the E2F family of transcription factors that are involved in
the control of the cell cycle. Unlike other E2Fs, E2F6 does not contain a transactivation
domain and is not regulated by pocket protein binding. This E2F has been shown to be a
repressor of transcription that seems to act through the recruitment of the Polycomb
Group complex. This complex is responsible for the maintenance of Hox gene
expression patterns during development and thus ensures the correct anterior-posterior
segmentation of the embryo. One of the most studied members of the PcG complex is
Bmil, which also has a role in cell cycle control through its negative regulation of
p 1 6 INK 4 Aand
pI 9 ARF
expression. In order to determine the biological function of E2F6,
we have generated and characterized E2f6-'- mice and mouse embryonic fibroblasts
(MEFs). The mutant mice are viable and survive into adulthood with no difference in
lifespan compared to their littermate controls. Furthermore, the E2f6 null MEFs are
indistinguishable from wild-type MEFs in asynchronous proliferation, cell cycle re-entry
from quiescence, senescence and E2F target genes expression levels. Instead, we found
that the loss of E2F6 results in posterior axial skeleton transformations that are
reminiscent of the Bmil-deficient mice defects. These findings suggest that E2F6 does
not play a major role in cell cycle control or that its function can be compensated by the
action of other factors. However, our data indicates that E2F6 participates in the
segmentation of the embryo during development providing proof that it is a bonafide
Polycomb Group protein.
75
INTRODUCTION
The E2F transcription factors are a family of key regulators of cell proliferation
and differentiation (Dyson, 1998; Nevins, 1998; Trimarchi and Lees, 2002). They
function by controlling the expression of genes that are necessary for S phase entry and
progression. Deregulation of these E2F target genes leads to inappropriate proliferation,
transformation and apoptosis. Recently, members of the E2F family have also been
implicated in the control of genes that are important for DNA repair and recombination,
mitosis and development (Cam and Dynlacht, 2003; DeGregori, 2002; Muller et al.,
2001; Stevaux and Dyson, 2002). The E2F family consists of 10 genes that can be
subdivided into two families, the E2Fs (1-8) and the DPs (1, 2). Functional E2F
complexes are formed when one E2F subunit and one DP subunit heterodimerize.
Although both subunits are required for E2F activity, the functional specificity is
conferred by the E2F moiety. The E2F proteins can be further subdivided into three
groups based on structural and functional similarities.
The first group is comprised of E2F1, E2F2 and E2F3a, which are strong
transcriptional activators that are able to drive quiescent cells into the cell cycle through
activation of E2F target genes (DeGregori et al., 1997; Kowalik et al., 1995; Lukas et al.,
1996; Verona et al., 1997). These proteins are not detected during quiescence but rather
their levels start to rise during GI and reach peak levels just before the G/S transition.
This E2F activity is exclusively regulated by the retinoblastoma protein (pRB) and
functions mainly by activation of genes that are responsible for cell cycle progression.
E2F4 and E2F5 are poor transcriptional activators and are unable to induce
quiescent cells to enter S phase (Lukas et al., 1996; Muller et al., 1997; Verona et al.,
76
1997). In fact, these proteins are thought to actively repress E2F target genes through
their ability to bind all pocket proteins and recruit histone deacetylases to the promoters
(Brehm and Kouzarides, 1999; Dyson, 1998). E2F3b does not share the same level of
structural similarity observed between E2F4 and E2F5 (He et al., 2000; Leone et al.,
2000) and appear to bind exclusively to pRB (Leone et al., 2000). Despite this, E2F3b
has been grouped with E2F4 and E2F5 due to its ability to repress target genes and
because its expression pattern through the cell cycle is reminiscent of the repressive E2Fs
(Aslanian et al., 2004; He et al., 2000; Leone et al., 2000).
The last group consists of the most novel members of the E2F family, E2F6, E2F7
and E2F8 (de Bruin et al., 2003; Logan et al., 2004; Maiti et al., 2005). These proteins
lack the characteristic transactivation and pocket protein domains found in the rest of the
E2F family members and are therefore unable to activate transcription and are not
regulated by pocket protein binding. Instead, they have been shown to repress the E2Fmediated transcription of target genes. However, there are differences between the
proteins of this group. E2F7 and E2F8 contain two DNA binding domains, lack a DP
dimerization domain and are able to bind DNA in the absence of DP. Incontrast, E2F6
contains both DNA binding and dimerization domains and requires DP for effective
interaction with DNA (Cartwright et al., 1998; Gaubatz et al., 1998; Morkel et al., 1997;
Trimarchi et al., 1998).
The mouse E2f6 gene produces two mRNAs through alternative splicing of exon
2 that give rise to two proteins (Kherrouche et al., 2001). E2F6a lacks this exon whereas
E2F6b is truncated at the N-terminal end of the protein and translation begins with exon
2(Dahme et al., 2002). E2F6 is widely expressed during embryogenesis and is present in
77
most, if not all, adult murine tissues (Dahme et al., 2002; Kherrouche et al., 2001). In
MEFs, E2F6 expression rapidly increases during the GO/G1 transition reaching its highest
level in mid-GI and remains relatively constant thereafter (Dahme et al., 2002;
Kherrouche et al., 2001). This is consistent with the finding that in primary human T
lymphocytes, E2F6 expression starts well before the S phase peak after PHA stimulation
(J.M.Trimarchi and J.A.Lees, unpublished results).
Several experiments indicate that E2F6 may have a role in cell cycle control.
Overexpression of E2F6 in asynchronous Saos2 or NIH3T3 cells leads to a modest
increase in the number of cells in GI and microinjection of E2F6 into quiescent NIH3T3
cells leads to a 50-60% reduction in the number of cells that are able to enter S phase
upon serum stimulation (Gaubatz et al., 1998). In contrast, transient expression of E2F6
in asynchronous U20S results in a small increase in the number of cells in S phase
(Cartwright et al., 1998). Moreover, the levels of the cell cycle inhibitor p19ARF are
decreased when E2F6 is overexpressed in wild-type MEFs arguing in favor of a positive
role for E2F6 in the regulation of the cell cycle (J.M.Trimarchi and J.A.Lees, unpublished
results). Finally, E2F6 has been recently found at E2F target promoters that are activated
during GI/S but does not associate with promoters of E2F target genes that are activated
during G2/M (Giangrande et al., 2004). This finding implies a role for E2F6 in
progression through the cell cycle by ensuring that the transcription of GI/S target genes
is extinguished before the activation of G2/M target genes. Therefore, the role of E2F6
in cell cycle regulation remains to be clarified since most studies were done by
overexpressing E2F6 and have yielded conflicting results.
78
E2F6 has been shown to function as a transcriptional repressor (Gaubatz et al.,
1998; Morkel et al., 1997; Trimarchi et al., 1998). At least when overexpressed, it can
inhibit the transcriptional activity of the other E2F complexes. It appears that this
repression does not occur via sequestration of DP molecules given that the repression is
not relieved by an excess of DP (Gaubatz et al., 1998; Trimarchi et al., 1998).
In an attempt to elucidate the mechanism of the E2F6-mediated repression, we
and others have identified proteins that interact with E2F6 (Attwooll et al., 2005; Ogawa
et al., 2002; Trimarchi et al., 2001). Surprisingly, E2F6 has been found in several
complexes containing proteins of the mammalian Polycomb Group (PcG). The PcG
proteins, first identified in Drosophila,form large multimeric complexes that are
responsible for the repression of the Hox genes, which determine the patterning of the
developing embryo (Kennison, 1995; Simon, 1995). In mammals, there seem to be many
more PcG proteins and at least two distinct PcG complexes (Otte and Kwaks, 2003). The
Eed-containing PcG (PRC2) initiates polycomb-mediated repression, whereas the Bmi-1containing PcG (PRC1) maintains the repression at later stages of development. Loss of
proteins of the PcG-PRC2 complex in mice generally result in early embryonic lethality
whereas PcG-PRC 1 mutant mice display posterior transformations of the axial skeleton
due to anterior shifts in Hox genes expression boundaries. Furthermore, several PcG
proteins have been implicated in the control of the cell cycle (Jacobs and van Lohuizen,
2002). For example, Bmi 1 regulates cell proliferation through its ability to repress the
4
And
tumor suppressors p 1 6INK
p1I9RF
(Jacobs et al., 1999).
A yeast two-hybrid screen has identified the PcG-PRC1 protein, RYBP, as a
direct E2F6 interactor (Trimarchi et al., 2001). Further biochemical analysis revealed
79
that E2F6 is found in association with several Polycomb Group proteins including Ring1,
Mel- 18, Mph 1 and the oncoprotein Bmi 1. In another study, an E2F6-containing complex
was purified from human HeLa cells (Ogawa et al., 2002). Mass spectrometry analysis
showed that several proteins are present in this complex including DPI, Mga and Max,
histone methyltransferases, HPly and several Polycomb Group proteins (RING 1, RING2,
MBLR, h-l(3)mbt-like protein and YAF2). The presence of E2F6, Max and HPly on
E2F- and myc-target promoters during GO but not at later stages of the cell cycle suggests
that this complex is involved in stable repression required for quiescence (Ogawa et al.,
2002). A third group has performed a yeast two-hybrid screen with the full length human
E2F6 and has identified the PcG-PRC2 protein, EPC1 (Enhancer of Polycomb 1), as a
direct interactor (Attwooll et al., 2004). This interaction mediates the association of
E2F6 with Sin3B and EZH2 specifically in proliferating cells.
Significantly, only a few PcG proteins have been shown to bind DNA directly
including Mel-18 and YY1 (Brown et al., 1998; Kanno et al., 1995). These cannot
account for all the Polycomb DNA binding activity suggesting that other DNA binding
factors may be responsible for targeting the PcG complexes to specific promoters. Since
E2F6 associates with PcG proteins and is able to directly bind DNA, it is reasonable to
speculate that E2F6 functions to recruit PcG complexes to target promoters and therefore
contributes to their biological function.
To test this hypothesis and to address the role of E2F6 in cell cycle control, we
have generated E2f6-deficient mice. If E2F6 were central to PcG function, we would
expect E2f6-'- mice to display phenotypes similar to those described for other PcG
mutants. Indeed, these mice display posterior axial skeleton transformations indicating
80
that E2F6 participates in the role of the PcG complex in segmentation of the developing
embryo. However, the analysis of the cell cycle properties of E2f6' MEFs revealed no
defects suggesting that E2F6 is dispensable for cell cycle control.
RESULTS
Generation of E2f6 mutant mice
In order to study the role of E2F6 in vivo, we generated E2f6-deficient mice. To
do so, we have mapped and cloned a region of the E2f6 genomic locus that encompasses
the translation initiation site through the stop codon (Fig. la). We constructed a vector
that targets most of the E2f6 gene including the exons that encode the DNA binding,
leucine zipper and marked box domains. This deletion should effectively abrogate
E2F6's ability to bind DNA, dimerize with DP and interact with RYBP and should
therefore constitute a null allele. Figure la shows the targeting vector construct, which
contains two regions of homology to the E2f6 genomic sequences upstream of the DNA
binding domain and downstream of the marked box domain. These homologous arms
flank a neomycin resistance cassette for positive selection of the recombinant embryonic
stem (ES) cells. The vector also includes a Herpes simplex virus tk gene for negative
selection of random integration events. E2f6"-ES cells were generated and used to
produce two independent lines of E2f6' mice and mouse embryonic fibroblasts (MEFs).
