The E4F protein is required for mitotic progression during embryonic cell cycles.
ABSTRACT The ubiquitously expressed E4F protein was originally identified as an E1A-regulated cellular transcription factor required for adenovirus replication. The function of this protein in normal cell physiology remains largely unknown. To address this issue, we generated E4F knockout mice by gene targeting. Embryos lacking E4F die at the peri-implantation stage, while in vitro-cultured E4F(-/-) blastocysts exhibit defects in mitotic progression, chromosomal missegregation, and increased apoptosis. Consistent with these observations, we found that E4F localizes to the mitotic spindle during the M phase of early embryos. Our results establish a crucial role for E4F during early embryonic cell cycles and reveal an unexpected function for E4F in mitosis.
- SourceAvailable from: shsmu.edu.cn[show abstract] [hide abstract]
ABSTRACT: The centromere is a chromosomal locus that ensures delivery of one copy of each chromosome to each daughter at cell division. Efforts to understand the nature and specification of the centromere have demonstrated that this central element for ensuring inheritance is itself epigenetically determined. The kinetochore, the protein complex assembled at each centromere, serves as the attachment site for spindle microtubules and the site at which motors generate forces to power chromosome movement. Unattached kinetochores are also the signal generators for the mitotic checkpoint, which arrests mitosis until all kinetochores have correctly attached to spindle microtubules, thereby representing the major cell cycle control mechanism protecting against loss of a chromosome (aneuploidy).Cell 03/2003; 112(4):407-21. · 31.96 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Gam1, an early adenoviral CELO protein, is required for viral replication. Consistent with its ability to inhibit histone deacetylation by HDAC1, Gam1 activates transcription. In this report, we identify the cellular transcription factor p120(E4F) as a Gam1 interaction partner. p120(E4F) is a low-abundance transcription factor that represses the adenovirus E4 promoter. Here we demonstrate that p120(E4F) interacts with HDAC1 in vivo and in vitro, and that E4F-mediated transcriptional repression is alleviated by the HDAC inhibitor trichostatin A or by overexpressing Gam1. A mutant E4 promoter unresponsive to E4F-mediated transcriptional repression is also not stimulated by Gam1. Moreover, our cofractionation experiments demonstrate that p120(E4F), HDAC1 and Gam1 may be concomitantly present in protein complexes. We conclude that Gam1 activates E4-dependent transcription possibly by inactivating HDAC1.Oncogene 06/2003; 22(17):2541-7. · 7.36 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: INCENP is a chromosomal passenger protein which relocates from the centromere to thel spindle midzone during the metaphase-anaphase transition, ultimately being discarded in the cell midbody at the completion of cytokinesis. Using homologous recombination, we have generated Incenp gene-targeted heterozygous mice that are phenotypically indistinguishable from their wild-type littermates. Intercrossing the hetero-zygotes results in no live-born homozygous Incenp -disrupted progeny, indicating an early lethality. Day 3.5 affected pre-implantation embryos contain large, morphologically abnormal cells that fail to fully develop a blastocoel cavity or thrive in utero and in culture. Chromatin and tubulin immunocytochemical stainings of these and day 2.5 affected embryos reveal a high mitotic index, no discernible metaphase or anaphase stages, complete absence of midbodies, micronuclei formation, morphologically irregular macronuclei with large chromosome complements, multipolar mitotic configurations, binucleated cells, internuclear bridges and abnormal spindle bundling. The phenotype is consistent with a defect in the modulation of microtubule dynamics, severely affecting chromosome segregation and resulting in poorly resolved chromatin masses, aberrant karyokinesis and internuclear bridge formation. These latter occurrences could pose a physical barrier blocking cytokinesis.Human Molecular Genetics 08/1999; 8(7):1145-55. · 7.69 Impact Factor
MOLECULAR AND CELLULAR BIOLOGY, July 2004, p. 6467–6475
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 14
The E4F Protein Is Required for Mitotic Progression during
Embryonic Cell Cycles
Laurent Le Cam,1† Matthieu Lacroix,2Maria A. Ciemerych,1‡ Claude Sardet,2
and Piotr Sicinski1*
Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical
School, Boston, Massachusetts 02115,1and Institut de Ge ´ne ´tique Mole ´culaire de Montpellier,
CNRS UMR 5535, IFR122, 34293 Montpellier, France2
Received 15 January 2004/Returned for modification 29 February 2004/Accepted 9 April 2004
The ubiquitously expressed E4F protein was originally identified as an E1A-regulated cellular transcription
factor required for adenovirus replication. The function of this protein in normal cell physiology remains
largely unknown. To address this issue, we generated E4F knockout mice by gene targeting. Embryos lacking
E4F die at the peri-implantation stage, while in vitro-cultured E4F?/?blastocysts exhibit defects in mitotic
progression, chromosomal missegregation, and increased apoptosis. Consistent with these observations, we
found that E4F localizes to the mitotic spindle during the M phase of early embryos. Our results establish a
crucial role for E4F during early embryonic cell cycles and reveal an unexpected function for E4F in mitosis.
