1 Wellcome Trust/Cancer Research UK Institute, University of Cambridge, Tennis
Court Road, Cambridge CB2 1QR, UK
2 LBNL, MS 84-171, 1 Cyclotron Road, Berkeley, CA 94720, USA
3 Laboratory of Lymphocyte Signaling, the Rockefeller University, 1230 York
Avenue, New York, NY 10021, USA
4 IMP, Dr. Bohrgasse 7, A-1030 Vienna, Austria
Author for correspondence (e-mail:
as10021{at}mole.bio.cam.ac.uk)
Accepted 23 May 2003
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SUMMARY |
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Key words: Polycomb, Ezh2, Histone methylation, X-inactivation, Pluripotency, Mouse
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INTRODUCTION |
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There are two mammalian homologues of enhancer of zeste: Ezh1,
which is expressed predominantly in adult tissues, and Ezh2, which is
expressed primarily in early development
(Laible et al., 1997).
Mutations of Ezh2 and Eed are very early embryonic lethal
(Faust et al., 1998
;
O'Carroll et al., 2001
;
Schumacher et al., 1996
).
Ezh2 is also essential for the derivation of pluripotent embryonic
stem (ES) cells (O'Carroll et al.,
2001
), and it is upregulated in prostate cancer
(Varambally et al., 2002
).
Furthermore, Ezh2 and Eed are implicated in the early events of imprinted
X-inactivation (Mak et al.,
2002
; Wang et al.,
2001b
), which may be a temporally regulated event
(Wutz and Jaenisch, 2000
).
Ezh2 and Eed are part of a single multimeric complex that includes histone
deacetylase 1 and 2 (Tie et al.,
2001
; van der Vlag and Otte,
1999
).
Ezh2 has a conserved SET (suppressor of variegation, enhancer of zeste and
trithorax) domain (Laible et al.,
1997), which is associated with histone methylation of lysine
tails (Strahl and Allis,
2000
). SET domains associated with Suv3-9H1/2
(Rea et al., 2000
), G9a
(Tachibana et al., 2002
), ESET
(Yang et al., 2002
), Set9/7
(Nishioka et al., 2002
;
Wang et al., 2001a
) and
PR-Set7 (Nishioka et al.,
2002
) all show histone methylation activities. Ezh2 also exhibits
histone methylation activity in Drosophila and human cells
(Cao et al., 2002
;
Czermin et al., 2002
;
Kuzmichev et al., 2002
;
Muller et al., 2002
). Histone
methylation can either repress or activate gene expression as it is present in
both euchromatin and in heterochromatin. The two best-studied sites of histone
H3 methylation are Lys9 (H3-K9), which is generally associated with repressive
chromatin, and Lys4 (H3-K4), which is found in transcriptionally active
DNA.
Little is known at present about the role of Ezh2 in very early mouse development. We first examined the role of maternally inherited Ezh2, and show that Ezh2 and Eed are involved in asymmetrical histone methylation of parental genomes in the early zygote. Depletion of maternal Ezh2 affects Ezh2-Eed colocalisation and histone methylation of the parental genomes, which results in a long-term effect on embryonic growth. At the blastocyst stage, we found that the Ezh2-Eed complex co-localises on the X chromosome in trophectoderm cells, where we detected H3-K27 methylation at the onset of X-inactivation; both the co-localisation of Eed and the histone methylation patterns were lost in the Ezh2 mutant blastocysts. We also detected a characteristic histone methylation pattern associated with the pluripotent epiblast cells in blastocysts, which was also abolished in the Ezh2 mutant embryos. Thus, Ezh2 is involved in early epigenetic events that regulate the histone methylation pattern in pluripotent epiblast cells and at the onset of differentiation of trophectoderm cells.
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MATERIALS AND METHODS |
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Genotyping of mice
Mice were genotyped by PCR or by standard Southern blot analysis with a 1
kb probe 1 on genomic tail tip DNA to identify wild type, targeted and deleted
Ezh2 alleles as described (Su et
al., 2003)
Embryo collection and activation
Embryos were obtained from superovulated B6CBAF1 female mice as described
(Hogan et al., 1994).
Fertilised or unfertilised oocytes were collected in PB1 medium containing 300
µg/ml hyaluronidase and incubated for a few minutes to allow the cumulus
cells to be shed. Pre-implantation embryos at other stages were usually
collected by flushing the oviducts and uteri. Oocytes and pre-implantation
embryos were cultured in T6/BSA under mineral oil at 37°C with 5%
CO2. Parthenogenetic embryos were obtained following incubation in
7% alcohol for 4.5 minutes (Cuthbertson,
1983
) followed by incubation with Cytochalasin B for 4 hours to
create diploid embryos.
