The atypical E2F family member E2F7
couples the p53 and RB pathways during
Ozlem Aksoy,1,2,3,6Agustin Chicas,1,3,6Tianying Zeng,4Zhen Zhao,1,3Mila McCurrach,3
Xiaowo Wang,4and Scott W. Lowe1,3,5,7
1Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA;2Watson School of Biological Sciences,3Cold
Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA;4MOE Key Laboratory of Bioinformatics, Bioinformatics
Division, TNLIST, Department of Automation, Tsinghua University, Beijing 100084, China;5Howard Hughes Medical Institute,
New York, New York 10065, USA
Oncogene-induced senescence is an anti-proliferative stress response program that acts as a fail-safe mechanism to
limit oncogenic transformation and is regulated by the retinoblastoma protein (RB) and p53 tumor suppressor
pathways. We identify the atypical E2F family member E2F7 as the only E2F transcription factor potently up-
regulated during oncogene-induced senescence, a setting where it acts in response to p53 as a direct transcriptional
target. Once induced, E2F7 binds and represses a series of E2F target genes and cooperates with RB to efficiently
promote cell cycle arrest and limit oncogenic transformation. Disruption of RB triggers a further increase in E2F7,
which induces a second cell cycle checkpoint that prevents unconstrained cell division despite aberrant DNA
replication. Mechanistically, E2F7 compensates for the loss of RB in repressing mitotic E2F target genes. Together,
our results identify a causal role for E2F7 in cellular senescence and uncover a novel link between the RB and p53
[Keywords: p53 target; mitosis; checkpoint; compensation; RB; E2F7]
Supplemental material is available for this article.
Received May 14, 2012; revised version accepted June 11, 2012.
Oncogene-induced senescence is a stable cell cycle arrest
program that limits the proliferation of cells that have
sustained oncogenic mutations, and as such provides an
important barrier to tumorigenesis (Mooi and Peeper 2006;
Campisi and d’Adda di Fagagna 2007). Initially described in
fibroblasts expressing oncogenic Ras (Serrano et al. 1997),
oncogene-induced senescence can be triggered by a variety
of oncogenic signals and appears to limit the proliferation
of developing tumor cells. Indeed, senescent cells have
been identified in a variety of premalignant human tu-
mors (Braig et al. 2005; Chen et al. 2005; Collado et al.
2005; Michaloglou et al. 2005), and mutations in genes
required for the successful execution of senescence are
frequently associated with tumor progression. Like other
companied by unique changes in cellular morphology and
physiology that are associated with increased expression
of senescence-associated b-galactosidase (SA-b-gal), as
well as dramatic changes in gene expression associated
with an overall repressive chromatin environment (Narita
et al. 2003). In addition to the cell cycle arrest program,
senescent cells secrete factors that modulate their micro-
environment and trigger immune surveillance, and these
processes may contribute to the tumor-suppressive prop-
erties of the program in a non-cell-autonomous manner
(Campisi and d’Adda di Fagagna 2007; Coppe et al. 2008;
Kuilman et al. 2008).
The p53 and retinoblastoma protein (RB) tumor sup-
pressor pathways control the cell-autonomous compo-
nents of the oncogene-induced senescence program, and
disruption of these pathways alone or in combination is
sufficient to bypass senescence and promote oncogenic
transformation (Shay et al. 1991; Voorhoeve and Agami
2003; Chicas et al. 2010). p53 is a sequence-specific DNA-
binding transcription factor that promotes senescence in
part through its ability to induce the cyclin-dependent
kinase (CDK) inhibitor p21CDKN1Aand the anti-prolifer-
ative microRNA miR-34 (Pantoja and Serrano 1999; He
et al. 2007; Chicas et al. 2010). In contrast, RB promotes
senescence by stimulating a repressive chromatin envi-
ronment that leads to the stable suppression of proliferative
genes, perhaps through the production of senescence-asso-
ciated heterochromatic foci (SAHF) (Narita et al. 2003;
6These authors contributed equally to this work.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.196238.112.
1546 GENES & DEVELOPMENT 26:1546–1557 ? 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org
Zhang et al. 2005) and/or by facilitating the accumulation
of repressive histone marks on proliferation-responsive
genes (Narita et al. 2003; Chicas et al. 2012).
Interestingly, RB is a member of a multigene family
consisting of RB, p107, and p130 (Burkhart and Sage 2008),
and these three proteins act to inhibit gene expression
partly through binding and modulation of the activity of
certain members of the E2F family of transcription factors.
Although RB family members can compensate for each
roleinits ability topromoteE2F targetgenerepression and
cell cycle arrest during senescence, particularly on E2F-
responsive genes required for DNA replication (Chicas
et al.2010).As RBis the only member of this family that is
frequently mutated or lost in cancer (Burkhart and Sage
2008), it has been argued that RB exerts its tumor sup-
pressor function in part by controlling cellular senescence
(Narita et al. 2003; Chicas et al. 2010).
The E2F family of transcription factors consists of
eight proteins that bind to the consensus E2F motif
(TTTCGCGC) (Zheng et al. 1999) that exists in many
genes involved in DNA synthesis, cell cycle progression,
and mitosis (Cam and Dynlacht 2003). Although in vivo
studies indicate that the roles and regulation of these
factors are complex (Trimarchi and Lees 2002), E2F1–3
are most commonly associated with transcriptional acti-
vation of genes involved in normal cell cycle transitions,
where their activities are restrained by their association
with RB family members in a manner that is relieved by
CDK-mediated hyperphosphorylation of
1998). E2F4 and E2F5 are most strongly linked to tran-
scriptional repression during quiescence (Chen et al.
2009), whereas E2F6 has been linked to polycomb-medi-
ated gene regulation (Trimarchi et al. 2001; Attwooll et al.
2005). E2F7/8 are transcriptional repressors with an atyp-
ical structure, having two DNA-binding domains and
lacking a dimerization domain, which is required for
association with dimerization partner (DP) proteins that
appear to be important for the sequence-specific binding
capacity of other E2Fs (Di Stefano et al. 2003; Logan et al.