The ES cells were genotyped by Southern blot and the mice and MEFs were genotyped
by PCR (Fig. lb and c). As expected, no E2F6 was detected in E2f6' MEFs by Western
blots (Fig. Id).
81
a
7 kb target
EooRI
P"
12
WOI EciaRi
5
4
3
6
8
7
5 kb
Ntl
Scal
RI
2.7
~
-
c
aI
4'ar
a3G-e
k3=
PI Pmel
RE
No
EcoRI
t
Fs
Epo
NO
F
ol
PGK-neo
Psti
A
12Prdb-
4 kb
+
b
c
4 kb-
4kb -
+
+
+
d
.
U_
U_
U
F6ai
Tubulin
Figure 1. Generation of E2f6-- mice. a. Wild-type E2f6 genomic locus (top), targeting
vector (middle) and targeted allele (bottom). b. Southern blot of genomic DNA from ES
cell lines digested with EcoRI and hybridized with the probe shown in a. c. PCR
genotyping of genomic DNA from ear punches. d. Western blot analysis of MEFs
lysates to confirm the absence of E2F6 protein. The asterisk denotes an unspecific band.
E2F6 is dispensable for mouse viability and survival
E2f6-deficient mice are viable and display no obvious morphological defects.
These animals survive into adulthood with no difference in growth or fertility compared
to their wild-type littermates (Table 1). E2f6'*, E2f6+' and E2f6~'- mice were compared
for lifespan and cause of death. We observed no difference in the percentage of animals
that were alive after 530 days (70%, 68% and 75%, respectively) (Figure 2a).
E2f6 +1+
E2/6 +/i
E2f5 -/-
Expected
58.25
116.5
58.25
Observed
60
103
70
Table 1. E2f6-deficient mice are viable. Heterozygous crosses were performed
generating a total of 233 animals.
Histological analysis of the dead animals was performed to determine the cause of
death and revealed a few interesting observations although their significance is still not
clear (See Table 2). E2f6f'- and E2f6'- mice seem to have a modest increase in the
incidence of certain tumor types insinuating the possibility that E2F6 may have a role in
the formation of certain tumors. First, 35% of E2f6"' mice and 20% of E2f6'- mice died
of blood tumors including lymphomas, hemangiosarcomas and histiocytic sarcomas.
Second, 20% of E2f6"' mice and 28% of E2f6' mice died of liver tumors. Third, 3 out of
5 E2f6-'- mice analyzed had developed odontomas, which normally arise as a consequence
83
a
110
100
90
80
--- E26-/70
--
a-E2f6+-
-a-E2)6
+/+
60
Ii,
50
40
30
20
10
0I
0
200
100
300
400
500
600
400
500
600
Age (days)
b
120
100
80
60
SE26
40
-.-
-
E2f6 +/-
-+--E2f6
+/+
20
0
0
100
200
300
Age (days)
Figure 2. Loss of E2F6 does not affect the lifespan of adult mice. a. Survival curves
of E2f6+' (blue, n=35), E2f6+'- (green, n=50) and E2f6' (red, n=53) mice. b. Survival
curves of E2f6+' (blue, n=14), E2f6*'- (green, n=13) and E2f6'- (red, n=18) mice treated
with 4 Grays of y-irradiation at 6 days of age.
of defective osteoclast function during tooth eruption (Amling et al., 2000). Furthermore,
20% of E2f6'- mice and 28% of E2f6'- mice displayed testicular atrophy. Finally, two
E2f6+'- mice and two E2f6'~ mice displayed glomerulonephritis whereas two E2f6-' mice
were found to have only one kidney. It is noteworthy that two other mice that were
homozygous mutant for E2f6 generated from a cross to Bmil-deficient mice also were
found to have a single kidney.
E216 +/+
E2f6 1-
E214 -/-
(n = 9)
(n=20)
(n=25)
Testicular atrophy
0
4
7
Missing kidney
0
0
2
Heart fibrosis
0
1
3
Glomerulonephritis
0
2
2
Liver tumors
0
4
7
Blood tumors
1
7
5
Odontoma
0
0
3
Table 2. Histological analysis of E2f6'+, E2f6'- and E2f6'- mice.
Mutation of genes that are required for resistance to oncogenic radiation leads to a
reduction in the latency of tumor formation following y-irradiation. To test the
possibility that absence of E2F6 may stimulate tumor formation through sensitization to
DNA damage, we performed a y-irradiation experiment. For this purpose, 14 E2f6+'+
mice, 13 E2f6+'- mice and 18 E2f6'- mice were treated with 4 Grays of y-irradiation at 6
days of age and monitored until moribund, then sacrificed and autopsied. We observed
no significant difference in the lifespan or tumor progression between all the genotypes
85
(Figure 2b; data not shown) implying that if E2F6 has a role in tumorigenesis it does not
involve the DNA damage response. These results are not consistent with the increase in
the frequencies of certain tumors in E2f6'- and E2f6-'- mice (Table 2). One possibility is
that this increase is not significant due to the small number of wild-type animals
analyzed. Another possibility may reflect the fact that the irradiated mice did not receive
the correct dose of y-irradiation due to problems in the calibration of the y-irradiator. To
definitively test the possibility that E2F6 behaves as a tumor suppressor we would have
to perform loss of heterozygosity studies on tumors isolated from E2f6'- mice.
E2f6 null mice display posterior transformations of the axial skeleton
Loss of Bmi 1 in mice results in posterior transformations along the entire axial
skeleton (van der Lugt et al., 1994). Since E2F6 has been found to associate with Bmil
(Trimarchi et al., 2001), we wished to determine if E2f6 mutant mice also displayed
transformations of the axial skeleton. We analyzed E2f6'*, E2f6'- and E2f6' mice at
post-natal day 3 (P3) and embryonic day 18.5 (E18.5) by staining of the skeletons with
Alcian blue and Alzarin red, which stain the cartilage in blue and the bone in red (Figure
3). E2f6' and E2f6'- mice display two types of posterior transformations that had been
previously described for the Bmil mutants. First, the thoracic vertebra T13 is
transformed into a lumbar vertebra Li as evidenced by degeneration or the complete lack
of ribs in 9% of E2f6'-and 67% of E2f6'- animals. Second, the lumbar vertebra L6 is
transformed into the sacral vertebra Si as evidenced by its association with the ilial bones
in 35% of E2f6'~ and 80% of E2f6'- mice (Table 3). These results suggest that E2f6
86
mutation results in dosage-dependent posterior transformations of the axial skeleton that
are reminiscent of the Bmil-deficient mice transformations.
E2f6 4-
E2f6 +/+
T13
L6
Figure 3. Posterior axial skeletal transformations of E2f6' mice. Ventral view of the
thoraco-lumbo-sacral region of E2f6' (left) and E2f6' (right) mice. The E2f6'- animals
have only 12 pairs of ribs compared to 13 in the wild-type control due to degeneration of
the thirteenth ribs. Also, the lumbar vertebra L6 has formed sacro-iliac joints, which are
typical of sacral vertebra Si.
Penetrance of skeletal
abnormalities (%)
E2j6 +/+
E2F6 +/-
E2/6 -/
(n = 11)
(n=23)
(n=15)
both sides
0
9
40
one side
0
0
27
both sides
0
13
53
one side
0
22
27
T3 -> LI
L6 - SI
Table 3. Skeletal transformation of E2f6-' and E2f6-- mice.
E2F6 is not required for cell proliferation and senescence
E2F6, a member of the E2F transcription family of cell cycle regulators, has been
implicated in the regulation of the cell cycle by several findings. First, it is expressed in a
cell cycle-dependent manner (Dahme et al., 2002; Kherrouche et al., 2001). Second, it is
found at the promoters of cell cycle-regulated genes (Giangrande et al., 2004; Ogawa et
al., 2002). Third, E2F6 overexpression leads to both a reduction or increase in the
percentage of cells in S phase depending on the cell type and conditions (Cartwright et
al., 1998; Gaubatz et al., 1998). Finally, E2F6 associates with Bmil, which has also been
implicated in cell cycle control through its negative regulation of the Ink4/Arf locus.
Bmil mutant MEFs have an impaired proliferation and premature senescence, which
4
A a
appear to be due to the up-regulation of both p 1 6INK
19 ARF
88
Based on these findings, it is reasonable to believe that E2F6 may function in the
control of cellular proliferation. In order to address this possibility, we generated E2f6'
MEFs and performed several cell cycle assays. In asynchronous proliferation assays as
well as on cell cycle re-entry experiments, E2f6-'- MEFs performed as well as their wildtype counterparts (Figure 4). No deregulation of E2F target genes (cyclin E, cyclin A,
E2F1, PCNA, p107) or p16INK4 Aand
PI
9
ARF
was observed (data not shown). Furthermore,
E2f6' MEFs underwent normal senescence when successively passaged in a 3T3
protocol (Figure 5). These results indicate that E2F6 is not required for normal
proliferation and senescence.
89
a
600000
+- E2f6 +/+
500000
sooo-
-i-
E2f6 +/+
-+-
E2f6 +/-
--
E2f6 +/-
400000
E2f6 -/+.--E2f6
-/-
S300000
200000
100000-
0
0
1
3
2
Tm
4
(days)
7
6
5
8
b
160000 -
140000 -
120000-
---
E2f6 +/+
-i-
E2f6 +/-
-+
E2f6 +/+-E2f6 -/-
2. 100000 -
0
E2f6
80000 -
60000-
40000 -
20000-
0
0
5
10
15
Time after serm release (h)
20
25
30
Figure 4. E2F6 is dispensable for normal proliferation of mouse embryonic
fibroblasts. a. Proliferation of asynchronously growing E2f6+'1 (blue), E2f6*'- (green)
and E2f6'~ (red) MEFs in medium with 10% serum. b. Thymidine incorporation of
E2f6+' (blue), E2f6*/- (green) and E2f6' (red) MEFs after serum arrest and release.
Curves are typical results obtained with different cell lines.
6
5
-+-- E2f6 +/+
-+--
E2f6 +/-
-.-
E2f6 -/-
4
32
1 -
0
4
6
8
10
12
14
16
18
20
22
Pasge Number
Figure 5. E2f6~'~ MEFs undergo normal senescence. E2f6+' (blue), E2f6*' (green) and
E2f6' (red) MEFs were passaged according to a standard 3T3 protocol. Fold replication
is the number of cells determined in each passage divided by the number of cells plated
(3x 105). Curves are typical results obtained with different cell lines.
DISCUSSION
The aim of this study was to determine the biological significance of E2F6. Since
E2F6 has been shown to associate with several PcG proteins, we anticipated that loss of
E2F6 would result in typical defects observed in the Polycomb mutants. Indeed, we have
found that E2f6' mice display posterior axial skeletal transformations that are reminiscent
of those found in Polycomb mutants. Moreover, E2f6'- MEFs were not defective in
proliferation or senescence as it might have been expected in light of the roles of some
PcG and E2F proteins in cell cycle control.