Immunofluorescence and chromosome paint
Embryos were fixed in 4% PFA for 15 minutes and washed three times with PBS
and permeabilised in AB-buffer (1% Triton-X100, 0.2% SDS, 10 mg/ml BSA in PBS)
for 30 minutes. They were then incubated in primary antibody diluted in
AB-buffer overnight at 4°C, followed by three AB-buffer washes of 10
minutes each and incubation in secondary antibodies (Alexa 564, Alexa 488,
Molecular probes) for 1-2 hours in AB-buffer at room temperature, followed by
AB-buffer washes as before and one wash in PBS. Finally the embryos were
incubated in 0.1 mg/ml RNase A (Roche) in PBS at 37°C for 30 minutes and
mounted on slides in Vectashield (Vector Laboratories) containing propidium
iodide.
Primary embryonic fibroblasts (PEFs) were allowed to settle down on
poly-L-lysine (Sigma)-coated slides after trypsination. Cells were rinsed in
PBS and fixed for 15 minutes in 4% PFA in PBS at room temperature, followed by
the protocol described above. X chromosome painting (Cambio) was performed as
described in the manufacture's manual. Prior to in-situ hybridisation, embryos
were treated as described (Costanzi and
Pehrson, 1998) and immunofluorescence performed as described
above. Immunofluorescence was visualised on a BioRad Radiance 2000 confocal
microscope.
The following antibodies and dilutions were used: Ezh2 (A. Otte, 1:150), Eed (A. Otte, 1:100), HDAC1 (New England Biolabs, 1:100), H3-me2-K4 (Upstate, 1:500), H3-me2-K9 (Upstate, 1:500), H3-4xme2-K9 (T. Jenuwein, 1:250), H3-me3-K9 (abcam, 1:150), H3-me1-K27 (abcam, 1:150), H3-me2-K27 (abcam, 1:150). Characterisation of H3-me3-K9, H3-me1-K27 and H3-me2-K27 are published elsewhere (http://www.abcam.com); H3-me3-K27 (T.J., unpublished). Antibodies were tested for their specificity in peptide competition assays (see supplementary Figures S1-S3 at http://dev.biologists.org/supplemental/).
Retroviral infection
Retrovirus was produced in Phoenix cells (gift from the Nolan laboratory)
as described (Jackson-Grusby et al.,
2001) and cells transfected with pMXpuro-CRE or pMXpuro-GFP using
MBS KIT (Stratagene) as described in the manual. 72 hours after transfection,
retrovirus containing supernatant was collected as described in pVPack vectors
instruction manual (Stratagene) and PEFs from 12.5 dpc embryos infected with
undiluted supernatant containing 4 µg/ml polyprene instead of 10 mg/ml
DEAE-Dextran.
Immunoprecipitation
ES cells (from 129aa/129aa mice) were cultured on feeder layers until 80%
confluency in LIF (1000 U/ml, ESGRO, Life Technologies) supplemented standard
growth medium, washed twice in PBS and spun down at 1300 g.
The cell pellet from five 15 cm dishes was lysed in low-salt IPH buffer (50 mM
Tris, pH 8.5; 100 mM NaCl; 0.5% NP40), vortexed and incubated on ice for 10
minutes. The lysate was spun down at 15,000 g at 4°C for
30 minutes and the supernatant incubated with Ezh2 antibodies or no antibodies
(mock) at 4°C for 2 hours before adding washed sepharose G and A beads
(1:1) (Pharmacia) and overnight incubation at 4°C. Beads were washed three
times with low-salt IPH and subjected to methyltransferase assay.
Methyltransferase assays
For methyltransferase assays, the immunoprecipitated proteins or the mock
precipitation were incubated with 10 µg of H3 peptides (H3 1-16:
ARTKQTARKSTGGKAPGGC and H3 24-32 AARKSAPATGGC, methylated on lysines as
indicated) together with 0.5 µCi of S-adenosyl-L-(methyl-3H) methionine
(NEN) in methyltransferase buffer (final concentration: 50 mM NaCl, 25 mM
TrisHCl pH. 8.8) for 1 hour at 30°C. Then 1 ml of binding buffer (20 mM
TrisHCl pH 8.0, 5 mM EDTA pH 8.0) was added to the supernatant and the peptide
was coupled to 25 µl of SulfLink beads (Pierce) for 1 hour. The beads were
washed three times and the radioactivity counted by liquid scintillation.