2004, 2005). Although little is known about their activity,
mice null for both E2f7 and E2f8 die during embryonic
development with phenotypes similar to Rb-deficient ani-
mals, suggesting a relationship between these proteins (Li
et al. 2008). Whether the effect is due to a direct association
with RB (Chen et al. 2009) or some parallel activity is not
p53 and RB act in a cooperative way to promote senes-
cence, and there are many complex interactions between
these pathways. The p53 target gene CDKN1A encodes the
protein p21, which, by inhibiting CDKs, prevents phos-
phorylation of the retinoblastoma family proteins, leading
to the activation of the RB family and repression of E2F-
driven transcription. On the other hand, inactivation of
the retinoblastoma proteins leads to up-regulation of
ARF, an E2F target gene, and ARF subsequently stabilizes
the p53 protein through MDM2 inhibition (Iaquinta et al.
2005). Interestingly, E2F3b, an isoform of E2F3 that re-
presses ARF transcription, may be crucial in this regulation
(Aslanian et al. 2004). The importance of this interplay for
the execution of the senescence program can vary de-
pending on context, and in some instances may be a
compensatory response to pathway perturbation. For
example, loss of RB can trigger p53 up-regulation via
ARF or other pathways, thereby providing a safeguard
that prevents evasion of senescence and malignant trans-
formation (White 1994; Chicas et al. 2010). Thus, in the
context of senescence, the nature of the cross-talk appears
to promote and reinforce the cell cycle arrest. In this study,
suppressive activity that provides a novel link between the
RB and p53 pathways during cellular senescence.
E2F7 levels increase during cellular senescence
We previously performed a large series of transcriptional
profiling experiments in order to identify genes that might
be selectively influenced by various RB family members in
oncogene-induced senescence relative to other growth
states (Chicas et al. 2010). IMR90 human normal diploid
fibroblasts, a paradigm model for the study of senescence
(Shay et al. 1991; Narita et al. 2003), were triggered to
senesce by oncogenic Ras in the presence of potent
shRNAs capable of individually repressing each RB family
member, and the resulting cells were subjected to tran-
scriptional profiling by Affymetrix U133 Plus 2.0 micro-
arrays. In that study, we noted that RB was unique among
RB family members based on its strict requirement for
the repression of a subset of E2F target genes, including
many involved in DNA synthesis. In contrast, p107 and
p130 could compensate for RB in repressing these genes
during normal proliferation or upon cell cycle exit into
In examining this transcriptional profiling data for the
expression of E2F family members, we noticed a marked
up-regulation of the atypical E2F family member E2F7
that was distinct for the senescent state (Fig. 1A). These
observations were confirmed by quantitative RT–PCR
(qRT–PCR) analysis using primers specific to each E2F
family member (Fig. 1B; Supplemental Fig. 1) and by im-
munoblotting using antibodies specific for E2F7 (Fig. 1C).
E2F7 transcription levels were also increased in cells trig-
gered to undergo senescence by replicative exhaustion
or treatment with the DNA-damaging agent etoposide
(Fig. 1B). In contrast, E2F7 was decreased as cells entered
quiescence through growth factor depletion, implying that
arrest. These results raise the possibility that E2F7 plays an
active role in the senescence program.
E2F7 is a direct p53 target gene
The most well-established transcriptional activator that
participates in senescence is p53, which is a sequence-
10-base-pair (bp) motif 59-PuPuPuC(A/T)(T/A)GPyPyPy-39
separated by 0–13 bp (el-Deiry et al. 1992; Wei et al. 2006).
Interestingly, E2F7 has a p53-binding site in its promoter
(Supplemental Fig. 2), and indeed, analysis of chromatin
E2F7 backs up RB during cellular senescence
GENES & DEVELOPMENT 1547
immunoprecipitation (ChIP)/next-generation sequencing
(ChIP-seq) data from IMR90 cells under different growth
conditions revealed a marked and specific binding of p53
to this site in senescence but not in growing conditions
(Fig. 2A). No specific p53 binding was detected in any of
the other E2F target genes, although E2F4 and E2F8 also
binds to the E2F7 promoter specifically during cellular
To determine whether p53 binding to the promoter of
the E2F7 gene influences transcription, we examined
whether the p53-binding site in the promoter of the E2F7
gene could confer p53 responsiveness to a heterologous
promoter and whether suppression of p53 would compro-
mise E2F7 induction during senescence. The p53-binding
site in the E2F7 promoter or a variant in which the p53-
binding site was mutated was cloned upstream of a lucif-
erase reporter and transfected together with a p53 expres-
sion construct in p53-null HCT116 cells, and luciferase
expression was assessed using bioluminescence. As
expected, cells transfected with the wild-type reporter
showed a marked increase in luciferase expression,
whereas those with the mutant did not (Fig. 2B). Similarly,
p53 was required for E2F7 induction in cells undergoing
senescence. While senescent IMR90 cells harboring a neu-
tral shRNA were capable of inducing E2F7 mRNA and
protein in response to oncogenic Ras, those expressing
a p53 shRNA were impaired in this effect (Fig. 2C,D).
Together, these data validate E2F7 as a bona fide p53 target
E2F7 cooperates with RB to promote senescence
Control of oncogene-induced senescence in IMR90 cells
involves a complex interplay between the RB and p53
Heat map of the expression patterns of E2F family members in
growing (G), quiescent (Q), and senescent (S) IMR90 cells. Each row
represents a biological replicate. (B) Histogram showing the levels of
E2F7 RNA in either growing (G) or quiescent (Q) cells or cells
triggered to undergo senescence by either RasV12 (Ras), replicative
exhaustion (RS, passage 34), or etoposide (Etop, 100 uM). The
quantity of RNAwas measured by real-time qPCR analysis (qPCR),
normalized to the expression of b-actin, and expressed as fold
change relative to growing IMR90 cells. The data represent the
mean (6the standard deviation) of three separate experiments. (C)
Immunoblots documenting the increase in E2F7 protein in cells
undergoing senescence. Protein lysates were from ER-Ras-infected
IMR90 cells at the indicated time after 4-hydroxy-tamoxifen
(4-OHT) treatment. Accumulation of p16INK4Aand p21CDKN1Aare
shown as an indication of Ras activation upon tamoxifen treatment.
Actin probing of a parallel blot was used as a loading control.