Viability and lifespan of E2f6'- mice
E2f61 mice are viable and display no difference in lifespan compared to their
control littermates. Furthermore, we did not observe a significant difference in the
response to y-irradiation. We did find a modest increase in the incidence of certain
tumors of liver and blood suggesting that E2F6 may function as a tumor suppressor.
However, it is important to note that these tumors were observed in mutant mice that
were over one year old and the number of wild-type animals analyzed at this age was
much smaller. In addition, most of the overexpression studies as well as E2F6's ability to
associate with the Bmil oncogene propose an oncogenic rather than a tumor suppressive
role for E2F6. In agreement with this, E2F6 has recently been shown to repress the
transcription of the tumor suppressor BRCA1 (Oberley et al., 2003). However, in order
to test the possibility that E2F6 is a tumor suppressor, a loss of heterozygosity study
should be performed on tumor samples from E2f6"' mice.
92
Significantly, 3 out 5 mice analyzed had developed odontoma. These are benign
tumors that arise in osteopetrotic mice as a consequence of defects in osteoclast function
or differentiation (Amling et al., 2000; Ida-Yonemochi et al., 2002; Ida-Yonemochi and
Saku, 2002; Oberley et al., 2003). A reduced osteoclast function during tooth
development leads to morphological abnormalities that result in the formation of
odontomas. The significance of this finding is not clear yet since we have not studied the
effects of loss of E2F6 in osteoclast function and differentiation. It would therefore be of
interest to isolate osteoclast precursor from peripheral blood mononuclear cells of E2f6T
mice and assay their differentiation and function.
E2f6+' and E2f6'- mice display testicular atrophy with low penetrance. However,
it is important to note that this is a common age-related defect and all the mice were at
least 500 days old. A histological analysis of males at different ages would be necessary
to determine if this atrophy develops earlier in the mutants. Significantly, while this
work was in progress, another laboratory reported the generation and characterization of
E2f6-'- mice and observed a reduction of spermatogenesis that did not affect the fertility of
these mice (Storre et al., 2002).
A few E2f6' mice were found to have a single kidney while the mutants that had
both kidneys did not exhibit any abnormalities. It is not clear if this observation reflects a
defect in kidney development or degeneration. In order to address this, a timed analysis
should be performed. However, the low penetrance of this phenotype would require the
generation of an enormous number of E2f6' animals. Significantly, the Hoxi1 paralog
group (HoxA 11, HoxC 1 and HoxD 11) are involved in the induction of ureter budding
from the nephric duct (Wellik et al., 2002).
93
Proliferation and senescence of E2f6- MEFs
We have shown here that E2F6 is dispensable for proliferation and senescence in
MEFs given that E2f6'~ MEFs are undistinguishable from their wild-type littermates.
This data is supported by similar findings by Storre et al., 2002. Previous data suggested
that E2F6 may repress the transcription of
p1
9 ARF
(J.M.Trimarchi and J.A. Lees,
unpublished results). However, we do not observe a deregulation of
pI 9 ARF
expression in
E2f6-'- MEFs. Together these findings suggest that E2F6 does not participate in the
Bmil-mediated repression of
p1
9 ARF.
Alternatively, other factors could compensate for the absence of E2F6.
Interestingly, although initial studies indicated that E2F6 was the only E2F able to
interact with RYBP, a subsequent report has suggested that E2F2 and E2F3 may also
associate with this protein (Schlisio et al., 2002; Trimarchi et al., 2001). This would
provide alternative proteins for targeting the PcG proteins to DNA in the absence of
E2F6. In agreement with this hypothesis, E2F3b has been found at the p1 9 ARF promoter
and has been implicated in its repression (Aslanian et al., 2004). Moreover, recent
findings suggest that E2F4 is able to compensate for the loss of E2F6 in MEFs
(Giangrande et al., 2004). In this study, although E2F6 was found at the promoters of
E2F target genes that are activated during G US, no deregulation was observed in E2f6'MEFs. However, when E2F4 levels were reduced using siRNAs, these genes were derepressed. Further studies are needed to determine if similar compensation for E2F6 loss
occurs at the pI 9 ARF and PcG target genes.
94
Posterior transformations in E2f6-- mice
Usually, the discovery of novel PcG proteins begins with the identification of a
protein that associates with other PcG proteins followed by its characterization as a
transcriptional repressor and its localization to Polycomb bodies on DNA. Nevertheless,
the ultimate proof that a protein is a member of the PcG complex has come, without
exception, from the observation of axial skeleton transformation in mutant mice. It has
been previously shown that E2F6 interacts with several PcG proteins (Attwooll et al.,
2004; Ogawa et al., 2002; Trimarchi et al., 2001) and that E2F6 may repress E2Fmediated transcription (Gaubatz et al., 1998; Morkel et al., 1997; Trimarchi et al., 1998).
Although E2F6 has been shown to be a nuclear protein, its expression seems to be diffuse
instead of localized to Polycomb bodies (J.M.Trimarchi and J.A.Lees, unpublished
results). Here we show that E2f6' mice display a subset of the posterior transformations
of the axial skeletons observed in Bmil-'- mice (van der Lugt et al., 1994) demonstrating
that E2F6 is a bonafide PcG protein. Although there is a difference in the penetrance of
these transformations, an independent analysis of E2f6'- mice revealed identical posterior
transformations of the axial skeleton (Storre et al., 2002).
However, the posterior transformations in the other PcG mutants are observed
throughout the entire length of the axial skeleton whereas the ones observed in E2f6'
mice are restricted to the more posterior region of the axial skeleton (Akasaka et al.,
1996; Core et al., 1997; Takihara et al., 1997; van der Lugt et al., 1994). Although we
have not examined the expression patterns of Hox genes in the E2f6-deficient mice, this
finding implies that E2F6 contributes to the PcG regulation of a subset of target genes.
95
EXPERIMENTAL PROCEDURES
Generation and genotyping of E2f6'- mice
The BAC clone b39J22 (Research Genetics) known to contain the E2f6 genomic
locus (Peterfy et al., 1999) was mapped and cloned to obtain the sequences necessary for
designing the targeting strategy (Figure la). The targeting vector described above was
introduced into 129Sv J1 ES cells by electroporation and the cells were selected with
G418 and Gancyclovir. 96 resistant clones were picked for genotyping. E2f6 +/- cells
were detected by Southern blot using external 5' and 3' probes as well as a neomycin
probe (Fig. la). Once the heterozygous clones were identified and verified to contain a
diploid genome by karyotyping, they were injected into C57/BLACK6 3.5 d.p.c.
blastocysts. The injected blastocysts were subsequently implanted into pseudo-pregnant
females and the chimeric progeny were identified by coat color. Mice with a high
contribution of agouti cells were mated to pure C57/BLACK6 mice. The agouti progeny
of these mice were genotyped by PCR of DNA obtained from ear or tail pieces using the
common primer 5'-ATCTCTGTCTGGTCTGATCC-3', the wild-type E2f6-specific
primer 5-GATGCCATCCAAGACATTGG-3', and the mutant targeting vector specific
primer 5-GCCGCATAACTTCGTATAGC-3' (Fig. 1c). The E2f6*'- mice were then
interbred to produce E2f6- mice.
Histological and skeletal analysis
Dead mice from our aging colonies were dissected and processed for histological
analysis. Soft tissues were fixed in 10% formalin and hard tissues were fixed in Bouin's
fixative. Paraffin sections were prepared and stained with hematoxylin and eosin.
Skeletal analysis was performed on 3-day old mice. After removing the skin and viscera,
96
the skeletons were fixed in acetone and stained with cartilage-specific Alcian Blue and
bone-specific Alzarin Red. Soft tissue was cleared with KOH.
Mouse embryonic fibroblasts
MEFs were prepared from 13.5 d.p.c embryos as previously described (Humbert
et al., 2000) and genotyped by PCR of DNA obtained from yolk sacs. Proliferation
curves were obtained by plating 2x10 4 MEFs in triplicate in 24-well plates. At the
indicated time points, MEFs were trypsinized and counted. For cell cycle re-entry assays,
2x10 5 MEFs were plated in triplicate in 6-well plates. After 2 days of growth in media
containing 10% serum, they were incubated in media containing 0.1% serum for 3-4
days. Re-entry into the cell cycle was induced by incubation in media containing 10%
serum and at the indicated time points, 5
[tCi
of 3 H-thymidine was added to the cells for 1
hour. Cells were then scraped from the plates and cell pellets were analyzed for 3Hthymidine incorporation using a scintillation counter. A 3T3 protocol was followed to
monitor senescence. 3x10 5 MEFs were plated in duplicates in 6-cm plates and re-fed 2
days later. On the third day, they were trypsinized, counted and replated. The fold
replication was determined by dividing the number of cells obtained at day 3 by 3x10 5.
Western blots were performed as described previously (Moberg et al., 1996) with 50-100
xg of whole cell lysates.
97
ACKNOWLEDGEMENTS
We are grateful to Scott Boyd, David Tuveson and Julien Sage from the laboratory
of Tyler Jacks for help with the generation of the E2f6' mice. We also thank Alicia
Caron and Roderick Bronson for the generation and analysis of histological sections and
Mark Garcia for help with colony maintenance and dissections. We would also like to
thank the members of the Lees lab for reagents and helpful discussions.
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102
ChapterThree
E2F6 and Bmil synergize in axial patterning
in a gene dosage-dependent manner
Maria Courel & Jacqueline A. Lees
The author contributed all text and figures. All mouse work and cell culture experiments
were done in the laboratory of Jacqueline A. Lees.
ABSTRACT
E2F6 is a member of the E2F family of transcription factors that does not contain
a transactivation domain and cannot be regulated by pocket protein binding. Instead, this
protein appears to repress transcription through the recruitment of the Polycomb Group
(PcG) complex. This complex participates in the anterior-posterior patterning of the
developing embryo by stably repressing Hox gene expression. Consequently, genetic
ablation of PcG proteins leads to posterior transformations of the axial skeletons.
Furthermore, other developmental defects are often observed in these mutants such as
hematopoietic, cerebellar and smooth muscle defects. Finally, the PcG complex has been
implicated in cell cycle control since at least one of its members, the oncoprotein Bmi 1,
appears to repress the transcription of p 16 INK4 A and
p1
9
ARF.
Consistent with E2F6's
association with PcG proteins, E2F6 loss results in posterior axial skeleton
transformations that are reminiscent of the Bmil-deficient mice defects. However, none
of the other commonly observed Polycomb mutant phenotypes have been observed in the
E2f6'- mice suggesting that E2F6 only participates in a subset of PcG functions. To
investigate the possibility that E2F6 may be involved in other aspects of PcG activity, we
have generated E2f6;Bmil compound mutant mice and MEFs. Our results confirm the
gene dosage effect of E2F6 on axial skeletal patterning and suggest that E2F6 may
possibly synergize with Bmil in mice viability and survival as well as cell proliferation
and senescence.