Western blotting
Supernatant (5 µl) and IP-beads (5 µl) were boiled in SDS loading
buffer for 10 minutes. Proteins were separated on 9% polyacrylamide gels,
blotted onto Immobilon-P membranes (Millipore), and probed for Ezh2 (1:1000),
Eed (1:1000) or HDAC1 (1:500) according to standard protocols. Primary
antibodies were detected with anti-mouse-HRP or anti-rabbit HRP (Amersham)
followed by ECL detection (NEN Life Sciences).
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RESULTS |
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Ezh2 depletion disrupts histone H3 methylation in the early
zygote
Histone H3-tails can potentially be methylated at H3-K4, H3-K9 and H3-K27.
As the Ezh2-Eed complex from ES cells showed histone methylation activity (see
below), we examined control zygotes for histone methylation (see Materials and
Methods; see supplementary Figures S1-S3 at
http://dev.biologists.org/supplemental/).
We found that mono-, di- and tri-H3-K27 methylation is strongly associated
with the female pronucleus and the second polar body at the onset of
development (Fig. 2A; left),
while staining of the paternal pronucleus was observed several hours later,
with a similar time course as shown for Ezh2
(Fig. 1B). The same situation
was observed with di- and tri-H3-K9 specific antibodies. In mutant zygotes
however, H3-K9 (not shown) and H3-K27 (Fig.
2A; right) was almost undetectable. When we tested H3-K4
methylation, the same asymmetry to the female pronucleus was observed but this
was unchanged in Ezh2 mutant zygotes (not shown). We conclude that Ezh2 has a
role in histone H3 methylation at least for H3-K27 and/or K9 methylation,
either during oocyte development or in the zygote.
|
Developmental consequences of the depletion of maternally inherited
Ezh2
To verify that Ezh2 is maternally inherited, we stained ovaries from
wild-type female mice with Ezh2-specific antibodies. We found that Ezh2 is
transcribed and stored as protein in the growing oocyte but not in the
surrounding adult ovarian somatic tissues
(Fig. 3A). To deplete the
oocytes of this maternal inheritance, we used an Ezh2 conditional
allele in which exons 8-11 of the Ezh2 gene were flanked by LoxP
sites so that parts of the C-terminus, including the conserved SET domain,
would be deleted in the presence of Cre-recombinase
(Su et al., 2003). As mice
inheriting two floxed alleles of Ezh2 (Ezh2F/F)
were viable and without any detectable phenotypic consequences
(Su et al., 2003
), we crossed
Ezh2F/F female mice with mice expressing Cre recombinase
driven by the Zp3 regulatory elements (ZP3-Cre), which is expressed
exclusively in the growing oocyte prior to the completion of the first meiotic
division (de Vries et al.,
2000
; Epifano et al.,
1995
). The Ezh2F/F-Zp3-Cre females did show
deletion of Ezh2 (see Fig.
1C).
|
However, unlike the Ezh2-null mice that show early embryonic
lethality (O'Carroll et al.,
2001), Ezh2DEL/F development proceeds to term,
probably because the wild type paternal allele is activated at the four-cell
stage and overcomes the early lethal phenotype observed in the homozygous
mutant embryos. Indeed, we never obtained homozygous
Ezh2Del/Del animals from any of our
Ezh2F/F crosses, which is consistent with our previous
studies (O'Carroll et al.,
2001
). By contrast, heterozygous Ezh2+/-
animals with normal maternal Ezh2 showed no detectable phenotype, which
demonstrates that the maternal Ezh2 is necessary for normal development.
Our attempts to delete the paternal Ezh2F/F allele
using the sperm-specific Cre line, Sycp1
(Vidal et al., 1998) were
unsuccessful. Therefore, we cannot rule out a function for Ezh2 during
spermatogenesis or test the effect of the paternally inherited Ezh2 null
allele. The phenotypic consequences of various genotypes with respect to
Ezh2 are summarised in Fig.
3C.
The role of Ezh2 during preimplantation development to the blastocyst
stage
During development of preimplantation embryos to the blastocyst stage from
a totipotent zygote, there is differentiation of trophectoderm cells and
development of the inner cell mass (ICM) with pluripotent epiblast cells.
Differentiation of trophectoderm (TE) is associated with the initiation of
paternal X chromosome inactivation. We therefore went on to examine how the
loss of Ezh2 affects these key epigenetic events.