E2F7 is up-regulated during cellular senescence. (A)
patterns of p53 to the E2F7 gene promoter shown as custom
tracks on the University of California at Santa Cruz (UCSC)
Genome Browser. p53-specific peaks are detected in the pro-
moter of the E2F7 gene (see magnified image of the highlighted
area below) specifically in senescent cells (S) but not growing
cells (G) or senescent cells expressing a p53 shRNA (S/shp53).
Peaks detected outside the promoter are insensitive to suppres-
sion of p53, suggesting that they are not specific. (B) Histogram
showing the luciferase activity induced by the indicated amount
of p53 on the reporter gene driven by the p53 response element
from the E2F7 gene. Luciferase activity was plotted as the
induction relative to basal luciferase activity. The data represent
the mean (6the standard deviation) of three separate experi-
ments. (C) Histogram showing the levels of E2F7 RNA in
growing or senescent cells expressing the indicated shRNA.
Expression is normalized to the expression of GAPDH and is
presented as the fold change relative to growing IMR90 cells.
Values are means 6 standard deviation of three independent
samples. (D) Immunoblot showing the levels of the E2F7 protein
in senescent cells expressing the indicated shRNA. Actin was
used as loading control. The figure is a representative blot of at
least three independent experiments.
E2F7 is a bona fide p53 target gene. (A) Binding
Aksoy et al.
1548 GENES & DEVELOPMENT
pathways such that cosuppression of the RB and p53
network components are required to fully bypass the
program (Shay et al. 1991; Voorhoeve and Agami 2003;
Chicas et al. 2010). Given our observation that E2F7 is a
direct p53 target gene that is up-regulated during senes-
cence, we set out to test whether E2F7 contributes to the
output of p53 signaling in this setting. IMR90 cell
populations were engineered to express two independent
shRNAs capable of suppressing E2F7 protein alone or in
tandem with a potent RB shRNA (Supplemental Fig. 3A,B)
and were examined for their ability to undergo oncogene-
induced senescence. Acute induction of senescence was
assessed by the ability of cells to express senescence mark-
ers or prevent DNA replication as assessed by incorpora-
tion of 5-bromo-29-deoxyuridine (BrdU) into DNA. Long-
term proliferative capacity was also assessed by the ability
of these cell populations to form colonies when plated at
As previously reported, suppression of RB impaired senes-
cence, as measured by the disappearance of senescence
markers such as SA-b-gal activity and SAHF formation
(Fig. 3A; Chicas et al. 2010), yet these cells still arrest, as
assessed by a reduction in BrdU incorporation and an
inability to form colonies (Fig. 3B,C). Suppression of E2F7
alone had little impact on certain senescence phenotypes,
although, in principle, the failure of E2F7 shRNAs to
produce a phenotype in this assay may reflect their
inability to completely suppress E2F7 protein expression.
Still, in marked contrast, cosuppression of E2F7 together
with RB was sufficient to bypass senescence, as measured
by various proliferation assays such as BrdU incorpora-
tion, colony formation, and cell counting (Fig. 3B,C;
Supplemental Fig. 3C). Similar results were observed in
another human fibroblast line (WI-38) (Supplemental Fig.
3D). On the other hand, in human BJ fibroblasts, a cell
type where senescence is mainly dependent on the p53
pathways due to weak p16INK4ainduction (Beausejour
et al. 2003), suppression of E2F7 alone was sufficient to
override Ras-induced senescence (Supplemental Fig. 3E).
Thus, E2F7 cooperates with the RB pathway to promote
Cosuppression of RB and E2F7 transforms primary
mouse embryonic fibroblasts (MEFs)
p53 can also control the senescence of MEFs in a manner
that directly restricts the ability of Ras to promote ma-
lignant transformation (Serrano et al. 1997; Azzoli et al.
1998). To examine the participation of E2F7 in this pro-
cess, we developed multiple shRNAs targeting mouse
E2f7 (Supplemental Fig. 4A) and introduced these into
early-passage MEFs in combination with oncogenic Ras.
The resulting cell populations were subsequently assessed
for their ability to undergo senescence in vitro or form
tumors in vivo following subcutaneous injection into
immunocompromised mice. Unlike MEFs expressing p53
shRNAs, MEFs expressing E2f7 shRNAs still underwent
senescence in response to oncogenic Ras, as assessed by
the accumulation of SA-b-gal activity, reduced BrdU in-
corporation, and an inability to form colonies (Fig. 4B; Sup-
plemental Fig. 4B). Accordingly, these cell populations
were incapable of forming tumors in vivo. Of note, these
negative results are not simply due to insufficient E2F7
knockdown, as MEFs derived from E2f7-null mice were
also arrested in response to oncogenic Ras (Supplemental
Fig. 4C). Thus, in this setting, disruption of E2F7 is unable
to fully recapitulate the effects of p53 loss on promoting
bypass of senescence in MEFs.
Acute RB inactivation enables bypass of an immediate
Ras-induced cell cycle arrest but, in contrast to p53 loss,
does not enable cellular immortalization, and Rb-deficient
MEFs are not transformed by Ras alone (Sage et al. 2003).
To assess whether, as occurs in human fibroblasts, E2F7
cooperates with RB to promote oncogene-induced se-
nescence in MEFs, we constructed a series of bicistronic
shRNAs capable of simultaneously targeting mouse E2f7
and Rb (Fig. 4A; Supplemental Fig. 4D) and examined
their ability to modulate the cellular response to onco-
genic Ras in the assays described above. As previously
reported (Sage et al. 2003), suppression of RB alone was
sufficient to impair senescence in MEFs, as measured by
the lack of senescent markers (e.g., a flat morphology and
SA-b-gal activity) and an increase in BrdU incorporation
shortly after introduction of oncogenic Ras (Fig. 4B; Sup-
plemental Fig. 4E). Nonetheless, these cells did not show
Micrographs showing SA-b-gal staining in senescent cells
expressing the indicated shRNAs. The numbers shown at the
bottom left of each micrograph represent the quantitation of SA-
b-gal-positive cells. The data represent means 6 standard de-
viation of three independent experiments. shE2F7-1 and shE2F7-2
are two independent shRNAs targeting E2F7. The same two
shRNAs were used in combination with shRB (shTan1 and
shTan2). An shRNA targeting Renilla is a neutral control
shRNA. Bar, 200 mm. (B) Quantification of BrdU incorporation
in senescent IMR90 cells expressing the indicated shRNAs. The
data represent means 6 standard deviation of three independent
experiments. Growing (G) cells were used as a control. (C)
Colony formation assay documenting the effect of suppressing
RB and E2F7 on the ability of H-RasV12expressing IMR90 cells
to grow at low density. Shown are representative crystal violet
stainings of 6-cm plates 2 wk after plating.