The products of the adenovirus early region 1A (E1A) gene
are potent oncoproteins. E1A was shown to subvert cell pro-
liferation in part by targeting essential cell cycle regulators
such as the retinoblastoma tumor suppressor protein. This, in
turn, leads to the release of the cellular transcription factors of
the E2F family, which then stimulate transcription of the ad-
enoviral E2 gene and of cellular genes that are critical for the
G13S-phase progression (21, 30, 35). In addition to the E2F
transactivators, the E1A 13S oncoprotein regulates the activity
of another cellular transcription factor, termed E4F, that was
shown to be required for transcription of the adenoviral E4
gene (18, 20). Unlike the much-studied E2Fs, the cellular func-
tion of E4F is poorly understood. The E4F gene encodes a
ubiquitously expressed 120-kDa protein, p120E4F, that is struc-
turally homologous to transcription factors of the GLI/Kruppel
family. Upon E1A expression, p120E4Fis proteolytically
cleaved, yielding a 50-kDa protein, p50E4F, which is believed to
represent a transcriptionally active form (7, 17, 18, 20). Al-
though both p50E4Fand p120E4Fcan recognize the same DNA
sequence in vitro (7), they very likely differentially regulate
gene expression in vivo. Thus, while p50E4Fis believed to act as
a transcriptional activator, overexpression of p120E4Fwas
shown to repress transcription of the viral E4 and E1A genes
(9) and of the cellular cyclin A2 gene (4). This repressive action
of p120E4Fmight rely on E4F’s direct interaction with histone
deacetylase 1 (2).
Unlike the well-described cellular roles of other E1A tar-
gets, the physiological function of E4F remains largely un-
known. It has been reported that ectopic expression of p120E4F
inhibits G13S-phase progression in various in vitro-cultured
cell lines (8). Importantly, this E4F-mediated cell cycle arrest is
reduced in pRB- or p53-deficient cells (5, 27), suggesting a
genetic interaction between E4F and these two tumor suppres-
sor pathways. Consistent with this notion, E4F was found to
physically interact with pRB (5) and p53 (27). Other reports
indicated that the ability of p120E4Fto block cell cycle pro-
gression might involve its physical interaction with p14ARF
(22), an increased expression of the p21Cip1and p27Kip1cell
cycle inhibitors (8), or transcriptional repression of the cyclin
A2 gene (4).
To probe the physiological functions of the E4F protein, we
inactivated the murine E4F locus by gene targeting in embry-
onic stem (ES) cells, and we generated E4F-null embryos. Our
analyses revealed that E4F knockout (KO) embryos die at the
peri-implantation stage and show mitotic progression defects,
chromosomal missegregation and increased apoptosis. We
found that these mitotic abnormalities correlate with E4F’s
association with the mitotic apparatus. Our results establish an
unexpected function for E4F in mitosis during early embryonic
MATERIALS AND METHODS
E4F gene targeting vector. Several overlapping genomic fragments encompass-
ing the mouse E4F gene were isolated by screening a lambda phage (provided by
A. McClatchey, Massachusetts General Hospital, Boston, Mass.) and a bacterial
artificial chromosome (Research Genetics) genomic library derived from the
mouse strain 129SvJ, with the full-length human E4F cDNA as a probe. Exons 3
to 14 of the mouse E4F gene were replaced with a phosphoglycerokinase (PGK)-
puromycin poly(A) resistance cassette placed in the orientation opposite to that
of E4F transcription. In addition to the resistance cassette, additional EcoRI and
NarI sites were introduced for screening purposes. The genomic fragments used
for homologous recombination were composed of a 3.8-kb XhoI-KpnI fragment
including part of the E4F promoter region, exons 1 and 2 for the 5? arm, and a
4.5-kb Avr II fragment located downstream of exon 14 of the E4F gene for the
3? arm. Our targeting vector also included a PGK-thymidine kinase-poly(A)
cassette to allow for enrichment of targeted ES cells with ganciclovir. The E4F
targeting vector was linearized with NotI and introduced into ES cells by elec-
troporation with a Gene Pulser (1 pulse of 0.4 kV and 25 ?F; Bio-Rad). The cells
were subsequently cultured for 3 days in the presence of puromycin (2 ?g/ml)
and ganciclovir (2 ?M) and then maintained only in puromycin for six additional
days. Resistant ES cell clones were maintained on a monolayer of triple resistant
(neomycin, puromycin, and hygromycin) feeders and cultured in conditioned ES
* Corresponding author. Mailing address: Dana-Farber Cancer In-
stitute, 44 Binney St., Boston, MA 02115. Phone: (617) 632-5005. Fax:
(617) 632-5006. E-mail: firstname.lastname@example.org.