The influence of Ezh2 on X-inactivation
To examine the role of Ezh2 during preimplantation development, we
generated embryos depleted of both maternally inherited product and embryonic
transcription (see Fig. 1C). To
do so, we used activated oocytes (Barton et
al., 2001; Hogan et al.,
1994
), thus avoiding the necessity for fertilisation and therefore
the inheritance of a wild-type Ezh2 allele from sperm. Diploid parthenogenetic
embryos (see Fig. 1A) have been
studied extensively and shown to develop appropriately at least up to the
blastocyst stage (Cuthbertson,
1983
). Furthermore, although it is the paternal X chromosome that
is preferentially inactivated in the trophectoderm in fertilised blastocysts
(imprinted X inactivation), one of the two maternal X chromosomes in
parthenogenetic embryos is selected to undergo inactivation in all embryos
(Kay et al., 1994
), which is
reminiscent of the normal `counting' mechanism involved in random X
inactivation in the embryo proper of normal fertilised embryos, indicating
that some of the mechanisms involved in X-inactivation are conserved in random
and imprinted X inactivation.
To determine if there was a marked effect on development on embryos completely devoid of Ezh2, we compared parthenogenetic embryos from normal oocytes (henceforth called PG+/+), with parthenogenetic embryos from Ezh2-deficient oocytes (henceforth called PG-/-). PG+/+ embryos, like fertilised embryos, will possess both the maternally inherited Ezh2, and the products of embryonic transcription, albeit from two maternal alleles. We found that both PG+/+ and PG-/- developed to the blastocysts stage in vitro at about the same rate (not shown).
We then went on to examine the distribution of Ezh2 and Eed in
trophectoderm cells, where they co-localise at the paternal X chromosome that
undergoes X-inactivation (Mak et al.,
2002). We found that
50% of normally fertilised embryos from
wild type animals (presumptive XX) showed this pattern (not shown). More
importantly, all of the PG+/+ blastocysts, which have two maternal
X chromosomes, showed a similar co-localisation of Ezh2 and Eed to one of the
two X chromosomes (Fig.
4A,B,D). This Ezh2-Eed co-localisation with the Xi was
confirmed by Eed staining which always overlapped with the strongest macro-H2A
signal, a histone variant, which is highly enriched on the Xi in
trophectoderm (Costanzi and Pehrson,
1998
) (Fig.
4E).
|
Ezh2-dependent histone methylation in trophectoderm cells
Next, we examined changes in histone methylation patterns at the blastocyst
stage, and found no detectable differences when comparing PG+/+ and
blastocysts resulting from normal fertilisation. Both sets of blastocysts are
referred to as control and compared with PG-/- blastocysts.
Immunostainings with an antibody raised against H3-me3-K9
peptides showed brightly stained foci in controls
(Fig. 5A, upper panel), which
was similar to the pattern observed with the `branched-K9' antibody, which
recognises higher-order H3-K9 methylated chromatin
(Maison et al., 2002). The
intensity and number of H3-me3-K9 foci in PG-/-
blastocysts were greatly diminished, especially in the TE cells
(Fig. 5A; far-right picture). A
more striking staining pattern was observed with H3-me2-K27
antibodies, which in controls showed a strong overall staining of the ICM
while TE cells showed a particularly intense staining concentrated in one spot
per nucleus, similar to the staining pattern we observed for Ezh2 and Eed at
this stage of development (Fig.
5B). This H3-me2-K27staining also co-localises to one
DNA dense region in trophoblast cells. Examining PG-/- blastocysts
we found that the accumulation of H3-me2-K27 in trophectoderm cells
was completely lost (Fig. 5B, far-right). We observed identical staining patterns in controls with a
me3-K27 specific antibody (Fig.
5C). By contrast, we did not observe any obvious differences in
staining pattern or intensity with the H3-me2-K9-specific antibody
when we compared PG-/- blastocysts with control embryos (see
supplementary Figures S1-S3 at
http://dev.biologists.org/supplemental/).