E2F7 cooperates with RB to promote senescence. (A)
E2F7 backs up RB during cellular senescence
GENES & DEVELOPMENT 1549
enhanced clonogenic capacity or form tumors in mice
(Fig. 4B–D), indicating that they eventually arrest.
Similarly, MEFs coexpressing E2f7 and Rb shRNAs did
not display any signs of senescence and continued to in-
corporate BrdU. However, in contrast to RB-deficient
cells, cells expressing shRNAs targeting both Rb and
E2f7 showed a marked increase in long-term proliferative
capacity and were potent at producing tumors in vivo
(Fig. 4B–D). Similar results were observed in MEFs de-
rived from E2f7-null mice after transduction with retro-
viruses expressing shRb and Ras (Supplemental Fig. 4F).
Together, these data indicate that E2F7 cooperates with
RB to control cellular senescence in MEFs, where it con-
tributes to a tumor-suppressive program.
E2F7 binds and represses a subset of E2F target genes
RB inactivation is unable to bypass oncogene-induced
senescence in part because RB loss triggers a second se-
nescence barrier involving p53 and its transcriptional
target, p21CDKN1A(Chicas et al. 2010). Our results iden-
tify E2F7 as a key target of p53 in senescence and suggest
barrier. Consistent with this view, E2F7 is hyperinduced
in IMR90 cells expressing RB shRNAs that were triggered
to senesce, leading to a corresponding increase in the
association of E2F7 with chromatin (Fig. 5A). To identify
specific genes targeted by E2F7 during senescence, we
performed ChIP-seq using an anti-E2F7 antibody in grow-
ing, quiescent, or senescent IMR90 cells expressing a con-
trol shRNA or an shRNA targeting RB. Sequence reads
were mapped back to the human genome, and the number
of reads corresponding to different genomic regions was
determined. We used MACS with a default P-value cutoff
of 1 3 10?5to identify genomic regions (ChIP-peaks) that
showed an enrichment of E2F7-specific binding. Although
the total number of called peaks was relatively similar
among growing, quiescence, and senescence, we noticed a
marked increase in E2F7-binding sites in senescent IMR90
cells expressing RB shRNAs (Supplemental Fig. 5A).
To determine whether E2F7 was associated with known
transcription factor-binding sites, we performed a de novo
motif analysis on the 300 most significant E2F7-enriched
regions. This analysis revealed a consensus motif highly
similar to the E2F consensus motif (P < 0.001) (Fig. 5B).
Accordingly, as has been reported by others (Di Stefano
et al. 2003), we detected E2F7 binding to known E2F target
genes (Supplemental Table 1), such as the E2F1 gene pro-
moter (Fig. 5C), a previously reported target of E2F7
(Panagiotis Zalmas et al. 2008; Westendorp et al. 2012).
Also consistent with our global analyses, E2F7 binding to
the E2F1 promoter was substantially higher in senescent
cells (Fig. 5C). These signals were specific to E2F7, were
not detected in parallel assays using a nonspecific anti-
body, and were substantially reduced in samples derived
from cells expressing E2F7 shRNAs. Nonetheless, we noted
other sites harboring apparently specific binding,raising the
possibility that E2F7 binds other genomic regions as well
(data not shown).
Based on the known binding of E2F7 to E2F target sites
and the nonbiased analysis described above, we focused
our subsequent analyses on 283 known E2F target genes,
as determined from having an E2F-binding site in the
promoter and/or experimental verification of promoter
sequences from most of these target genes (Zhao et al.
2005). Compared with growing and quiescent conditions,
E2F7 binding to E2F target genes was generally increased
during senescence and further enhanced in RB-deficient
cells (Supplemental Fig. 5B). In addition, more genes were
bound under these conditions; hence, while ;50 E2F tar-
get genes showed significant association under growing
and quiescent conditions, this number was doubled in
senescent cells and tripled in cells triggered to senesce in
the absence of RB (Supplemental Fig. 5C). Approximately
30 E2F target genes were bound under all four conditions,
with very few scoring as unique to growing (four), qui-
escent (five), or senescent (three) conditions. However, a
substantial number of known E2F target genes (47)
showed significant binding of E2F7 only in RB-deficient
senescent cells (Supplemental Fig. 5C). While this in-
crease may reflect qualitative differences in E2F7 binding
between conditions, it is possible that the increase in the
signal to noise ratio produced by more E2F7 binding
tion. (A) Immunoblots from lysates of mouse liver progenitor
cells documenting the efficiency of the indicated shRNAs in
suppressing RB and E2F7. shTan is the bicistronic shRNA
targeting RB and E2F7, and shRen is a neutral control shRNA.
Actin was used as a loading control. (B, top) Micrographs
showing SA-b-gal staining in MEFs undergoing senescence in
the presence of the indicated shRNAs. Growing cells were used
as a control. (Bottom) Colony formation assays documenting
the effect of suppressing RB and E2F7 on the ability of H-RasV12-
expressing MEFs to grow at low density. Shown are representa-
tive crystal violet stainings of 6-cm plates 2 wk after plating. (C)
Representative images of immunocompromised mice 20 d after
subcutaneous injections of 1 3 106MEFs expressing H-Ras and
the indicated shRNA. Tan1 and Tan2 are two different hairpins
targeting both RB and E2F7. (D) Quantification of tumor
volumes. Each point represents mean tumor volume 6 SEM
(n = 6). Tumor size was measured once a week.
Cosuppression of RB and E2F7 promotes transforma-
Aksoy et al.
1550 GENES & DEVELOPMENT
enhanced the statistical power for calling a particular
region as a peak. Regardless, since E2F7 exerts its most
prominent effects when RB is suppressed, we focused
more closely on the subset of E2F targets that showed the
binding of E2F7 to their promoters in these conditions.