† Present address: Institut de Ge ´ne ´tique Mole ´culaire de Montpel-
lier, CNRS UMR 5535, IFR122, Montpellier, France.
‡ Present address: Department of Embryology, Institute of Zoology,
Warsaw University, 02-096 Warsaw, Poland.
medium composed of ES Dulbecco’s modified Eagle’s medium (KO DMEM;
GIBCO BRL) supplemented with leukemia inhibitory factor (550 ng/ml; Chemi-
con), 15% heat-inactivated fetal bovine serum (HyClone), 2 mM L-glutamine
(GIBCO), 0.1 mM nonessential amino acid solution (GIBCO BRL), penicillin G
(100 U/ml), streptomycin sulfate (100 ?g/ml), and 50 ?M ?-mercaptoethanol.
Homologous recombination was verified by Southern blotting of genomic DNA
prepared from puromycin-resistant ES cell clones, by using external digests and
external probes as indicated in Fig. 1B. Several correctly targeted E4F?/?ES cell
clones that were confirmed to carry a single copy integration at the E4F locus and
displayed a normal karyotype were subsequently injected into C57BL/6 blasto-
cysts and gave rise to germ line-transmitting chimeric mice.
Blastocyst culture and genotyping of preimplantation embryos. All embryos
were generated by natural mating of E4F heterozygote animals. The morning of
the day on which a vaginal plug was detected was designated as day E0.5.
Embryos were collected on E3.5 or E4.5 by flushing the uteri with M2 medium
(Sigma). Embryos were then either fixed immediately or cultured in ES complete
medium for the appropriate time.
For genotyping, individual embryos were lysed by incubation at 55°C overnight
in 5 ?l (for the blastocysts) or 100 ?l (for E9.5 embryos) of PCR lysis buffer (10
mM Tris-HCl, pH 8.0, 50 mM KCl, 2 mM MgCl2, 0.45% NP-40, 0.45% Tween
20, 0.2 mg of proteinase K/ml). To detect the wild-type (WT) and mutant E4F
alleles, PCRs were performed with the following primers: E4F WT allele, primer
?192 (5? AGGTCTGCTAGGGTATGAGG) and primer ?236 (5? GCCCTAG
CCTGCTCTGCCATC); E4F mutant allele, primer ?22 (5? CACTGCCTTGG
AGGACTTTG) and primer ?227 (5? CCTCTGTTCCACATACACTTCAT
TC). The amplification protocol included an initial incubation step at 94°C for 5
min, followed by 35 cycles with each cycle comprised of 1-min denaturation at
94°C, 1-min annealing at 55°C, and 1 min of elongation at 72°C, with AmpliTaq
Gold polymerase (Applied Biosystems).
Immunocytochemistry and terminal deoxynucleotidyltransferase-mediated
dUTP-biotin nick end labeling (TUNEL) staining. Embryos were washed two
times in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS
for 30 min at 4°C, and then permeabilized for 20 min at room temperature in
PBS containing 0.3% Triton X-100 and 1.5% bovine serum albumin. For BrdU-
treated embryos, DNA was denatured after the permeabilization step with 2 N
HCl–0.5% Triton X-100 for 20 min at room temperature and then washed
extensively in PBS with 1.5% bovine serum albumin. Embryos were incubated
under mineral oil with specific primary antibodies overnight at 4°C. The anti-
Bub1 (clone 4B12) and anti-BubR1/Mad3 (clone 5F9) monoclonal antibodies
and the polyclonal anti-CENP-E antibody were kindly provided by F. McKeon
(Harvard Medical School, Boston, Mass.) and by D. Cleveland (University of
California, San Diego). The polyclonal anti-phospho-(ser10) histone H3 anti-
body was from Upstate Biotechnology, the anti-?-tubulin (clone T9026) was
from Sigma, and the monoclonal anti-BrdU antibody was from Becton Dickin-
son. The affinity-purified anti-E4F polyclonal antibody was raised against resi-
dues 358 to 784 of the human E4F protein (5). Cy-3 or fluorescein isothiocya-
nate-conjugated secondary antibodies (Jackson Immunoresearch Laboratory)
and DAPI (4?,6?-diamidino-2-phenylindole) were incubated for 4 h at room
temperature. DNA fragmentation associated with apoptosis was detected with an
in situ cell death detection kit (Roche).