104
INTRODUCTION
E2F6 is a recently identified member of the E2F family of transcription factors
that has significant homology to other family members within the DNA binding,
dimerization and marked box domains (Cartwright et al., 1998; Gaubatz et al., 1998;
Morkel et al., 1997; Trimarchi et al., 1998). However, it is truncated at the C-terminus
and therefore lacks transactivation and pocket protein binding domains. Consistent with
its structure, E2F6 can bind DNA as a heterodimer with DP, is not able to activate
transcription of target genes and is not regulated by pocket protein binding.
E2F6 is widely expressed during embryogenesis and is present in all adult mouse
and human tissues examined (Cartwright et al., 1998; Dahme et al., 2002; Kherrouche et
al., 2001; Trimarchi et al., 1998). The expression of this protein in MEFs rapidly
increases during the GO/GI transition reaching its highest level in mid-GI and remaining
relatively constant thereafter (Dahme et al., 2002; Kherrouche et al., 2001). In agreement
with this, in primary human T lymphocytes, E2F6 expression starts well before the S
phase peak after PHA stimulation (J.M.Trimarchi and J.A.Lees, unpublished results).
The role of E2F6 in the cell cycle remains elusive although several pieces of data
suggest that it may stimulate cell cycle progression. First, transient transfection of E2F6
in asynchronous U20S cells leads to a modest increase in the number of cells in S phase
(Cartwright et al., 1998). Second, overexpression of E2F6 in MEFs results in the
downregulation of the cell cycle inhibitor pI9ARF (J.M.Trimarchi and J.A.Lees,
unpublished results). Finally, E2F6 has been proposed to allow cell cycle progression
through S phase by repressing the transcription of E2F target genes that are activated
during the GI/S transition (Giangrande et al., 2004). Thus, E2F6 would ensure that these
105
GI/S-specific genes are no longer expressed before the next phase of the cell cycle
begins. In contrast, a reduction in the number of S phase cells was reported when E2F6
was microinjected into quiescent NIH3T3 cells before re-stimulation to enter the cell
cycle by serum addition (Gaubatz et al., 1998). These differences may simply reflect
distinct functions of E2F6 during different phases of the cell cycle, may be the result of
overexpression of E2F6 at artificially high levels, or may be due to cell type specificity.
However, the analysis of E2f6-'- MEFs has revealed no cell cycle defects arguing against a
role for E2F6 in the cell cycle (Chapter 2; Storre et al., 2002). An alternative explanation
is that E2F6's function in the cell cycle can be compensated by other factors. In fact,
Giangrande et al (2004) have reported that E2F4 can compensate for E2F6 loss in the
regulation of the G/S genes repression during S phase. Another candidate is E2F3b,
which has recently been implicated in the repression of p1 9 ARF (Aslanian et al., 2004).
E2F6 has been shown to act as a dominant-negative transcriptional repressor by
binding to E2F-target promoters and blocking the access of other activating E2Fs
(Gaubatz et al., 1998; Morkel et al., 1997; Trimarchi et al., 1998). Overexpression of
E2F6 can counteract the E2F1-mediated transcriptional activation of a reporter gene even
in the presence of non-limiting concentrations of DP. More recently, however, it has
been suggested that the E2F6-mediated repression may be the result of active recruitment
of repressive complexes to E2F target genes (Attwooll et al., 2004; Ogawa et al., 2002;
Trimarchi et al., 2001). Three independent studies have reported that E2F6 is present in
complexes that contain members of the mammalian Polycomb Group (PcG) as well as
chromatin remodeling enzymes.
106
The PcG proteins were first identified in Drosophilaas a group of proteins that
are necessary for the maintenance of stable repression of Hox genes during development
(Kennison, 1995; Simon, 1995). Subsequently, many mammalian orthologs have been
discovered revealing a similar function despite the higher level of complexity (Gould,
1997). The correct spatial and temporal expression of Hox genes during development is
essential for the normal anterior-posterior segmentation of the embryo. To achieve the
repression of these genes, the PcG proteins form large multimeric complexes that are
thought to act by changing the local chromatin structure (Pirrotta, 1997).
In mammals, the PcG proteins form part of at least two different complexes (Otte and
Kwaks, 2003). The PRC2 complex contains Eed, Enxl/Ezh2 and Enx2/Ezhl and is
responsible for initiating the Polycomb-mediated repression (Hobert et al., 1996; Laible
et al., 1997; Schumacher et al., 1998; Sewalt et al., 1998; van Lohuizen et al., 1998).
Deletions of these genes in mice result in early embryonic lethality underscoring the
importance of their products during development (O'Carroll et al., 2001; Pasini et al.,
2004). The PRC1 complex contains Bmil, Mel-18, M33, Ringla and RYBP among
others (Gunster et al., 1997; Hemenway et al., 1998; Satijn et al., 1997; Schoorlemmer et
al., 1997; Tagawa et al., 1990; van Lohuizen et al., 1991). This complex maintains the
repression established by PRC2 at later stages of development. In contrast to PRC2
mutants, loss of most PRC1 proteins leads to anterior shifts in the Hox genes expression
boundaries and the corresponding posterior transformations of the axial skeleton
(Akasaka et al., 1996; Akasaka et al., 1997; del Mar Lorente et al., 2000; van der Lugt et
al., 1994). These relatively mild defects observed in PRC1 mutants may reflect the
functional redundancy between PcG proteins. In fact, in flies which do not have as many
107
PcG proteins, loss-of-function mutations of these genes lead to widespread abnormalities
that are not restricted to a few segmental shifts as in mice.
Furthermore, several PcG proteins have been implicated in hematopoiesis (Jacobs
and van Lohuizen, 2002). First, the expression of PcG genes is highly regulated in
hematopoietic differentiation (Fukuyama et al., 2000; Lessard et al., 1998; Lessard et al.,
1999; Peytavi et al., 1999; Raaphorst et al., 2001; Raaphorst et al., 2000). Second, all
PcG mutant mice display defects in different hematopoietic compartments and have
severely reduced body weights and sizes (Akasaka et al., 1996; Core et al., 1997; KatohFukui et al., 1998; Kimura et al., 2001; Lessard et al., 1998; Takihara et al., 1997; van der
Lugt et al., 1994). Finally, at least the hematopoietic defects observed in BmiP' mice
have been attributed to a deficiency in the self-renewal capacity of the hematopoietic
stem cells (Lessard and Sauvageau, 2003; Park et al., 2003)).
Apart from the common defects in axial patterning and hematopoiesis observed in
all PRC1-PcG mutants, specific defects were also reported including decreased cerebellar
cellularity in Bmil' mice, smooth muscle atrophy in Mel-18-'- mice, male-to-female sex
reversal in M33' mice and neural crest defects in Mph-' mice (Akasaka et al., 1996;
Core et al., 1997; Katoh-Fukui et al., 1998; Takihara et al., 1997; van der Lugt et al.,
1994). Interestingly, it has also been proposed that the cerebellar proliferation defects of
Bmil' mice are due to decreased proliferation of neural stem cells (Leung et al., 2004;
Molofsky et al., 2003; Zencak et al., 2005) suggesting a general role for Bmil in the selfrenewal of adult stem cells.
The proliferation defects observed in vivo are consistent with a role for the PcG
proteins in the control of the cell cycle. Indeed, Bmil', Mel-18' and M33' MEFs are
108
impaired in proliferation (Core et al., 1997; Jacobs et al., 1999a). The tumorigenic
properties of Bmi 1 have made it a major focus of interest in the field. The Bmil gene
was originally identified as an oncogene that could cooperate with Myc in the formation
of B-cell lymphomas in mice (Haupt et al., 1991; van Lohuizen et al., 1991).
Subsequently, Bmil was shown to transform rodent fibroblasts in vitro (Cohen et al.,
1996). In harmony with its oncogenic capacity, loss of Bmil in MEFs resulted in
4
A and
impaired proliferation and premature senescence due to an up-regulation of p 16 INK
p1 9 ARF (Jacobs et al., 1999a). In fact, loss of the Ink4a/Arf locus at least partially rescues
the proliferation defects in MEFs and hematopoietic and neural stem cells (Bruggeman et
al., 2005; Jacobs et al., 1999a; Jacobs et al., 1999b; Park et al., 2003).
We and others have previously shown that E2F6 is a bonafide member of the
mammalian PRC1-Polycomb complex (Chapter 2; Storre et al., 2002). The finding that
E2F6 was found in association with several PcG proteins, led to the hypothesis that E2F6
may act by recruiting these proteins to, at a mininum, a subset of their target promoters
(Attwooll et al., 2004; Ogawa et al., 2002; Trimarchi et al., 2001). The investigation of
E2f6-deficient mice is consistent with this model. E2f6-'- mice display some of the same
axial skeletal transformations observed in Bmil-' mice suggesting that E2F6 only
participates in the repression of a subset of Hox genes (Chapter 2; Storre et al., 2002).
Unlike Bmil-'-, E2f6-'- mice do not exhibit hematopoietic or other developmental defects.
Finally, E2f6'- MEFs present normal proliferation and sensescence properties. These
findings suggest that the function of E2F6 is restricted to the correct patterning of the
thoraco-lumbo-sacral region of the axial skeleton. Other possibilities are that E2F6 loss
109
is compensated by other factors as discussed above or that E2F6 has a minor role in these
processes resulting in subtle defects that have not been detected.
Compound mutants of PRC1-PcG proteins have been generated in flies and mice
resulting in dramatic synergistic effects (Adler et al., 1991; Akasaka et al., 2001; Bel et
al., 1998; Kwon et al., 2003). The transformations and mortality frequencies were more
severe in the double mutants suggesting a strong gene dosage effect. In an attempt to
uncover further unknown roles for E2F6 in development and proliferation and investigate
the extent of cooperation between E2F6 and Bmi 1, we have generated mice and MEFs
that are deficient for both E2f6 and Bmil genes. The most striking observation is a
significant increase in the penetrance of posterior axial skeleton transformations in the
compound mutant mice compared to the single knockout littermates. Furthermore,
although more experiments are required, our initial observations suggest synergistic
effects between E2F6 and Bmil in mouse viability as well as in cell proliferation and
senescence.
RESULTS
Viability and survival of compound mutants
To further characterize E2F6 function, we have generated E2f6'-;Bmil-'-mice
from double heterozygous crosses. We had previously shown that E2f6 deletion alone
does not affect the viability and survival of mice (Chapter 2) while it has been reported
that Bmil deficiency leads to a reduction in the survival of newborn mice (van der Lugt et
al., 1994). Although BmilP mice are born at the expected frequency, only approximately
50% survive into adulthood. Those that do survive are significantly smaller than their
110
wild-type littermates and display poor health that result in early lethality (3 to 20 weeks).
In agreement with the published data, only 51% of Bmil~'~ mice survived to weaning at 3
weeks of age in our crosses (Table 1). E2f6-'-;BmiL-'- mice were underrepresented at both
weaning age and E18.5 (38% and 46% of expected numbers, respectively). Like the
a. 3 weeks of age
E2t6 +,+
E2f6 +/+
Bmil +/+ BudI -/-
E2f6 +1-
E2f6 -I-
Bmil -/-
Bmil -I-
Expected
15.625
15.625
31.25
15.625
Observed
16
8
24
6
% of expected
102
51
77
38
E2f6 +W-
E2f6 --
Bmil --
Bmii --
b. E18.5
E.2f6 +/+
E2f6 +/+
Bmil +/+ BmiJ -I-
Expected
6.5625
6.5625
13.125
6.5625
Observed
5
8
7
3
122
53
46
%of expected 76
Table 1. Offspring from intercrosses of double heterozygous mice. a. Results
obtained from 250 animals genotyped at 3 weeks of age. B. Results obtained from
105 animals genotyped at E18.5.