Gene ontology (GO) analysis revealed that E2F target
genes bound by E2F7 in cells harboring compromised RB
were enriched for those participating in mitosis (Fig. 5D,E;
Supplemental Fig. 5D). For instance, we identified binding
of E2F7 to the MAD2L1 and BUB3 genes (Fig. 5D,E), each
of which encodes proteins involved in the spindle check-
point (Musacchio and Salmon 2007). Unlike binding to
the E2F1 gene, which is detected in senescent cells with
or without RB, binding of E2F7 to MAD2L1 and BUB3
promoters only achieved significance in RB-repressed cells
triggered to senesce. Similar patterns were observed at the
promoters of other ‘‘mitotic’’ genes (Supplemental Table 2)
and were different from those observed on DNA replica-
tion genes where E2F7 binds equally to these promoters in
bothconditions(Supplemental Fig. 5E,F).This result raises
the possibility that E2F7 might repress genes that are in-
volved in mitosis, particularlyincells compromisedfor RB
E2F7 compensates for RB loss in repressing
mitotic cell cycle targets
Our previous work indicated that, during senescence, RB
is uniquely required for the repression of E2F targets
involvedinDNA replication,asthis class ofgenes remains
derepressed in RB-deficient cells triggered to senesce; in
contrast, other E2F targets (e.g., many involved in cell
cycle progression and mitosis) were silenced (Chicas et al.
2010). To identify genes whose expression was influenced
by E2F7, we performed transcriptional profiling analysis
on the cell populations described above, paying particular
attention to genes synergistically impacted by the com-
bined inactivation of E2F7 with RB. RNA was extracted
fromgrowing orsenescent cellsexpressing shRNAs target-
ing either luciferase as control, RB, E2F7, or both RB and
E2F7 and hybridized to the Affymetrix U133 Plus 2.0
microarrays. Genes identified as differentially expressed
between conditions (Supplemental Table 3) were then
subjected to GO analysis to identify putative functional
categories of genes controlled by E2F7.
Consistent with our previous work, suppression of RB
led to the up-regulation of genes categorized by their role
targets during senescence. (A) Immunoblot
showing E2F7 protein levels in chromatin-
bound fractions of growing (G), senescent
(S), or shRB-expressing senescent (S/shRB)
IMR90 cells. Core histones were used as
loading control. (B) Position weight ma-
trix of the motif significantly enriched in
the DNA associated with E2F7. This se-
quence is highly similar to the E2F motif
(TTTCGCGC). (C) Binding patterns of
E2F7 to the E2F1 gene promoter shown as
custom tracks on the UCSC Genome
Browser. The green bar at the bottom of
the figure corresponds to CpG islands in the
promoter of the E2F1 gene. Notice the in-
crease in E2F7 binding in senescent cells
with or without RB expression compared
with growing and quiescent conditions.
(D,E) Binding patterns of E2F7 to the BUB3
and MADL2 gene promoters shown as cus-
tom tracks on the UCSC Genome Browser.
Notice the increase in E2F7 binding in se-
nescent cells suppressed for RB (shRB). (G)
Growing; (S) senescent; (shRB) shRNA
E2F7 binds to a subset of E2F
E2F7 backs up RB during cellular senescence
GENES & DEVELOPMENT 1551
in the ‘‘cell cycle’’ (P = 2.1 3 10?27) and ‘‘DNA replica-
tion’’ (P = 7.1 3 10?27) (Supplemental Table 4), but also
genes with functions in mitosis (P = 1.1 3 10?15for the
GO term ‘‘mitosis,’’ and P = 8.3 3 10?16for the GO term
‘‘M phase of mitotic cell cycle’’). In contrast, many genes
categorized by a role in DNA replication were not af-
fected by suppression of E2F7 (hence, ‘‘replication’’ was
not detected in the top 25 GO categories), suggesting that
the requirement for E2F7 in repression of this subset of
genes is distinct from that imposed by RB (Supplemental
Table 4). However, genes up-regulated by suppression
of E2F7 showed enrichment in GO categories such as
‘‘mitotic cell cycle’’ (P = 7.7 3 10?9), ‘‘M phase of mitotic
cell cycle’’ (P = 2.7 3 10?8), and ‘‘mitosis’’ (P = 2.0 3 10?8)
(Supplemental Table 4). The expression of the mitotic
genes in RB-deficient or E2F7-deficient cells was mini-
mal, but became robust when both RB and E2F7 were
inactivated (Fig. 6B). Thus, while E2F7 is dispensable for
the repression of DNA replication genes, it appears to
cooperate with RB in the repression of mitosis-related
Similar conclusions were obtained by hierarchical clus-
tering of our gene expression data (Fig. 6A–C) and by direct
immunoblotting for E2F target proteins (Fig. 6D). Hence,
cells with suppressed RB were clearly distinguishable from
normal senescent cells by virtue of their up-regulation of
a cluster of genes linked to DNA replication. In marked
contrast, cosuppression of E2F7 with RB produced a gene
expression state characterized by the up-regulation of both
the mitotic gene cluster and the DNA replication gene
cluster. These effects were underscored by the changes in
sample proteins corresponding to the members of each
gene category. Thus, while the DNA replication factor
MCM3 was highly expressed in RB-deficient cells, its
expression is not affected by suppression of E2F7 (Fig.
6D). Components of the mitotic cell cycle machinery
such as Cyclin A, Cyclin B, and CDC2 were highly ex-
pressed only in cells deficient of both RB and E2F7 (Fig.
6D). Of note, this association between E2F7 and mitotic
cell cycle gene repression is not an indirect consequence
of cell cycle proliferation, as our ChIP-seq analysis shows
these results indicate that E2F7 compensates for loss of RB
in the repression of mitotic cell cycle genes.