Generation of E4F-deficient mice. We cloned the mouse
E4F gene and determined its exon-intron structure. The hu-
man and murine E4F genes, located on chromosomes 16p13.3
and 17, respectively (24, 26), share a highly conserved genomic
organization composed of 14 exons spread over the distance of
FIG. 1. Targeting strategy of the E4F locus. (A) Structure of the mouse E4F locus, the targeting vector, and the targeted allele after the homologous
recombination. Solid boxes denote exons. Only restriction sites relevant to the targeting construct and to the screening strategies are indicated.
(B) Southern blot analysis of a representative E4F?/?ES cell clone. Genotypes are shown above each lane. Homologous recombination on both ends
was verified using external digests and external probes. For each digestion (NarI or EcoRI), the bands representing the WT and mutant alleles are
indicated. The genomic fragments used as probes are shown in panel A. PGK, phosphoglycerokinase promoter; TK, thymidine kinase.
6468LE CAM ET AL.MOL. CELL. BIOL.
approximately 12 kb (Fig. 1A). In our gene targeting strategy,
we replaced exons 3 to 14 of the murine E4F gene (which
together encode amino acids 104 to 784 of the E4F protein) by
a puromycin resistance cassette (Fig. 1A). This large deletion
removes all C2H2 zinc-finger domains which are responsible
for E4F DNA binding and functional activities (5, 8, 25).
The E4F targeting construct was electroporated into J1 em-
bryonic stem (ES) cells, and puromycin-resistant clones were
screened for homologous recombination by Southern blotting,
as indicated in Fig. 1B. E4F?/?ES cells were injected into
C57BL/6 blastocysts and gave rise to germ line-transmitting
chimeric mice. These mice were backcrossed to C57BL/6 ani-
mals, yielding E4F heterozygotes. The results reported below
were obtained with two independently derived E4F-deficient
E4F is essential for early embryogenesis. Heterozygous
E4F?/?mice were phenotypically normal, healthy, and fertile
with no developmental or histological abnormalities detectable
over a 20-month observation period (data not shown). In con-
trast, no homozygous E4F-null animals were detected among
366 live births from E4F?/?intercrosses (Table 1), indicating
that one functional E4F allele is sufficient to support full em-
bryonic and adult tissue development, whereas inactivation of
both alleles leads to embryonic lethality.
To assess the time of E4F KO developmental failure, em-
bryos from heterozygote intercrosses were collected at differ-
ent times of gestation and genotyped by PCR. No homozygous
mutant embryos were recovered at E7.5 or beyond (Table 1).
However, we observed empty deciduae at an approximately 1
in 4 ratio, suggesting that E4F-deficient embryos died after the
implantation (Table 1). To verify this notion, the entire uteri
containing E5.5 embryos were processed for histological anal-
yses. Embryos were laser captured from the sections and were
genotyped by PCR. We found that E4F?/?embryos were ei-
ther completely resorbed or markedly abnormal. In the latter
case, mutant embryos exhibited greatly reduced size and dis-
played no detectable layer organization (Fig. 2A, lower pan-
We also collected embryos at day E4.5 by flushing the uteri
of pregnant females, and we analyzed their appearance. We
found that E4F-deficient E4.5 embryos were severely growth
retarded compared to control littermates (Fig. 2A, upper right
panel). In contrast, mutant blastocyst stage embryos flushed
out at E3.5 (n ? 68) were virtually indistinguishable from WT
(n ? 164) or heterozygous (n ? 81) littermates (Fig. 2A, upper
left panel). Visualization of cell nuclei by DAPI staining re-
vealed that all E3.5 blastocysts were composed of 53 ? 8 cells,
irrespective of their genotype (n ? 10 for each genotype).
Collectively, these observations revealed that E4F-deficient
embryos fail at the peri-implantation stage.