These results indicate that the Xi is associated with H3-K27
methylation and that Ezh2, either directly or indirectly, mediates this
methylation. Furthermore, in control embryos, H3-me3-K27
co-localises with Eed, the interacting partner of Ezh2
(Fig. 5C). This H3-K27
methylation is associated with one X chromosome as shown by immunostaining in
combination with X chromosome-specific fluorescence immunohybridisation in TE
cells (Fig. 5D). As H3-K27
methylation co-localises with Eed and Ezh2 in TE cells, we can conclude that
the Xi is associated with H3-K27 methylation at the onset of
X-inactivation. Again, we observed a few cells in the ICM that were positive
for H3-me2-K27 and H3-me3-K27, which were largely
unaffected in PG-/- blastocysts.
|
Ezh2 and histone methylation patterns in the ICM
With the H3-K27 antibodies (me1-K27, me2-K27,
me3-K27), we obtained a very intense staining in the ICM of
controls, whereas the trophectoderm cells showed only a weak overall staining
except for the X chromosome (Fig.
5B,C; Fig. 6A; see
supplementary Figures S1-S3 at
http://dev.biologists.org/supplemental/).
This striking difference in histone methylation staining shows that there is
an epigenetic difference between cells of the pluripotent ICM and
differentiated TE cells.
|
To test whether Ezh2 is also necessary in cells from later embryos, we
obtained primary embryonic fibroblasts (PEFs) with a null and floxed allele
for Ezh2 and infected them with Cre or GFP expression retroviral
supernatant (Jackson-Grusby et al.,
2001). When the floxed allele was excised by Cre to generate PEFs
lacking Ezh2, we could not detect a significant effect on proliferation in
these cells compared with the controls (see
Fig. 6C), even though Ezh2
protein levels were highly reduced in Cre-infected cells as early as 3 days
after infection (Fig. 6D).
Thus, Ezh2 is essential for early developmental events and in pluripotent ES
cells, but not in differentiated cells.
The Ezh2/Eed complex has histone methyltransferase activity for K9
and K27
Histone modifications as judged by antibody staining suggest that Ezh2 has
histone methyltransferase activity (HMTase), particularly for K9 and K27,
which is abolished in Ezh2 depleted embryos. To verify this observation, we
assayed for HMTase activity in pluripotent murine ES cells. We
immunoprecipitated Ezh2 from total ES cell extract and tested the antibody
bound fraction for HMTase activity. Using differentially methylated peptides
as substrates (Fig. 7A), we
observed HMTase activity specifically for H3-K9 and for H3-K27, with the
highest activity for H3-K27 (Fig.
7B). Furthermore, we confirmed by western blotting the presence of
Ezh2, Eed and HDAC1 in the precipitated fraction
(Fig. 7C). Our data strongly
indicate that the Ezh2/Eed complex has an intrinsic H3-K9/K27 specific HMTase
activity that is probably associated with Ezh2 that has the SET domain
characteristic. Recent findings from Drosophila and human cells
support our findings (Cao et al.,
2002; Czermin et al.,
2002
; Kuzmichev et al.,
2002
; Muller et al.,
2002
).
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DISCUSSION |
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The purpose and importance of the epigenetic asymmetry between parental
genomes has been considered in the context of genomic imprinting
(Surani, 2001;
Ferguson-Smith and Surani,
2001
). It is thought that the oocyte cytoplasm has played a
significant role in discriminating between parental genomes during evolution,
for example in the selective elimination of paternal chromosomes in some lower
organisms (Werren and Hatcher,
2000
). The epigenetic differences between parental genomes in the
mouse zygote are not observed in lower vertebrates and may have arisen
according to the genomic conflict hypothesis that was proposed to explain
genomic imprinting (Haig and Graham,
1991
; Reik and Walter,
2001
). In this context, disruption of imprinting in interspecific
hybrids of the deermouse Peromyscus maniculatus, may be due to
nuclear-cytoplasmic incompatibility (Vrana
et al., 1998
). As the neonates recover from the growth deficiency
after weaning, it is possible that the absence of Ezh2 may affect the
placenta. It has been shown, for example, that specific disruption of
imprinting of Igf2 in the placenta has a marked effect on foetal
growth, which is subsequently restored in neonates after weaning
(Constancia et al., 2002
). We
propose in the future to examine whether the absence of Ezh2 in oocytes has an
effect on placental development, on physiological functions and on the status
of imprinted genes.