E2F7 prevents normal cell cycle progression
in RB-compromised cells
Our combined ChIP-seq and gene expression profiling
suggests that E2F7 represses the expression of a subset of
E2F target genes that would otherwise enable cell cycle
progression and mitosis in cells with compromised RB
function. To determine the impact of E2F7 suppression on
cell cycle progression, we examined the effect of E2F7 and/
or RB shRNAs on the DNA content of IMR90 cells fol-
Inaddition, theimpact of E2F7 and RB suppression on cell
cycle distribution in response to oncogenes was exam-
ined in WI38 diploid fibroblasts (Supplemental Fig. 6A)
and a Ras-transformed hepatoblast line expressing a regu-
latable p53 shRNA that can be triggered to senesce by re-
establishing wild-type p53 expression (Fig. 7C; Xue et al.
Consistent with our previous results (Chicas et al.
2010), suppression of RB caused a greater than a twofold
accumulation of cells with a 4N and 8N DNA in senesc-
ing IMR90 cells (Fig. 7A,B). Interestingly, the cells that
eventually arrest retained high levels of cyclin E with low
levels of cyclin B and phospho-histone H3, a phenotype
consistent with a mitotic failure and endoreplication
rather than a G2 or M arrest (Supplemental Fig. 6B–D;
see also Chicas et al. 2010). In marked contrast, simulta-
neous suppression of RB and E2F7 alleviated this effect,
preventing the appearance of polyploid cells and re-
establishing a normal cell cycle profile (Fig. 7A,B). Sup-
pression of E2F7alone alsoreduced thepercentage of cells
with 4N DNA content that accumulate in the presence
of RB (Fig. 7A,B), suggesting that it normally restricts
mitotic progression in a manner that is exacerbated by RB
loss. Similar results were also observed in WI38 fibroblasts
(Supplemental Fig. 6A) and Ras-transformed hepatoblasts
following reactivation of p53 (Fig. 7C). Therefore, in both
human and mouse cells, E2F7 controls a cell cycle check-
point that compensates for RB loss by preventing aberrant
E2F family members are involved in various cellular
processes, including cell cycle progression, DNA replica-
tion, DNA repair mechanisms, apoptosis, differentiation,
sion during senescence. (A) Heat map of the expression patterns
expressed genes among various groups (P < 0.001; 2486 probes)
highlighting the E2F target genes. (B,C) Magnification of gene
clusters. (B) Mitotic cell cycle gene clusters. (C) DNA replica-
tion factors clusters. (D) Immunoblots for representative DNA
replication (MCM3) or mitotic cell cycle (CDC2, Cyclin A, and
Cyclin B) proteins. Actin was used as a loading control. (G)
Growing; (S) H-RasV12-expressing cells.
E2F compensates for RB in controlling gene expres-
Aksoy et al.
1552 GENES & DEVELOPMENT
and development (Rowland and Bernards 2006). In our
study, we identified a role for E2F7 in cellular senescence,
a function that was not previously appreciated. We show
that E2F7 is a direct p53 target gene that cooperates with
RB in the repression of E2F target genes during oncogene-
induced senescence. In cells with compromised RB, E2F7
is required to repress genes involved in mitosis and as
such acts as a final safety net to prevent uncontrolled pro-
liferation, immortalization, and, in at least murine cells,
In fibroblasts and most other cell types, cellular senes-
cence is triggered by the combined action of the RB and
tribution of each network to the execution and main-
tenance of the program can be context-dependent
(Courtois-Cox et al. 2008). Here we see that in human
fibroblasts, E2F7 acts as a key effector of p53 in the pro-
gram, since like disruption of p53, suppression of E2F7
cooperates with RB to bypass oncogene-induced senes-
cence. E2F7 can also be induced acutely by cytotoxic
agents in osteosarcoma cells with intact p53 (Panagiotis
Zalmas et al. 2008) and in cells triggered to senesce by
DNA-damaging agents or replicative exhaustion (see Fig.
1B), suggesting that its roles and regulation may extend
beyond the oncogene-induced senescence program de-
scribed here. Of note, suppression of p21, a canonical p53
target, also cooperates with RB loss to bypass senescence,
thus displaying a phenotype similar to cells with sup-
pressed E2F7. During senescence and more acute DNA
damage checkpoints, p21 acts to inhibit the activity of
various CDKs, thereby preventing RB hyperphosphoryla-
tion and the release of more canonical E2Fs to drive
induction of S-phase genes. In contrast, E2F7 apparently
acts further downstream directly on E2F target pro-
moters, perhaps to reinforce the effects of p53. Both p53
effectors are crucial for the program, highlighting how
p53 can coordinate cellular processes through its action
on multiple effectors.
Although our studies clearly show that E2F7 is a direct
p53 target gene in human cells, it seems likely that its
regulation is more complex. Hence, we do not detect
E2F7 activation in response to Ras in MEFs, although it is
induced in MEFs following RB suppression (data not
shown). We also noted consensus NF-kB- and E2F-binding
sites in the E2F7 promoter, and accordingly, studies
demonstrate that E2F7 can be transcriptionally activated
by E2F1 (Di Stefano et al. 2003). Whether NF-kB sites
contribute in a meaningful way to E2F7 remains to be
in the presence of the indicated shRNA. The X axis shows the DNA content, whereas the Y axis shows the number of cells. (B) Bar
graph showing the percentages for G1 and G2/M cells. The data shown are the mean value 6 standard deviation from three
independent experiments. (C) Cell cycle profiles of hepatoblasts triggered to undergo senescence by reactivation of p53 in the presence
of the indicated shRNAs. The DNA content was measured by flow cytometry 8 d after doxycycline treatment.
E2F7 enforces a second cell cycle checkpoint that backs up RB. (A) Cell cycle profiles of IMR90 cells undergoing senescence
E2F7 backs up RB during cellular senescence
GENES & DEVELOPMENT1553
determined, but it is noteworthy that our group and
others have recently described a role for NF-kB in medi-
ating various aspects of cellular senescence, including the
the impact of senescent cells on the tissue microenviron-
ment (Chien et al. 2011; Jing et al. 2011). Moreover, E2F7
may alsoreinforce p53 activation duringsenescence, as we
noted a modest reduction in p53 phosphorylation levels
following E2F7 depletion in IMR90 cells triggered to se-
nescence (data not shown). Similar results have been ob-
served for the p53 target genes miR-34a and PML during
senescence (de Stanchina et al. 2004; He et al. 2007),
further underscoring the compensatory mechanisms at
play in this tumor suppressor network.