To further characterize the developmental abnormalities of
E4F-deficient embryos, we isolated E3.5 blastocysts derived
from E4F?/?? E4F?/?crosses and cultured them in vitro for
several days. As expected, WT or heterozygous embryos ex-
panded after 24 h of culture and later hatched from their zonae
FIG. 2. E4F disruption results in early embryonic lethality. (A) The
appearance of mutant embryos. Upper panels, E3.5 and E4.5 embryos
were flushed out from the uteri and were photographed under bright
field conditions. E3.5 blastocysts were subsequently genotyped by
PCR. Note normal appearance of mutant embryos at E3.5 and severe
growth retardation at E4.5. Lower panels, the appearance of E5.5
embryos developing in utero, as revealed by hematoxylin and eosin
staining of histologic sections. A typical picture of a WT and of a not
yet fully resorbed E4F?/?embryo is shown. (B) Impaired in vitro
development of E4F-deficient embryos. Blastocyst stage embryos were
flushed from the uterus at E3.5, cultured in vitro for several days, and
subsequently genotyped by PCR. While all the littermates displayed
similar morphology at E3.5 (upper left panel), E4F?/?embryos ap-
peared growth retarded after 24 h of culture (upper right panel). After
72 h of in vitro culture, WT and E4F?/?embryos developed out-
growths composed of the inner cell mass (ICM) surrounded by a single
layer of trophoblast giant cells (TGC) (lower left panel), whereas
E4F?/?embryos degenerated inside the zonae pellucidae (lower right
TABLE 1. Genotypes of progeny from E4F heterozygous matinga
No. per genotype
aNA, not applicable; dpc, days postcoitum.
VOL. 24, 2004E4F PROTEIN IS REQUIRED FOR MITOTIC PROGRESSION6469
pellucidae to form a prominent inner cell mass outgrowth on a
flat patch of trophectoderm cells. In contrast, none of the
E4F?/?embryos hatched, and they degenerated inside the
zonae pellucidae after 3 days of in vitro culture (Fig. 2B). The
difference between WT (and E4F?/?) versus E4F?/?embryos
was already obvious after 24 h of in vitro culture (i.e., at a stage
roughly corresponding to E4.5). At this time point, E4F?/?
embryos were invariably smaller than heterozygous or WT
littermates (Fig. 2B, upper right panel) and were composed of
only 88 ? 10 cells (n ? 9), compared with 126 ? 13 cells (n ?
9) in WT embryos. Hence, our in vivo and in vitro analyses
indicate that the E4F gene is critically required during early
Cell cycle progression of E4F?/?embryonic cells. We next
used the in vitro blastocyst cultures to ascertain whether the
developmental failure of E4F-deficient embryos was the con-
sequence of cell proliferation defects. In the initial set of ex-
periments, E3.5 blastocysts that had been cultured in vitro for
24 h were pulse-labeled for 20 min with 5-bromo-2-deoxyuri-
dine (BrdU), and BrdU-positive cells were quantitated by in-
direct immunofluorescence. As shown in Fig. 3, we detected on
average 45% ? 3% of BrdU-positive cells in E4F?/?embryos
(n ? 6), compared to 54% ? 4% in control littermates (n ? 9).
Although statistically significant (Mann-Whitney test, P ?
0.05), this small difference cannot fully explain the 30% de-
crease in cell number observed in E4F-deficient blastocysts
after 24 h of in vitro culture, given that the cell division dou-
bling time at that developmental stage is close to 20 h (10).
These data suggested to us the existence of additional abnor-
malities in E4F-deficient embryos.
Mitotic progression defects in E4F-deficient embryos. We
next turned our attention to the M-phase progression in E4F-
deficient cells. To this end, we immunostained WT and
E4F?/?blastocysts with an antibody against histone H3 phos-
phorylated on serine 10 (HH3 P-Ser10), a common mitotic
marker used to mark M-phase cells (11). We found that all
freshly isolated E3.5 blastocysts showed a very similar propor-
tion of phosphohistone H3-positive (i.e., mitotic) cells, irre-
spective of their genotype (approximately 3% of cells; Fig. 4B).
In contrast, after 24 h of in vitro culture, the mitotic index of
E4F-deficient embryos was threefold higher than that of WT
littermates (Fig. 4A and B).
Careful examination of mitotic figures in E4F?/?embryos
revealed that an unusually large proportion of them corre-
sponded to the prometaphase stage, a phase during which
chromosomes are already fully condensed but not yet aligned
on the metaphase plate (68% of all mitotic figures analyzed in
E4F-deficient, n ? 88, versus 32% in WT embryos, n ? 66; Fig.