111
Bmil' mice, the compound mutants eventually became severely anemic and had to be
sacrificed. Interestingly, the frequency of E2f6*';Bmil' mice is higher than that of both
Bmil' and E2f6-'-;Bmil' mice at weaning age but not at E18.5. This does not fit our
hypothesis of gene dosage effects but rather suggests an alternative unknown mechanism
whereby removing only one allele of E2f6, but not both, would confer a survival
advantage after birth. However, it is important to note that the total number of animals
analyzed is relatively small (n=250) resulting in low statistical power that could explain
these results.
The E2f6';Bmi-' mice, as the Bmil' mice, were significantly smaller than their
wild-type littermates as evidenced by their body weight at 2-3 months of age (Figure 1).
Yet, as discussed before, the number of animals analyzed is not enough to establish
whether there is a significant difference in weight between these mice.
Neurological abnormalities of compound mutants
The Bmil-deficient mice develop ataxia at the age of 2-4 weeks as a result of
decreased cell density in the different layers of the cerebellum. Our results show that
E2f61;BmiL' mice also develop ataxia. We examined animals from our double
heterozygous crosses that survived to 2 months of age. Unlike Bmil'* mice, 60% of
Bmil', 65% of E2f6"-;BmiL' and 70% of E2f6';Bmi]' mice developed ataxia by 2
months of age. We are currently analyzing histological sections of E2f6;BmiL' brains
to assess the state of the cerebellum defect compared to Bmil' brains. Although it is
difficult to quantify the level of ataxia in the different genotypes, we could attempt to
112
a
45 -
40
35-
"
E2f6+/+;Bmil+/+
" E2f6+/-;Bmil+/+
* E2f6-/-;Bmil+1+
* E2f6+/+;Bmil+/0 25
* E2f6 +/-;Bmil +/* E2f6-/-;Bmil +/-
P
20
E2f6+/+;Bm1-/* E2f6+/-;Bmil-/E2f6-/-;Bmi1-/
15
10-
5-
0
b
45
40
35
* E2f6+/+;Bmil+/+
* E2f6+/-;Bmil +/+
30
* E2f6-/-;Bmi1+1+
* E2f6+/+;Bmil +/-
25
* E2f6+/-;Bmil +/* E2f6-/-;Bmil +/-
S.
20
E2f6+/+;Bmi1-/E2f6+/-;Bmi1-/-
15
* E2f6-/-;Bmil-/-
10
5 -
0 '
Figure 1. Weight distribution of 2-3 month old animals. a. Females and b. males of
all genotypes were weighed at 2-3 months of age. Blue dots represent animals that are
wild-type for Bmil; green dots, Bmil heterozygous animals; and red/orange dots, Bmil
knock-out animals (all irrespective of E2f6 genotype).
measure the severity of this phenotype by determining the age of onset of the ataxia or by
performing a Rotarod test where mice are tested for their ability to stay on a rotating rod.
Interestingly, we have observed that the ataxia development is correlated with the
body weight of the animals. Specifically, animals that had lower body weights
consistently developed ataxia whereas bigger mice did not (Figure 2). It is unclear if any
of these defects is a consequence of the other or if they merely indicate that some mutants
are more affected in all phenotypes.
45 -
40* Ataxic females
35 -
* Non-ataxic females
AAtaxic males
, Non-ataxic males
30
co 25
-CI
20
*
15
*
*
A0
A
A
10
5
0-
Ataxia
Figure 2. Ataxia and weight correlation for 2-3 month-old females and males.
Shown in red are the weights of animals that displayed the ataxic phenotype whereas blue
represents the weights of animals that did not develop ataxia. The animals included in
this chart are mice that are heterozygous or homozygous mutant for Bmil.
Axial skeletal transformations of E2f6-'-;Bmil-' mice
Bmil' mice display several morphological abnormalities along the anteroposterior axis
(van der Lugt et al., 1994). These changes indicate posterior transformations of vertebra
identity and consist of (1) an extra piece of bone rostral to the cervical vertebra C1, (2) a
CI to C2 conversion, (3) a C7 to TI (thoracic vertebra) conversion evidenced by the
presence of ribs at C7 which then fuse on the ventral side with the ribs associated with
T1, (4) a T7 to T8 conversion resulting in only six instead of seven vertebrosternal ribs,
(5) a T13 to Li (lumbar vertebra) conversion shown by the absence or degeneration of
ribs at T13 and (6) a L6 to SI (sacral vertebra) conversion evidenced by the joints
between L6 and the iliac bones. As expected, we also observed all of these
transformations, which are illustrated in Figure 3. E2f6-deficient mice exhibit the T13 to
Li and L6 to S I conversions with a penetrance of 67% and 80%, respectively (Chapter 2;
Storre et al., 2002). Data from both Bmil-'- and E2f6'mice indicate that the severity of
the defect is dependent on the gene dosage.
We have examined the axial skeletons of all genotypes generated from a double
heterozygous cross and noted a higher penetrance of all except one of the BmiT-'- defects
in mice doubly-deficient for E2f6 and Bmil (Table 2). Specifically, our results showed
no effect on the penetrance of the T7 to T8 conversion by additional deletion of E2f6.
For the remaining transformations, deletion of only one allele of E2f6 was enough to
increase the penetrance of each abnormality. Further deletion of the remaining E2f6
allele led to an even higher penetrance, at least in the Bmil]'
background, suggesting a
gene dosage effect. In the Bmil-/- background, deletion of both copies of E2f6 appears
to result in a lower penetrance of the certain transformations. However, only two animals
115
a
C1
C2
4
C7T1
b
T13
Ll
L6
S1
Figure 3. Posterior transformations of the axial skeletons of Bmil-deficient
mice. a. Lateral view of the cervical and upper thoracic region of wild-type (left) and
Bmil' (right) skeletons. b. Ventral view of the thoraco-lumbo-sacral region of wildtype (left) and Bmil' (right) skeletons. See text for explanation.
were analyzed for this genotype and the remaining data is consistent with a gene dosage
effect. Still, more animals should be analyzed to confirm this observation. Significantly,
the conversions of Ci to C2 and C7 to TI were detected in all of the E2f6'-;Bmi-'-and
E2f6'-;BmiL-'- mice analyzed. In contrast, the T13 to Li and L6 to Si conversions were
observed in 100% of E2f6*';Bmi1-'~ mice and only in 50% of E2f6'-;BmiP-'-mice
analyzed. These results suggest that E2F6 and Bmi1 act synergistically in the
anteroposterior segmentation of the axial skeleton.
Penetrance of skeletal abnormalities (%)
E2/6
Bmi/
WT HET KO WT HET KO XVT HET KO
WT WT WT HET HET HET KO KO KO
Extra
0
0
0
0
4
0
20
60
50
C1 toC2
0
0
0
0
4
0
60
100
100
C7 to TI
0
0
0
31
35
50
60
100
100
T7 to T8
0
0
0
0
4
0
20
20
0
T13 toL
0
9
67
39
58
50
20
100
50
L6 to S1
0
35
80
62
76
75
40
100
50
Total#
11
23
15
13
26
4
5
5
2
Table 2. Penetrance of axial skeletal transformations.
Cell cycle properties of double mutant MEFs
Bmil'-MEFs exhibit impaired proliferation and premature senescence due to an
up-regulation of p16 INK4Aand
pI9 ARF
protein levels (Jacobs et al., 1999a). In contrast,
117
E2f6'- MEFs display no defects in either proliferation or senescence (Chapter 2; Storre et
al., 2002). Since E2F6 and Bmil share a common and synergistic role in axial
patterning, we wished to determine if these proteins could also cooperate in cell cycle
control. To do so, we have compared wild-type, Bmil-'-, E2f6'- and E2f6'-;Bmi1-'-MEFs
in several cell cycle assays.
Our analyses of Bmil-'- MEFs revealed the same defects that had been previously
reported (Figures 4 and 6). In addition, we found that serum deprived Bmil-'- MEFs were
impaired in their ability to re-enter the cell cycle following the re-addition of serum
(Figure 5). The asynchronous proliferation rate of three out of four E2f6-'-;BmiP-' cell
lines were even lower than that of Bmil-'- MEFs (Figure 4). However, only one out of
four double mutant cell lines displayed a stronger impairment in S phase re-entry than the
Bmil single mutant (Figure 5). Finally, in a standard 3T3 protocol, two out of four E2f6';Bmil-'-cell lines exhibited an earlier senescence than observed in the Bmil-'- MEFs
(Figure 6). These results suggest that E2f6 deletion affects the cell cycle defects of Bmil
mutant MEFs. However, more cell lines need to be examined to corroborate these
findings.
To further investigate these observations, we have collected RNA samples from
the experiments described above for real-time PCR analysis of target gene expression.
These experiments are currently underway. Specifically, we wish to determine whether
an increase in p16INK4 Aand
pI9 ARF
levels correlates with the stronger impairment in
proliferation and cell cycle re-entry as well as the earlier senescence in the double mutant
MEFs. If so, this would argue in favor of a synergism between E2F6 and Bmi 1 in the
repression of the Ink4a/Arf locus. However, if no changes are observed in the levels of
118
p 1 6 INK 4 A and pI 9
ARF
between Bmil-'- and E2f6-'-;BmiL-'- MEFs, this would suggest that
E2F6 modulates the cell cycle in the Bmil-deficient background through a different
mechanism. Alternatively, E2F6 and Bmil could be cooperating without synergizing in
the repression of the Ink4a/Arf locus. In fact, this result would be expected if these
proteins were present in one complex that regulates the expression of p16 INK 4 Aand
pI9 ARF
119
a
700000
--
E216+/+;BmiI1++
--- E2f6-/-;Bmil +/+
600000
-
A-
--
E2f6-/-;Bmi1-/-E2f6+/+;Bml1-/-
-
E2f6-/-;Bmil-/-
500000
400000
E
300000
-
200000
100000
0
0
1
2
4
5
Time (days)
3
6
7
8
9
b
800000-
700000
-I-
-EMf-/-;Smil
600000
-
-l-
-o-E2f6+/+;Bmi1+/+
E2f6+/+;8miI-/-
500000E
2 400000-
300000
-
200000-
100000
-
0
0
1
2
3
4
Days
5
6
7
8
Figure 4. Proliferation of asynchronously growing MEFs. a. and b. Two separate
experiments using different cell lines of the following genotypes: wild-type (blue), E2f6'
(green), Bmil-'-(green) and E2f6'-;BmiL' (red).
a
160000---
E2f6+/+;Bmil+/+
-- a-E2f6-/-;Bml1+/+
140000
-*-- E2f6+/+;Bmil-/-a-E2f6+/-;Bmi1-/-
120000-
- -E2f6-/-;Bmi1-/-
o
100000-
80000-
E
60000-
40000-
20000
0
5
0
10
15
20
25
30
25
30
Time after serum release (hours)
b
120000E2f6-/-;Bmi1-/E2f6+/+;Bmi1-/--- E2f6-/-;Bmi1-/-E2f6+/+;Bmil+/+
*-E2f6+/+;Bmil-/-100000
80000
60000E
40000-
20000-
0
0
5
20
15
10
Time after serum release (h)
Figure 5. Cell cycle re-entry of MEFs. a. and b. Two separate experiments using
different cell lines of the following genotypes: wild-type (blue), E2f6'-(green), Bmil
(green) and E2f6'~;Bmi1' (red).
a
14-
12 -
----
E2f6+/+;BmiI+1+
---
E2f6-/-;Bmi1 +/+
E2f6+/-;BmiI-/-
10
E2f6-/-;Bmil-/-
C
6-
4-
2-
0
4
5
6
7
8
9
Passage Number
10
11
12
13
b
14
-&-
12
-+-
10
E2f6-/-;Bmil/E2f6+/+;BmIi-1E2f6-/-;Bmil1-E2f6+/+;BmiI +/+
-a- E2f6+/+;Bmil1--
C0
8
4-
2
4
6
8
7
Passage number
9
10
11
Figure 6. Senescence properties of MEFs. a. and b. Two separate experiments using
different cell lines of the following genotypes: wild-type (blue), E2f6' (green), Bmil'
(orange) and E2f6-';BmiL-'~ (red).