Mammalian development is also marked by the early activation of the
embryonic genome. Ezh2, Eed and other epigenetic modifiers may establish a new
higher-order chromatin structure that may be important for this purpose
(Fig. 8). As with DNA
methylation, establishment, maintenance and heritability of histone
methylation patterns may be distinct events requiring different modifiers. For
example, HMTases, including G9a and Ezh2, are required early in development
whereas others, such as Suv3-9H, have a more subtle effect on early
development (O'Carroll et al.,
2001; Peters et al.,
2001
; Tachibana et al.,
2002
). The Ezh2-Eed complex contains HDAC1/2 as well as the
intrinsic HMTase activity, which are two significant chromatin modifications
that have the potential for establishing new gene expression patterns. There
is continuing genome-wide DNA demethylation during preimplantation
development, which reaches the lowest point at the blastocyst stage
(Carlson et al., 1992
;
Howlett and Reik, 1991
;
Kafri et al., 1993
;
Monk et al., 1987
). It is
perhaps at this time that the Ezh2-Eed complex might play a crucial role in
establishing a new epigenetic pattern in the epiblast and the trophectoderm.
It is possible that the epigenetic patterns established by histone methylation
may be followed by appropriate DNA methylation patterns, as seems to be the
case in Neurospora (Tamaru and
Selker, 2001
) and possibly in Arabidopsis
(Jackson et al., 2002
).
The role of Ezh2-Eed in the initiation of X-inactivation in
trophectoderm cells
We have shown that the Ezh2-Eed complex in blastocysts colocalises to the
Xi, an event that is disrupted in the Ezh2 mutant embryos
accompanied by the loss of H3-K27 methylation. The recruitment of the Ezh2-Eed
complex to the Xi is restricted to early development only, but
X-inactivation is stably propagated thereafter. We propose that the Ezh2-Eed
complex has the enzymatic properties to establish an early mark for
X-inactivation in the form of H3-K27 methylation prior to H3-K9 methylation.
Once H3-K27 methylation is established, we suggest two alternative pathways
for establishing stable X-inactivation. Either H3-K27 methylation recruits
further factors or complexes with H3-K9 HMTase activity, or H3-K9 methylation
on the Xi is also catalysed by Ezh2. In contrast to H3-K27
methylation, we do not see a strong co-localisation of Ezh2 and H3-K9
methylation. However, if we assume that H3-K9 methylation follows H3-K27
methylation at the blastocyst stage, the time window of co-localisation
between Ezh2-Eed and H3-K9 could be very short.
As stated above, we have made similar observations in both PG+/+
and normal blastocysts. Since PG+/+ have two maternal X
chromosomes, there would be a random choice of which chromosome undergoes
inactivation, as suggested from previous studies
(Iwasa, 1998). It is therefore
also likely that the same mechanism involving Ezh2-Eed must operate during
random inactivation in XX embryonic cells.
Ezh2 and pluripotency
Early mammalian development differs from that in other vertebrates because
of the necessity to generate extraembryonic trophectoderm cells, as well as
the pluripotent epiblast cells, from which both the embryo proper and germ
cells are derived. Oct4 is clearly essential for pluripotency
(Rossant, 2001). It is
noteworthy that the presence of Oct4 is always accompanied by the presence of
Ezh2 and Eed, which is the case in the zygote, early blastomeres, epiblast and
pluripotent stem cells. It is important to note a characteristic and distinct
staining pattern for H3-K27/9 histone methylation in pluripotent epiblast
cells compared with trophectoderm cells, which coincides with the expression
of Oct4-GFP. We know that both Ezh2 and Oct4 are also highly expressed in
pluripotent ES cells, and they are both crucial for the propagation of the
pluripotent state; their levels decline after the onset of differentiation.
Loss of function of either of these two genes results in the loss of these
pluripotent cells (Nichols et al.,
1998
). The Ezh2-mediated H3-K27/K9 methylation in pluripotent
epiblast cells is clearly affected in Ezh2 depleted blastocysts. These results
strongly argue that Ezh2 is important for maintaining the epigenetic
plasticity of pluripotent epiblast and of ES cells. It is also noteworthy that
in both the zygote and in germ cells major epigenetic reprogramming events
occur against a background of high Ezh2 and Oct4
(Hajkova et al., 2002
;
Lee et al., 2002
). In summary,
we have shown here that the early histone modifications in the zygote, the
trophectoderm and in epiblast cells all depend on Ezh2. Thus, Ezh2 seems to
mediate histone modifications in totipotent/pluripotent cells, and it may be
crucial for early cell fate determination. Our study may, in the future,
provide a link between histone modifications, epigenetic gene regulation by
PcG genes and the establishment of epigenetic marks that are apparently
crucial for early development and for pluripotency.
Very recent publications on Ezh2 support our findings and conclusions that
the Xi is associated with H3-K27 methylation
(Silva et al., 2003;
Plath et al., 2003
).
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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* To whom requests for mice carrying conditional Ezh2 allele should be
sent
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