Our studies suggest that E2F7 can regulate gene ex-
pression during senescence and is most important for
the control of E2F target genes involved in cell cycle
progression and mitosis. Specifically, we see that E2F7
associates with various E2F target gene promoters in a
manner that is substantially enhanced by loss of RB and
that, under these circumstances, the direct targets of E2F7
correspond to genes that are significantly enriched for
those controlling cell cycle progression and mitosis. Ac-
cordingly, disruption of E2F7 under circumstances where
RB is compromised prevents repression of various cell
cycle progression and mitotic genes and is sufficient to
abrogate a checkpoint that otherwise prevents proper
mitosis in cells undergoing oncogene-induced senescence.
of this checkpoint.
Although E2F7 and RB bind overlapping gene sets, the
consequence of this binding differs. Hence, while RB is
strictly required for the suppression of replication-asso-
ciated E2F targets during senescence, the requirement for
E2F7 in gene repression only becomes evident after sup-
pression of RB. Apparently, E2F7 and RB compensate for
each other in repressing genes involved in mitosis, but
not those involved in DNA replication, explaining why
only the mitotic gene cluster remains silenced after sup-
pression of RB or E2F7 alone. In this manner, E2F7 func-
tionally behaves like a fourth RB family member involved
in E2F target gene repression.
RB represses gene expression by sequestering activated
E2Fs and recruiting histone-modifying activities that pro-
duce a repressive chromatin context (Morris and Dyson
2001; Nijwening et al. 2011; Chicas et al. 2012). How
E2F7 acts to repress gene expression remains to be de-
termined. In contrast to the regulation of canonical E2Fs,
it seems unlikely that E2F7 acts by corecruitment of RB
family members to E2F target promoters because E2F7
lacks the ‘‘pocket protein’’-binding domain required for
RB family member association. Also, E2F7 binds and
represses its targets in the absence of functional RB.
Furthermore, we noted that cosuppression of p107 and
p130 are not able to cooperate with RB loss to bypass
oncogene-induced senescence (A Chicas and SW Lowe,
unpubl.) Perhaps, instead, E2F7 competes with the acti-
vating E2Fs (E2F1–3) for promoter occupancy of E2F target
genes topreventthe transcriptionalactivation.Thismodel
is consistent with observations in Drosophila, where the
activator E2F (dEF2F1) and the repressor E2F (dE2F2) act
antagonistically (Frolov et al. 2001). Further studies will be
required to test this possibility.
The crucial role of E2F7 during senescence, and the
now established role of senescence as a barrier to tumor-
igenesis, raises the possibility that E2F7 is a tumor sup-
pressor gene. Consistent with such an activity, E2F7
inactivation can, together with RB loss, promote malig-
nant transformation of murine fibroblasts. E2F7is located
in chromosome 12q21, a region that is frequently deleted
in pancreatic cancer, and patients harboring this deletion
are associated with a poor prognosis (Kimura et al. 1998).
Interestingly, the genetics of pancreatic cancer suggest
that this is a setting where oncogene-induced senescence
restricts tumorigenesis, as activating KRAS mutations
frequently co-occur with mutations targeting the INK4A/
ARF locus or p53 in later stages of the disease. Under-
expression of E2F7 is also associated with platinum re-
sistance and reduced survival in ovarian cancer patients
(Reimer et al. 2007). Still, somatic mutations in E2F7 have
yet to be described, raising the possibility that its activity
is not rate-limiting for tumorigenesis. Studies in mouse
models will further test its tumor-suppressive role. Re-
gardless of their outcome, our studies establish a clear
functional role for E2F7 in cellular senescence and high-
light the importance of compensatory mechanisms in
modulating the RB tumor suppressor network.
Materials and methods
Vectors, cell culture, and gene transfer
The following retroviral vectors were used in this study: pWZL-
Hygro (H-rasV12) (Serrano et al. 1997) and pLNCX2-neo
(ER:RasV12) (Young et al. 2009). shRNAs targeting RB were
previously described (Chicas et al. 2010). shRNAs targeting
mouse and human E2F7 were generated using the method
previously described (Zuber et al. 2011). Briefly, the 10 top-
scoring siRNA predications were obtained using BIOPREDsi,
and the siRNAwas incorporated intothe miR-30 backbone (Silva
et al. 2005). The polycistronic shRNAvectors were cloned in two
steps as described (Chicas et al. 2010). Luciferase and RB shRNAs
were described before (Chicas et al. 2010). Human E2F7 hairpins
2 corresponds to shE2F7.1190. Mouse E2f7 hairpins were as
follows: shE2f7-1 corresponds to shE2f7.771, and shE2f7-2 corre-
sponds to shE2f7.5220. The human RB shRNA that was used
throughout the study corresponds to shRB.698. The sequences of
all shRNAs will be provided on request.
Human diploid IMR90 fibroblasts and WI38 (American Type
Culture Collection) were cultured in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal bovine serum and anti-
biotics. MEFs were isolated from embryonic day 13.5 (E13.5)
embryos from E2f7?/?mice (O Aksoy, A Chicas, and SW Lowe,
unpubl.) derived from embryonic stem cells generated by the
trans-NIH Knock-Out Mouse Project (KOMP) and obtained from
the KOMP Repository (http://www.komp.org). At least two lit-
termate controls were used for comparison. Retroviruses were
packed using Phoenix cells (G. Nolan, Stanford University, CA)
and infections were performed as described elsewhere (Narita
et al. 2003). The infected population was selected using either
2 mg/mL puromycin (Sigma) for 2 d, 500 mg/mL neomycin for 3 d,
or 75 mg/mL hygromycin B (Roche) for 3 d. For coinfection, cells
Aksoy et al.
1554 GENES & DEVELOPMENT
were sequentially selected with puromycin, and then hygro-
mycin or neomycin. Post-selection day 7 refers to 7 d after the
conclusion of puromycin selection.
Senescence was induced by etoposide (100 mM; Sigma) treatment
or infection of IMR90 cells with oncogenic H-RasV12 as de-
scribed (Narita et al. 2003). Replicative senescent IMR90 cells
were established by culturing the early-passage cells until pas-
sage 34. At this point, the cells were not proliferating and showed
all of the senescent markers, including being SA-b-gal-positive.