DISCUSSION
E2F6 has been previously shown to play a role in PcG-mediated axial patterning
during murine development (Chapter 2; Storre et al., 2002). Although data from
overexpression as well as promoter occupancy studies implicated E2F6 in the regulation
of the cell cycle (Cartwright et al., 1998; Gaubatz et al., 1998; Giangrande et al., 2004;
Ogawa et al., 2002), the examination of the proliferation and senescence properties of
E2f6-deficient MEFs suggests otherwise (Chapter 2; Storre et al., 2002).
Bmi 1 deficiency leads to posterior axial skeletal transformations, reduced
newborn survival, ataxic gait and hematopoietic defects in the mouse (van der Lugt et al.,
1994). Furthermore, the Bmil-' MEFs exhibit impaired proliferation and premature
sensescence due to up-regulation of p 1 6 INK 4 Aand
pI9 ARF
(Jacobs et al., 1999a). This
suggests that Bmi 1 normally represses the transcription of these cell cycle inhibitors.
However, very few PcG proteins are able to directly bind DNA, including Bmil (Brown
et al., 1998; Kanno et al., 1995). Since E2F6 is a transcription factor that associates with
DNA at E2F sites (Cartwright et al., 1998; Gaubatz et al., 1998; Morkel et al., 1997;
Trimarchi et al., 1998) and is associated with Bmil, it is possible that BmiI is recruited to
E2F sites in the p19ARF promoter (Bates et al., 1998) through the action of E2F6.
The purpose of this study was to test whether E2F6 functions in cooperation with
Bmil in cell cycle control as well as in axial skeletal patterning. To do so, we have
generated E2f6;Bmil compound mutants. In both flies and mammals, the deletion of two
PcG proteins leads to stronger axial skeletal transformations suggesting a synergistic
effect (Akasaka et al., 2001; Bel et al., 1998). Indeed, we observe a higher penetrance of
these defects as the E2f6 dosage decreases in the Bmi 1-deficient background. Moreover,
123
our preliminary analyses of the double mutant cells indicate that E2F6 may have a role in
PcG function that is not restricted to axial patterning.
Viability and lifespan of compound mutant mice
The E2f6';Bmi-' mice arise at lower frequencies and seem to be smaller than
Bmil single mutants although the analysis of a larger number of mutants would be
necessary to reach statistical significance. Similarly, the M33-'-;Bmil' and Mel-18'
;Bmil' mice also arise at lower frequencies than the Bmil-/- mice (Akasaka et al., 2001;
Bel et al., 1998). Bmil-/- mice are known to be born at expected frequencies and die
perinatally (van der Lugt et al., 1994). Our data is consistent with a similar trend
although we did not find 100% of expected E2f6;BmiP' embryos at E18.5. This could
mean that the E2f6';BmiP' mice die before birth suggesting an even stronger phenotype.
It would be helpful to look at earlier time points in development to determine if, in fact,
the E2f61;BmiT' embryos have reduced viability. Alternatively, our numbers may be too
small and if we increase them, we might discover that E2f61,;BmiU' mice are born at the
expected Mendelian frequency as well.
We have not observed a significant difference in the lifespan of the E2f6';BmiJ'
and Bmil' mice that reach adulthood, indicating that loss of E2F6 does not alter the
survival potential of the Bmil-/- animals. Furthermore, the double mutant animals
exhibit poor health, have lower body weights and develop an ataxic gait. Eventually,
they become severely anemic and need to euthanized. Histological analysis of these
animals reveals no other defects except for decreased hematopoiesis in the bone marrow
and atrophic spleen.
124
Neurological abnormalities of E2f6~';Bmil-'- mice
The Bmil-'- mice have been described to develop ataxia between 2-4 weeks of age
due to a decreased cellularity of all the layers in the cerebellum, which is the major motor
coordination center (van der Lugt et al., 1994). The ataxic phenotype appears to be more
penetrant in E2f6;BmiJi' mice. At the moment, we have not been able to determine if
the severity of this phenotype is also increased. We are in the process of analyzing
histological sections of the cerebellum to determine if the E2f6--;Bmi1i' mice display a
more severe defect. In addition, we plan to determine the age of onset of the ataxic gait
in an attempt to quantify the severity of the defect.
Posterior transformations of E2f6;Bmil double mutant mice
Bmil' mice exhibit six different transformations along the entire anteriorposterior axis (van der Lugt et al., 1994). In contrast, E2f6' mice display defects
restricted to the thoraco-lumbo-sacral region indicating that E2F6 controls the expression
of no more than a subset of Hox genes. In this study, we have shown that reducing the
E2F6 dosage in the Bmil-deficient background leads to a progressively higher
penetrance of five out of the six transformations observed in Bmil' mice. This finding
strongly supports a model in which E2F6, like M33 and Mel-18, synergizes with Bmil in
a dose-dependent manner in axial patterning. However, unlike the M33-';Bmi-' and
Mel-18b;Bmil/ studies, we have not detected any novel transformations in the E2f6b
;Bmil' mice. This result further supports the hypothesis that E2F6 plays a limited role in
the PcG-mediated anteroposterior segmentation during embryogenesis. Since E2F6 is
125
found in association with several PcG proteins, it would be interesting to determine
whether this protein synergizes with other members of the PcG complex.
Proliferation and senescence of E2f6-'-;Bmil'- MEFs
E2F6 appears to be dispensable for normal proliferation and senescence given that
E2f6'- MEFs are indistinguishable from their wild-type littermates (Chapter 2; Storre et
al., 2002). Although previous data suggested that E2F6 may repress the transcription of
p19ARF (J.M.Trimarchi and J.A. Lees, unpublished results), we do not observe a
deregulation of this protein in E2f6'- MEFs by western blot analysis. Together, these
findings suggest that E2F6 does not participate in the Bmil-mediated repression of
p1 9 ARF. Alternatively, other factors could compensate for the absence of E2F6. Our
working model indicates that E2F6 participates in Polycomb function through its ability
to target PcG complexes to appropriate target genes. Thus, a factor that replaces E2F6 in
the recruitment of PcG proteins to target genes has to have similar DNA binding
properties making other E2Fs the most likely candidates.
Indeed, E2F4 has been shown to associate with GI/S gene promoters during S
phase only in the absence of E2F6 (Giangrande et al., 2004). In agreement with this
finding, these genes were deregulated exclusively in E2f6'- MEFs in which E2F4 had
been knocked-down by RNAi. Moreover, E2F3b has been found at the p1 9 ARF promoter
and has been implicated in its transcriptional inhibition (Aslanian et al., 2004). We had
originally reported that E2F6 was the only E2F capable of RYBP association (Trimarchi
et al., 2001). However, another study indicated that E2F2 and E2F3, but not the other
E2Fs including E2F6, associate with this PcG protein (Schlisio et al., 2002). These
126
discrepancies may be due to subtle differences in the experimental protocols or could
reflect the well-established diverse compositions of the PcG complexes in different cell
types and conditions. Indeed, we have now been able to detect RYBP association with
E2F1-6 (P.J.Iaquinta and J.A.Lees, unpublished results) in agreement with the knowledge
that it is the well-conserved Marked box domain of E2F that mediates the direct
interaction with RYBP (Trimarchi et al., 2001).
We analyzed four different E2f6'-;BmiP-'-MEF cell lines for their properties in
asynchronous proliferation, cell cycle re-entry and senescence. First, three double mutant
cell lines displayed a stronger proliferation impairment than the Bmi] single mutant
MEFs. Second, only one E2f6-'-;Bmi1-'- MEF cell line exhibited a more severe cell cycle
re-entry defect. Third, two compound mutant MEF cell lines underwent senescence even
earlier than the Bmil single mutant. Although more cell lines need to be analyzed, these
results support that E2F6 participates in regulating the cell cycle in cooperation with
Bmi 1. It is a distinct possibility that these proteins work together in the repression of the
Ink4a/Arf locus. Therefore, we are in the process of examining the expression levels of
p16INK4 and pL9ARF by real-time PCR in the E2f6;BmiJ-'-MEFs.
The results described in this chapter indicate that E2F6 and Bmil act
synergistically in the anteroposterior patterning of the developing embryo. Futhermore,
they are consistent with our model that E2F6 recruits the PcG complex to a subset of
target genes. Moreover, although more experiments are needed, our work raises the
possibility that E2F6 may have a role in the control of proliferation and tumorigenesis.
127
EXPERIMENTAL PROCEDURES
Histological and skeletal analysis
Dead mice from our aging colonies were dissected and processed for histological
analysis. Soft tissues were fixed in 10% formalin and hard tissues were fixed in Bouin's
fixative. Paraffin sections were prepared and stained with hematoxylin and eosin.
Skeletal analysis was performed on 3-day old mice. After removing the skin and viscera,
the skeletons were fixed in acetone and stained with cartilage-specific Alcian Blue and
bone-specific Alzarin Red. Soft tissue was cleared with KOH.
Mouse embryonic fibroblasts
MEFs were prepared from 13.5 d.p.c embryos as previously described (Humbert
et al., 2000) and genotyped by PCR of DNA obtained from yolk sacs. Proliferation
curves were obtained by plating 2x10 4 MEFs in triplicate in 24-well plates. At the
indicated time points, MEFs were trypsinized and counted. For cell cycle re-entry assays,
2x10 5 MEFs were plated in triplicate in 6-well plates. After 2 days of growth in media
containing 10% serum, they were incubated in media containing 0.1% serum for 3-4
days. Re-entry into the cell cycle was induced by incubation in media containing 10%
serum and at the indicated time points, 5
tCi
of 3 H-thymidine was added to the cells for 1
hour. Cells were then scraped from the plates and cell pellets were analyzed for 3 Hthymidine incorporation using a scintillation counter. A 3T3 protocol was followed to
monitor senescence. 3x10 5 MEFs were plated in duplicates in 6-cm plates and re-fed 2
days later. On the third day, they were trypsinized, counted and replated. The fold
replication was determined by dividing the number of cells obtained at day 3 by 3x10 5.