4C and D). Consequently, metaphase, anaphase, and telophase
mitotic figures were observed less frequently in E4F-deficient
embryos than in the WT counterparts (Fig. 4D), suggesting a
mitotic progression failure in the absence of E4F.
To further ascertain whether the observed accumulation of
cells in mitosis was indeed the result of a mitotic arrest, we
examined the ability of E4F-deficient cells to exit mitosis. To
this end, we cultured embryos in vitro for 24 h (a time point
when E4F KO embryos exhibit a fully penetrant phenotype)
and then subjected them to 10 Gy of gamma irradiation, in
order to activate their DNA damage checkpoint. In embryonic
cells, this checkpoint does not trigger a mitotic arrest (16) but
mainly induces a G2block (12, 15, 28, 31). Therefore, cells that
are in the M phase at the time of irradiation are expected to
exit mitosis, progress through the cell cycle, and finally accu-
mulate at the next G2phase. As expected, upon gamma irra-
diation of WT embryos, we observed a strong decrease of the
number of HH3 P-Ser 10-positive cells, revealing exit of cells
from the M phase. In contrast, the proportion of mitotic cells
remained unchanged in E4F-deficient embryos after the irra-
diation (Mann-Whitney test, P ? 0.05) (Fig. 4E). These results
suggest that in E4F?/?conceptuses, these HH3 P-Ser 10-pos-
FIG. 3. S-phase progression in E4F-deficient embryos. (A) S-phase progression was gauged by determining BrdU incorporation in blastocysts
recovered from E4F?/?intercrosses. Embryos were cultured in vitro for 24 h, pulsed for 20 min with BrdU, and processed for BrdU immuno-
staining and DAPI counterstaining. (B) Series of Z-plane images were stacked and analyzed by deconvolution microscopy to precisely quantify the
percentage of BrdU-positive cells in WT and E4F?/?blastocysts after 24 h of culture. Error bars indicate standard deviations.
6470LE CAM ET AL.MOL. CELL. BIOL.
itive cells are unable to exit mitosis but are instead arrested or
“trapped” at the prometaphase stage. Altogether, our data
suggest that E4F is critically required for M-phase progression
in embryonic cells.
We next tried to address the molecular basis of the mitotic
arrest seen in E4F-deficient embryos. We hypothesized that
E4F?/?cells were blocked at the prometaphase stage due to
the constitutive activation of their spindle checkpoint. The
core components of this checkpoint were first identified in
yeast and are highly conserved in mammals. These proteins,
which include the protein kinases Bub1, BubR1 (also called
Mad3), and the motor protein CENP-E (for a review, see
reference 1), can be detected by immunofluorescence in mi-
totic cells with an activated spindle checkpoint (33).
In order to assess whether E4F?/?cells have activated their
spindle checkpoint, we stained in vitro-cultured E4F-deficient
CENP-E. Indeed, we observed that the kinetochores of
E4F?/?mitotic cells were strongly labeled by anti-Bub1 (Fig.
5A), anti-BubR1 (Fig. 5B), and anti-CENP-E (data not shown)
antibodies, indicating that E4F?/?cells are arrested in pro-
metaphase with an activated spindle checkpoint.
When we analyzed the cellular localization of E4F in the
early embryos by indirect immunofluorescence, we detected
endogenous E4F protein in the nucleus of interphasic cells
(data not shown), a localization consistent with the previously
described function of E4F as a transcription factor. Notably, in
mitotic cells, we noticed immunolabeling of E4F on the mitotic
spindle which colocalized with ?-tubulin (Fig. 6). In contrast,
no staining of E4F was observed on the condensed mitotic
chromosomes. Importantly, both interphasic and mitotic stain-
ing patterns were strongly reduced in E4F?/?blastocysts. In
addition, preincubation of the anti-E4F antibody with recom-
binant full-length E4F protein completely abolished the stain-
ing in WT embryos (data not shown), demonstrating that this
immunoreactivity is specific.