Senescent IMR90 cells (post-selection day 7) were plated on
coverslips coated with 0.1% gelatin and fixed in 4% formalde-
hyde. For cell cycle arrest analyses, cells were labeled with BrdU
(100 mg/mL; Sigma) and 5-fluor-29-deoxyuridine (10 mg/mL;
Sigma) for 4 h. Nuclei incorporating BrdU were visualized by
immunostaining with anti-BrdU antibodies (1:400; BD Pharmin-
gen) as described previously (Narita et al. 2003). For colony
formation assays, 1250, 2500, or 5000 cells were plated in each
well of a six-well plate, cultured for 14 d, fixed with 4% form-
aldehyde, and stained with crystal violet. Detection of SA-b-gal
activity was performed as described previously at pH 6.0 for
IMR90 cells and pH 5.5 for mouse cells. Briefly, adherent cells
were fixed with 0.5% gluteraldehyde in phosphate-buffered
saline (PBS) for 15 min, washed with PBS supplemented with
1 mM MgCl2, and stained for 5–6 h in PBS containing 1 mM
MgCl2, 1 mg/mL X-Gal (Roche), and 5 mM each potassium
ferricyanide (Sigma) and potassium ferrocyanide (Sigma).
Immunoassays and flow cytometry
Immunoblotting was carried out as described previously (Narita
et al. 2003). Isolation of chromatin-bound proteins was per-
formed as described (Chicas et al. 2010). The following anti-
bodies were used: anti-E2F7 (1:500; gift from Kristian Helin),
anti-p16INK4a (1:1000; H-156, Santa Cruz Biotechnology), anti-
p53 (1:1000; Do-1, Oncogene), anti-Cyclin A (1:1000; Sigma),
anti-Cyclin B (1:1000; Cell Signaling), anti-actin (1:5000; ac-15,
Sigma), anti-Rb antibody (1:500; G3-245, Pharmingen) together
with XZ-55 hybridoma supernatant (1:50), anti-MCM3 (1:1000;
gift from Bruce Stillman), anti-p21 (1:200; C-19, Santa
Cruz Biotechnology), and anti-cdc2 (1:500; B-6, Santa Cruz
For flow cytometry, cells were collected, washed with PBS,
resuspended in 100 mL of PBS plus 900 uL of cold methanol, and
stored overnight at +4°C. Cells were washed in PBS with bovine
serum albumin (0.5%) and Tween-20 (0.5%) and resuspended in
500 mL of PI/RNase staining buffer (BD Pharmingen). Data were
collected on an LSRII flow cytometer (BD Bioscience) and
analyzed with Flowjo software (TreeStar).
Gene expression analysis and RT–qPCR
Total RNA was isolated using the RNeasy minikit (Qiagen), and
cDNA was obtained using the TaqMan reverse transcription
reagents (Applied Biosystems), used for qPCRs or for cRNA
preparation (Message AmpII [Ambion]), and hybridized to U133
Plus 2.0 microarray (Affymetrix) according to the manufacturer’s
instructions. Data analyses were performed as previously de-
scribed (Chicas et al. 2010). Gene-specific primer sets were
designed using Primer Express 1.5 (sequences available on re-
quest). Real-time PCR was carried out in triplicate using SYBR
Green PCR Master Mix (Applied Biosystems) on the Roche IQ5
iCycler. GAPDH or b-actin served as endogenous normalization
Microarray data processing
We used the GCRMA function implemented in the R Biocon-
ductor Affymetrix package (Robust Multi-Array Averaging with
GC content correction) to subtract background, normalize in-
tensities, and summarize gene expression levels (Wu and Irizarry
2004), and the R program combat was used to perform batch
effect normalization. The absolute probe set intensity differ-
ences between replicates were calculated and ranked according
to descending order, and the difference values of the top 0.1%
were used as the threshold to select differentially expressed
probe sets that were used for hierarchical clustering analysis to
study the global gene expression patterns. Gene expression values
were clustered and visualized using the Cluster and TreeView
packages (Eisen et al. 1998).
Known E2F targets and GO analysis
Known E2F target genes were extracted from TRED (Zhao et al.
2005). For this study, we used 283 genes with confident evidence
(‘‘known’’ or ‘‘likely,’’ corresponding to evidence of binding or
additional functional assays). The significance of overlap be-
tween E2F target genes and other gene lists was evaluated by
Fisher’s exact test. The enrichment of GO terms in a particular
gene list was performed using the online tool DAVID (Dennis
et al. 2003). P-values reported were corrected using the Benjamini-
The ChIP experiments were done as previously described (Chicas
et al. 2010). The immunoprecipitated DNA was prepared for
GA sequencing tags were mapped to the unmasked human
reference genome (NCBI version 36, hg18) using the program
Bowtie with the parameter setting ‘‘-t -q -a –best –strata -m 1.’’
ChIP-seq peak regions were determined using MACS (Zhang
et al. 2008) to find ChIP-enriched regions compared with the
input DNA control with default parameter settings and a signif-
icance threshold (P # 1 3 10?5). We associated ChIP-enriched
regions to a target gene if it localized within the region from 3 kb
upstream of to 1 kb downstream from the transcription start site
of the gene. The gene coordinates were extracted according to
RefSeq gene annotation (hg18) downloaded from the University
of California at Santa Cruz Genome Browser.
We are indebted to Kristian Helin for kindly providing us with
the E2F7 antibody used throughout this study. We thank Charles
Sherr for helpful scientific discussions and editorial suggestions,
as well as Jessica Bolden for editorial help, Thomas Kitzing for
help with the preparation of the figures, and Meredith J. Taylor
for technical support. We also thank current and former mem-
bers of the Lowe laboratory for helpful comments and techni-
cal support. This work was supported by funds from NBRPC
(2012CB316503) and the NSF of China (60905013, 91019016,
31061160497) to X.W., a post-doctoral fellowship from NIH to
A.C. (5F32AG027631), and an RO1 grant (AG16379) from the
NIH to S.W.L. O.A. was supported by George A. and Marjorie H.
Anderson Fellowship. S.W.L. is an investigator in the Howard
Hughes Medical Institute.
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