128
Western blots were performed as described previously (Moberg et al., 1996) with 50-100
tg of whole cell lysates.
129
ACKNOWLEDGEMENTS
We are grateful to Marteen van Lohuizen for the generous gift of the Bmil-'- mice. We
also thank Alicia Caron and Roderick Bronson for the generation and analysis of
histological sections and Mark Garcia for help with colony maintenance and dissections.
We would also like to thank the members of the Lees lab for reagents and helpful
discussions.
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135
ChapterFour
Conclusions
Despite the in vitro evidence that suggests a role for E2F6 in cell cycle control as
well as its interaction with the PcG complex, the biological significance of this protein
had not been analyzed in vivo. This work unequivocally shows that E2F6 is a bonafide
member of the PcG complex that functions in the anteroposterior segmentation of the
developing embryo. Furthermore, our results allude to a broader role for E2F6 in PcG
that extends to control of cell proliferation. In chapter two, we have established that loss
of E2F6 results in posterior transformations of the axial skeleton that are reminiscent of
defects seen in other PcG mutants providing genetic evidence of a PcG function for
E2F6. However, our study of E2f6-deficient MEFs did not reveal a clear role for E2F6 in
cell cycle control. In chapter three, we further examined the axial skeletal
transformations of E2f6-'-;Bmil-'-mice and found a clear gene dosage effect. In addition,
our preliminary evidence suggests that E2F6 may participate in the control of cell
proliferation in MEFs and cerebellar neurons by Bmi 1. Although it would be expected
that the E2f6'- mice and MEFs would also display these proliferative defects albeit with
decreased severity, we cannot rule out the possibility that the E2F6 plays a minor role in
the cell cycle and therefore, ablation of this gene alone has no effect. If that was the case,
it is possible that in the presence of another mutation, the effects of E2f6-deficiency
become apparent. Taken together, our data show that E2F6 is an important effector of
PcG function.
E2F6 in axial patterning
The identification of PcG proteins as E2F6 interactors led to the hypothesis that
E2F6 may play a role in the anterior-posterior patterning of the developing embryo
137
(Attwooll et al., 2004; Ogawa et al., 2002; Trimarchi et al., 2001). The transcriptional
repressive property of E2F6 is compatible with a role in the PcG complex, which
negatively regulates Hox genes expression (Gaubatz et al., 1998; Kennison, 1995; Morkel
et al., 1997; Trimarchi et al., 1998). However, in order to prove that a protein is a bona
fide member of the PcG complex, it is essential to demonstrate that it is a functional
component of this complex. In order to do this, we and others have generated E2f6deficient mice and analyzed their axial skeletons (Chapter 2; Storre et al., 2002). These
mice display two types of posterior transformations of the axial skeleton with dosedependent penetrance. First, the thoracic vertebra T13 is transformed into the lumbar
vertebra Li as evidenced by the absence or degeneration of the ribs normally associated
with T13. Second, the lumbar vertebra L6 is transformed into the sacral vertebra SI as
indicated by the presence of sacral-iliac joints. These identical defects are present among
several transformations along the entire axial skeleton of in Bmil-'- mice suggesting the
E2F6 and BmiI may function together at least in some regions (van der Lugt et al.,
1994). Significantly, similar results have been reported by an independent study (Storre
et al., 2002).
The T13-to-LI transformation is found in 9 %of E2f6/- mice and in 67% of
E2f6'- mice whereas the L6-to-S 1 transformation is found in 35% of E2f6*'- mice and in
80% of E2f6'l mice. These observations indicate a clear gene dosage effect and imply
that E2F6 loss has a more profound effect in the more posterior regions of the axial
skeleton. It is possible that E2F6 has a more prominent role in the repression of those
Hox genes that specify the identities of the more posterior vertebra. The analysis of the
expression patterns of Hox genes during embryogenesis in E2f6-' mice would provide
138
further insight into the role of E2F6 in axial patterning. Of particular interest would be
the HoxlO and Hox9 paralog groups, which are known to have their anterior expression
boundaries at the lumbosacral and thoracic region respectively (Burke et al., 1995).
Another good candidate would be Hoxc8 since its deletion, leads to anterior
transformation of the lumbar vertebra Li into a thoracic vertebra T13 (Le Mouellic et al.,
1992). Interestingly, the expression of Hoxc8 is among the affected ones in Bmi-' mice
suggesting that lack of Hoxc8 activity leads to anteriorization of Li to T13, whereas
ectopic expression of Hoxc8 results in the posteriorization of T13 to LI (van der Lugt et
al., 1996). Based on these observations, it would be reasonable to speculate that E2F6
only participates in the repression of specific Hox genes that determine the identity of the
more caudal structures.
The fact that the transformations observed in the E2f6- mice are also found in the
Bmil' animals implies that these proteins may act in synergism in the thoracic/lumbar
and lumbar/sacral transitions. In agreement with this model, we have shown in chapter
three that the posterior transformations observed in the Bmil mutants are found with a
higher penetrance as the E2f6 gene dosage decreases. Specifically, we have shown that
five out of six defects are enhanced in the compound mutants. For example, the
penetrance of the C7-to-T 1 transformation went up from 31% in Bmil'- to 50% in E2f6'
;Bmil'-and from 60% in Bmil' to 100% in E2f6'-;Bmi'1. These results strongly
support a synergistic and dose-dependent genetic interaction between E2F6 and Bmi .
139
E2F6 in viability and survival
E2f6-deficient mice are viable, survive into adulthood and display no gross
morphological defects, growth abnormalities or fertility problems. Moreover, their
lifespan is not significantly different from that of their littermate controls even when
treated with y-irradiation at 6 days of age. In contrast, whereas E2f6'-;Bmil-'-mice are
underrepresented at E18.5 and 3 weeks of age, the Bmil~'- animals are underrepresented
only at 3 weeks of age. This result argues in favor of a synergistic relationship between
E2F6 and Bmil in viability and survival. However, it is important to note that our
numbers are still relatively small and may not reflect statistically significant results (250
animals were genotyped at 3 weeks of age whereas 105 embryos were analyzed at
E18.5). We are currently attempting to increase the total number of mice examined.
In chapter two, we have analyzed by histology all the deceased mice from our
aging colonies in an attempt to determine the cause of death of the E2f6-' mice. The
examination of these animals revealed a few interesting observations although further
studies are required to determine their significance.
Among the causes of death of the E2f6*/ and E2f6-'- aging mice, liver tumors and
several blood tumors were the most prominent suggesting a potential tumor suppressor
role for E2F6. However, the tumors observed in E2f6 mutant mice may be merely the
result of old age since the mice that developed these tumors were over one year of age.
In fact, based on the biochemical and cell cycle data, it is more likely that E2F6 behaves
as an oncogene through the repression of important negative regulators of the cell cycle.
For example, it has recently been shown that E2F6 is found at the promoter of the tumor
suppressor BRCA1 gene (Oberley et al., 2003). Furthermore, the association and
140
synergism of E2F6 with the Bmil oncogene suggests that it may function in an analogous
manner by repressing the Ink4a/Arf locus (Jacobs et al., 1999). The discovery of E2F
target sites in the p1 9 ARF promoter supports this hypothesis and led to the proposition that
E2F6 is responsible for Bmil recruitment to p1 9ARF promoters (Bates et al., 1998). If this
were the case, we would expect p1 9 ARF levels to be similarly up-regulated in Bmil-'-, E2f6'- and E2f6-'-;BmiP-'-MEFs since removal of one or the other should be enough to abrogate
the repression of this gene. However, although overexpression of E2F6 in MEFs leads to
down-regulation of p 1 9 ARF, E2f6'- MEFs do not exhibit increased levels of this cell cycle
inhibitor (J.M.Trimarchi and J.A.Lees, unpublished results; Chapter 2). We are currently
in the process of determining the p19ARF levels in E2f6';Bmi1-'-MEFs. Additionally,
overexpression studies would address the role of E2F6 in oncogenesis. First, if E2F6
functions in a similar manner to Bmil, overexpression of E2F6 and other known
oncogenes should result in transformation of primary cells (Cohen et al., 1996; Jacobs et
al., 1999). Second, if Bmil requires E2F6 to bind the p19ARF promoter, it would be
expected that overexpression of Bmil would not be able to transform E2f6'- MEFs.
Third, transgenic mice that overexpress E2f6 should cooperate with other oncogenes in
tumor formation (Alkema et al., 1997; Haupt et al., 1991; Haupt et al., 1993; van
Lohuizen et al., 1991b). Finally, E2f6'-;Ep-Bmi] mice should not cooperate with E1 tmyc mice in lymphomagenesis.
Another interesting finding is that three out of five mice analyzed had developed
odontoma. These benign tumors of the tooth germs arise in osteopetrotic mice as a
consequence of defects in osteoclast function or differentiation (Amling et al., 2000; IdaYonemochi et al., 2002; Ida-Yonemochi and Saku, 2002; Tiffee et al., 1999). Defects in
141
bone remodeling during tooth development results in morphological abnormalities that
lead to the formation of odontomas. Significantly, Hoxc8 represses the transcription of
two proteins produced by osteoblasts, Osteoprotegerin (OPG) and Osteopontin (OPN).
OPG is a secreted decoy receptor that binds to osteoclast differentiation factor and
inhibits osteoclast maturation (Wan et al., 2001) whereas OPN facilitates bone resorption
by decreasing bone mineralization (Lei et al., 2005). It is reasonable to speculate that in
the absence of the appropriate PcG proteins, in this case E2F6, deregulated expression of
Hoxc8 inappropriately inhibits OPG and OPN expression resulting in an impaired bone
resorption. Therefore, it would be very interesting to study the function and
differentiation of osteoclasts isolated from E2f6-'- mice as well as to determine the
expression levels of OPG and OPN in E2f6 mutant osteoblasts.
A low percentage of E2f6-deficient mice exhibited testicular atrophy, a common
age-related defect. Although the mice that displayed testicular atrophy were at least 500
days old, it is interesting to note that another group reported a reduction of
spermatogenesis in E2f6' mice (Storre et al., 2002). Surprisingly, our colony of E2f6'
mice exhibits no fertility problems.
Finally, we have noticed that two E2f6' mice as well as one E2f67;BmiU'-mouse
and one E2f6' ;BmiL' mouse have only a single kidney. It is not clear if this is a result of
failure to develop a second kidney or of the degeneration of an existing kidney. To
address this question, a timed analysis of mice of different ages should be performed but
given the low penetrance of this phenotype this would require the generation of a large
number of animals. The phenotype in the kidney establishes another connection between
E2F6 and the PcG complex. In fact, the Hox genes have been implicated in kidney
142
development. The Hoxi1 paralog group (HoxAl1, HoxCi1 and HoxD11) are involved
in the induction of ureter budding from the nephric duct (Wellik et al., 2002). In additon,
mutant mice for HoxalO and Hoxd]O display altered kidney placement and size (Lin and
Carpenter, 2003). In fact, the expression of 37 Hox genes was analyzed in the developing
kidney at E12.5, E13.5 and E17.5 (Patterson and Potter, 2003; Patterson and Potter,
2004). Multiple Hox genes were found to be expressed in kidney and the colinearity of
expression boundaries and the location of the genes in the clusters was conserved. It
appears that these Hox genes regulate the expression of renal morphogens. Therefore, it
would be interesting to study the expression levels of these Hox genes in E2f6' kidneys.