FIG. 4. M-phase progression defects in E4F-deficient embryos. (A) Increased mitotic index in E4F?/?embryos. Shown are representative WT
and mutant littermates that were cultured in vitro for 24 h and then fixed and stained with antibody against phospho-(Ser 10) histone H3 (HH3
P-Ser10), a marker of mitotic cells. Nuclei were counterstained with DAPI. (B) The mitotic index in freshly isolated E3.5 embryos (T ? 0) or in
embryos cultured in vitro for 24 h (T ? 24) was calculated as the mean number of HH3 P-Ser10-positive cells per embryo divided by the mean
number of cells per embryo, times 100% (n ? 15 for each genotype). (C and D) E4F?/?cells are blocked at the prometaphase stage. (C) Typical
prometaphase figures (arrows) observed after DAPI staining of cultured E4F-deficient embryos. (D) Mitotic figures were identified in cultured WT
and E4F?/?embryos based on the HH3 P-Ser10 immunostaining and were classified into the appropriate mitotic stage. The data are presented
as percentages of these various stages among all M-phase cells. (E) Defective mitotic exit in E4F?/?embryos. Embryos were isolated at E3.5,
cultured for 24 h, gamma irradiated, and left in culture for six additional hours. Embryos were then fixed, processed for the HH3 P-Ser10
immunostaining, and subsequently lysed individually for genotyping by PCR analysis. The number of mitotic cells (HH3 P-Ser10 positive) in
nonirradiated (black circles) or irradiated embryos (open circles) is plotted according to their genotype.
VOL. 24, 2004 E4F PROTEIN IS REQUIRED FOR MITOTIC PROGRESSION 6471
Strikingly, we frequently noticed abnormal mitotic figures in
E4F-deficient embryos, where one or two misaligned chromo-
somes were positioned far outside of the metaphase plate (Fig.
7A). In a total of more than 100 mitotic figures analyzed, 18%
displayed abnormalities with misaligned chromosomes. Such
abnormal mitotic figures were extremely rarely observed in
normal embryos (1 in 100 analyzed).
Altogether, our data suggest that the lack of E4F results in
the chronic activation of the spindle checkpoint and the arrest
of E4F-deficient mitotic cells at the prometaphase stage and
leads to chromosomal misalignment.
Increased cell death in E4F?/?embryos. We further hypoth-
esized that the chromosomal segregation defects seen in E4F
KO cells might eventually result in increased cell death in
FIG. 5. Embryonic cells that lack E4F are arrested in prometaphase with an active spindle checkpoint, as revealed by Bub1 and BubR1
immunostaining. Examples of WT embryos, representing various phases of the M-phase progression, and representative E4F-deficient embryos
(KO) were costained in panel A with anti-HH3 P-Ser10 antibodies, anti-Bub1 antibodies, and DAPI, or in panel B, with anti-HH3 P-Ser10
antibodies, anti-BubR1 antibodies, and DAPI.
6472LE CAM ET AL.MOL. CELL. BIOL.
mutant conceptuses, leading to the death of the entire embryo.
Consistent with this hypothesis, DAPI staining revealed the
presence of numerous condensed and fragmented nuclei, a
hallmark of apoptotic cells, in in vitro-cultured E4F-deficient
embryos. We further confirmed this observation by TUNEL
staining. Our analyses revealed that apoptotic cells were rarely
detected in freshly isolated E3.5 blastocysts, irrespective of the
embryo genotype (data not shown). However, after 24 h of in
vitro culture, TUNEL-positive cells were readily detected in
E4F?/?embryos but were rarely observed in their heterozy-
gous or WT littermates (Fig. 7B). Collectively, these results
suggest that cell death is responsible, at least in part, for the
demise of E4F-deficient embryos.
In this study, we generated a mouse mutant for the E4F
gene. The product of this gene was originally identified as a
cellular protein activated by the viral E1A oncoprotein during
adenoviral infection (13, 18, 20). Our analyses of E4F-deficient
embryos demonstrated a critical and nonredundant function
for E4F during early murine embryogenesis between days E3.5
and E5.5. Normal development of E4F?/?embryos prior to
day E3.5 might suggest that E4F is dispensable at these very
early embryonic stages. Alternatively, we reason that the nor-
mal development of E4F-deficient embryos up to the blastocyst
stage may rely on protein stocks of maternal origin.
By using an in vitro outgrowth model that recapitulated the
in vivo developmental failure of E4F-deficient embryos, we
showed that E4F?/?conceptuses exhibit mitotic progression
defects. Our analyses revealed that E4F-deficient cells are
blocked at the prometaphase stage with an activated spindle
checkpoint. It is interesting that several mouse strains engi-
neered to lack proteins involved in the spindle checkpoint also
die at the peri-implantation stage, with mitotic abnormalities
resembling the ones found in E4F-deficient embryos. These
strains include the Mad2 (32), the survivin (34), the Incenp (3),
and CENP-E mutant mice (19). However, our results clearly
indicate that in contrast to the Mad2- and CENP-E-null em-
bryos, E4F-deficient blastocysts display an increased mitotic
index, supporting the idea that E4F?/?embryos have a func-
tional spindle checkpoint. One possibility is that—in a manner
similar to survivin-depleted cells (14)—E4F?/?cells cannot
FIG. 6. E4F is localized on the mitotic spindle. WT blastocysts were immunostained with a specific anti-E4F antibody. The mitotic spindle was
visualized by ?-tubulin immunostaining. Nuclei were counterstained with DAPI.