E2F6 in the control of cell proliferation
Several observations indicate that E2F6 may function in the cell cycle control.
First, E2F6 is a member of the E2F transcription factor family, which have been known
to play an important role in the control of cell proliferation (Trimarchi and Lees, 2002).
Second, the cell cycle-dependent expression of E2F6 probably reflects a differential
requirement for E2F6 function at different stages of the cell cycle (Dahme et al., 2002;
Kherrouche et al., 2001; J.M.Trimarchi and J.A.Lees, unpublished results). Third,
overexpression studies have implicated E2F6 in the control of S phase entry (Cartwright
et al., 1998; Gaubatz et al., 1998). Fourth, E2F6 overexpression in MEFs results in
down-regulation of p19ARF , an important cell cycle inhibitor. Finally, E2F6 has been
found at the promoters of E2F target genes that are activated during GUS (Giangrande et
al., 2004).
143
In contrast to these observations, E2f6-'- MEFs exhibit no proliferation, senescence
or target gene expression abnormalities arguing against a role for E2F6 in cell cycle
control (Chapter 2; Storre et al., 2002). However, it is possible that E2F6 may participate
in the control of cell proliferation yet the E2f6' MEFs display no phenotypes. In fact,
E2f-'- and E2f4-'- MEFs also present normal cell cycle properties despite their known role
in cell cycle regulation (Humbert et al., 2000a; Humbert et al., 2000b).
One possibility is that compensation by other E2F family members may occur. In
support of this model, E2F4 appears to compensate E2f6 deficiency in the regulation of
GUS genes repression during S phase (Giangrande et al., 2004). E2F4 was found at the
promoters of Gu/S-activated genes during S phase in MEFs lacking E2F6 whereas in
wild-type MEFs E2F4 is found in association with DNA exclusively during GO
(Takahashi et al., 2000). Another potential candidate for E2F6 loss compensation is
E2F3b, which has recently been implicated in the regulation of the p19ARF gene
expression (Aslanian et al., 2004). This could explain why p19ARF is not up-regulated in
E2f6'- MEFs. Interestingly, like E2F6, E2F3 has also been shown to associate with the
PcG protein RYBP suggesting that it may participate in the recruitment of the PcG
complex to target gene promoters (Schlisio et al., 2002). It would therefore be interesting
to determine the cell cycle properties of MEFs doubly deficient for E2f6 and E2f4 or E2f6
and E2f3b.
Another explanation for the lack of detectable phenotypes in E2f6' MEFs is that
E2F6 plays a minor role in this cell type and therefore its loss does not have an effect.
However, it is possible that by combining the E2f6 and Bmil mutations, the Bmil-'- MEFs
phenotypes are exacerbated. This would be reminiscent of the effects that E2F6 loss in
144
the Bmil mutant background has on axial patterning. In this case, while the analysis of
E2f6' mice has indicated that E2F6 participates in the establishment of the correct
identities of only the more posterior vertebrae, when E2f6/Bmil compound mutant mice
were analyzed, the role of E2F6 in axial patterning was extended to the entire axial
skeleton. This suggests that certain roles of E2F6 cannot be uncovered by deletion of
E2f6 alone. Similarly, we could speculate that the analysis of E2f6;Bmil' MEFs could
reveal a function for E2F6 in the cell cycle. Our preliminary analysis of four such MEF
lines suggests that the cell cycle defects observed in Bmil' MEFs are exacerbated by
deletion of E2f6. We are currently in the process of studying the expression levels of
several E2F-target genes including pI 9 ARF to further understand how E2F6 participates in
the control of cell proliferation.
The role of Bmil in the cell cycle through the regulation of
1 6 NK4A
and
pI 9
ARF
levels appears to be the cause for the cerebellar and hematopoietic defects found in
Bmil' mice. In recent years, it has become apparent that Bmi 1 is required for the selfrenewal and proliferation of both neural and hematopoietic stem cells (NSCs and HSCs)
(Lessard and Sauvageau, 2003; Molofsky et al., 2003; Park et al., 2003; Zencak et al.,
2005). It has also been shown that Bmil plays a crucial role in the maintenance and
expansion of immature granule cell precursors (Leung et al., 2004).
In the cerebellum, the proliferative defects in both NSCs and the more committed
cerebellar precursor cells lead to a progressive loss of cerebellar neurons in Bmi] mutant
mice that results in the development of ataxia (Leung et al., 2004; Molofsky et al., 2003;
van der Lugt et al., 1994). In addition, as a consequence of the HSCs and progenitor cells
self-renewal defects, Bmil' mice exhibit several hematopoietic abnormalities which
145
affect the more immature compartments more severely (Lessard and Sauvageau, 2003;
Park et al., 2003; van der Lugt et al., 1994). Significantly, Ink4a/Arf locus deletion
partially rescues both the cerebellar and hematopoietic defectsdefects of Bmil' mice
(Jacobs et al., 1999). It is important to note that while the proliferative defects of NSCs
and cerebellar precursors could explain the cerebellar defects and ataxic phenotype of
Bmil' mice, other possibilities are also worth investigating. It has been shown that the
mutation of certain Hox genes lead to problems in motor neurons which could also lead
to motor coordination defects (Lin and Carpenter, 2003).
The E2f6;Bmil compound mutant mice develop ataxic gait with a modestly
higher penetrance than the Bmil mutant mice (70% versus 60%). However, we have not
quantified yet the level of ataxia. This could be done in at least three ways. First, we
could measure the average time each mutant is able to stay on a rotating rod (Rotarod
test). Second, we could determine the latency period for the onset of the ataxia for each
mutant. Third, we could determine the extent of the cerebellum defect by histological
analysis. We are currently pursuing the last two options.
We are also in the process of examining the hematopoietic defects of E2f6';BmiTmice. Like the Bmil-'- mice, the double mutants that survive into adulthood are
significantly smaller than their wild-type littermates and exhibit poor health throughout
their lives. Eventually, they become very anemic and need to be euthanized. We are
performing FACS analysis on the peripheral blood, bone marrow, spleen and thymus of
these animals to determine if the hematopoietic defects observed in the Bmil-'- mice are
exacerbated by additional loss of E2F6.
146
It woud be interesting to study the self-renewal capacity of E2f6';BmiL' HSCs
and NSCs. However, it is important to note that the defects observed in Bmil' mice are
so profound that it might prove hard to detect a significant exacerbation of such a strong
phenotype.
Mode of action of E2F6
Our working model proposes that E2F6 is responsible for recruiting PcG
complexes to a subset of target genes. Only a few PcG proteins have been shown to bind
DNA in a sequence-specific manner suggesting that the recruitment could be mediated by
additional factors (Brown et al., 1998; Dejardin et al., 2005; Mohd-Sarip et al., 2002;
Mulholland et al., 2003; Poux et al., 2001; Srinivasan and Atchison, 2004). However,
most of this work has been performed in Drosophilawhere the discovery of PcG
responsive elements (PREs) has allowed the study of PcG-DNA binding. PREs are large
and complex regulatory elements that are able to repress reporter genes in a PcGdependent manner (Chan et al., 1994; Christen and Bienz, 1994; Simon et al., 1993)
To confirm our model, it would be important to establish that E2F6 1, regulates
the expression of some PcG target genes; 2, directly binds the promoters of those genes;
and 3, is required for PcG recruitment to DNA and repression.
First, it would be interesting to examine the Hox gene expression patterns during
the development of E2f6' embryos. A few good candidates are those Hox genes known
to be deregulated in Bmil' mice. These include Hoxa4, Hoxc4, Hoxa5, Hoxc5, Hoxb6,
Hoxc6, Hoxc8 and Hoxc9 (Brunk et al., 1991; van der Lugt et al., 1996; van der Lugt et
al., 1994; van Lohuizen et al., 1991a; van Lohuizen et al., 1991b). Other good candidates
147
are the Hox paralog families Hoxl0 and Hox1l, which regulate the identity of the
vertebrae in the lumbar and sacral regions of the axial skeleton which are affected in the
E2f6' mice (Burke et al., 1995). In addition, although we do not observe an upregulation of
pI 9ARF
in E2f6' MEFs, it would be interesting to analyze the p1 9 ARF levels
in E2f6';BmiL' MEFs. A more unbiased approach to identify E2F6 target genes would
be to do microarrays using E2f6' MEFs. In fact, such an analysis has been recently
reported and this study shows that E2F6 represses the transcription of a-tubulin 3 and CCtubulin 7 (TUBA3 and TUBA7) in all organs except in the male gonads (Pohlers et al.,
2005). Significantly, no deregulation of E2F target genes was observed in E2f6' MEFs
in agreement with previous data (chapter 2; Giangrande et al., 2004).
Second, we would like to determine if E2F6 is found at Hox genes as well as the
p19ARF promoters. So far, we have not been able to consistently detect E2F6 at any of the
promoters tested and the lack of a known E2F6 target gene to use as a positive control
has made it impossible to interpret these results (P.J.Iaquinta and J.A.Lees, unpublished
results). Recent studies have shown that E2F6 binds and regulates the expression of
certain tumor suppressor as well as GI/S-activated E2F target genes (Giangrande et al.,
2004; Oberley et al., 2003). Therefore, it would now be possible to use these known
E2F6 target genes as positive controls. Due to the little information available on Hox
gene regulatory elements in mammals, it is impossible to examine E2F6's binding to
these promoters at the present time.
Finally, it is important to establish E2F6's role in the recruitment of the PcG
proteins to target genes. If we could detect Bmi 1 and E2F6 at the pI 9 ARF promoter, we
could test this in a very simple manner as we should not detect Bmil at this promoter in
148
E2f6' MEFs. An alternative in vitro approach has been used in Drosophila(Mulholland
et al., 2003). A recruitment assay was developed using the promoter sequences of the
Ubx Hox gene to demonstrate that the GAGA factor, which binds to several GAGA
sequences in the Ubx promoter, can recruit the PcG complex to DNA. This DNA was
assembled into chromatin and pre-incubated with either buffer alone or GAGA factor
prior to the addition of the purified PRC1 core complex. Subsequent Western blot
analysis revealed an increase in the PRC1 binding in the presence of GAGA factor. A
similar experiment could be envisaged by using E2F binding sites to recruit E2F6 to
DNA. One caveat to this experiment is that we would need to purify the mammalian
PRC 1 complex and demonstrate that it can associate with E2F6.
Taken as a whole, our studies have demonstrated a clear role for E2F6 in PcG
function in the anterior-posterior patterning of the developing embryo. The part that
E2F6 plays in cell cycle control remains unresolved although preliminary evidence
supports its participation in the proliferation control by Bmi 1. Therefore, this work has
uncovered at least one biological function of E2F6 and, at the same time, has opened the
field to a number of interesting questions.
149
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