FIG. 7. E4F?/?embryos exhibit abnormal mitotic figures and in-
creased cell death. (A) Mitotic cells in in vitro-cultured blastocysts were
identified by HH3 P-Ser10 immunostaining. Examples of abnormal mi-
totic figures seen in E4F-deficient embryos are shown. Arrows indicate
misaligned chromosomes. (B) Increased apoptosis in E4F-deficient em-
bryos. Merged image obtained from representative WT and E4F?/?blas-
tocysts cultured for 24 h and processed for DAPI and TUNEL staining.
VOL. 24, 2004 E4F PROTEIN IS REQUIRED FOR MITOTIC PROGRESSION6473
permanently sustain this mitotic arrest, and they finally exit
mitosis in an aberrant manner. Consistent with this model, we
frequently observed abnormal mitotic figures with misaligned
chromosomes in E4F-deficient embryos.
While it is our prediction that the increased cell death seen
in E4F-deficient embryos is the consequence of the mitotic and
chromosomal abnormalities observed in E4F-null mutants, our
data do not allow us to formally rule out an independent
function for E4F in controlling apoptosis in embryonic cells.
Surprisingly, while E4F overexpression-based experiments
suggested an important role for this transcription factor at the
G1/S transition, we have not detected any major abnormalities
in the ability of E4F KO embryos to incorporate BrdU. How-
ever, we found that the knockdown of the E4F mRNA by RNA
interference in in vitro-cultured NIH 3T3 fibroblasts resulted
in a strong inhibition of BrdU incorporation (data not shown).
These results suggest that E4F may have different functions in
early embryonal cells versus those in cells at later stages of
development. Interestingly, a recent report revealed that over-
expression of p120E4Fin fibroblasts led to the formation of a
subset of tetraploid and multinucleated cells (23), raising a
possibility that E4F may also play roles in mitosis in these cells.
The availability of an E4F conditional KO strain should greatly
facilitate analyses of E4F’s function in various cell types.
We do not know at present whether the function of E4F in
cell cycle progression of early embryonic cells involved E4F’s
transcriptional activities. In this regard, E4F target genes that
may play a role in mitotic progression remain unknown. The
work of Fajas et al. indicated that E4F acts as a transcriptional
regulator of the cyclin A2 gene (4). However, this target gene
is likely not involved in the observed embryonic defect since
our analyses (data not shown), as well as that of other labora-
tories (36), demonstrated that cyclin A2 is not expressed in
blastocysts. Based on the localization of the E4F protein to the
mitotic spindle in M-phase cells, we propose that E4F plays a
direct role in mitosis, possibly in chromosome congression.
This function is likely independent from the role of E4F as a
transcription factor. Consistent with this hypothesis, while this
paper was under review, Fenton et al. reported that p120E4F
physically interacts with the tumor suppressor gene product
RASSF1A (6). RASSF1 was recently demonstrated to be in-
volved in controlling mitotic progression through the inhibition
of the ubiquitin ligase activity of the Cdc20–anaphase-promot-
ing complex (29). Collectively, these findings point to an in-
triguing possibility that E4F may be also involved in the regu-
lation of anaphase-promoting complex functions. Further
experiments will be required to address this hypothesis.
We thank Rod Bronson for help with histopathological analyses, F.
McKeon and D. Cleveland for plasmids and reagents, J. Alberta and
M. Donohoe for technical assistance, and A. Le Cam, T. Makela, L.
Kim, and R. Hipskind for critical reading of the manuscript. We are
grateful to all members of the Sicinski’s laboratory for their support
and technical help and to C. Bouchard for help with statistical analyses.
This work was funded by the Susan Komen Breast Cancer Founda-
tion grant to P.S. L.L. was supported by postdoctoral fellowships from
La Ligue contre le Cancer and the Human Frontier Science Program,
M.L. by a predoctoral MNRT fellowship, and M.A.C. by International
Agency for Research on Cancer and Kosciuszko Foundation fellow-
ships. C.S. is funded by the French Ligue Nationale contre le Cancer
(Equipe Labelise ´e 2003).
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