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Molecular and Cellular Biology, July 2001, p. 4330-4336, Vol. 21, No. 13
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.13.4330-4336.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Polycomb-Group Gene
Ezh2 Is Required for Early Mouse Development
Dónal
O'Carroll,1
Sylvia
Erhardt,2
Michaela
Pagani,1
Sheila C.
Barton,2
M. Azim
Surani,2 and
Thomas
Jenuwein1,*
Research Institute of Molecular Pathology
(IMP), A-1030 Vienna, Austria,1 and
Wellcome/CRC Institute of Cancer and Developmental Biology,
Cambridge CB21QR, United Kingdom2
Received 22 December 2000/Returned for modification 1 February
2001/Accepted 28 March 2001
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ABSTRACT |
Polycomb-group (Pc-G) genes are required for the
stable repression of the homeotic selector genes and other
developmentally regulated genes, presumably through the modulation of
chromatin domains. Among the Drosophila Pc-G genes,
Enhancer of zeste [E(z)] merits special
consideration since it represents one of the Pc-G genes most conserved
through evolution. In addition, the E(Z) protein family contains the
SET domain, which has recently been linked with histone
methyltransferase (HMTase) activity. Although E(Z)-related proteins
have not (yet) been directly associated with HMTase activity, mammalian
Ezh2 is a member of a histone deacetylase complex. To investigate its
in vivo function, we generated mice deficient for Ezh2.
The Ezh2 null mutation results in lethality at early
stages of mouse development. Ezh2 mutant mice either cease developing after implantation or initiate but fail to complete gastrulation. Moreover, Ezh2-deficient blastocysts
display an impaired potential for outgrowth, preventing the
establishment of Ezh2-null embryonic stem cells.
Interestingly, Ezh2 is up-regulated upon fertilization
and remains highly expressed at the preimplantation stages of mouse
development. Together, these data suggest an essential role for
Ezh2 during early mouse development and genetically link Ezh2 with eed and YY1, the
only other early-acting Pc-G genes.
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INTRODUCTION |
Polycomb-group (Pc-G)
genes are required for the stable repression of the homeotic selector
genes and other developmentally regulated genes, thereby providing
differentiation programs with a "transcriptional memory" (reviewed
in reference 25). Among the Drosophila Pc-G
genes, Enhancer of zeste [E(z)] (19,
20) and extra sex combs (esc)
(12) are exceptional, since both genes are required early
during development, in contrast to other Drosophila Pc-G
genes that appear to have later functions. For example, E(z) is involved in the repression of some of the early-acting
segmentation-gap genes (24, 26). E(z) and
esc are the Pc-G genes most highly conserved throughout
evolution, being the only Pc-G genes found in the Caenorhabditis
elegans genome (17, 21). E(z) function also influences chromosome integrity during the rapid nuclear divisions
in the first 2 h of development (19). Moreover,
E(z) null alleles have been shown to reduce immunostaining
of several Pc-G proteins in polytene chromosomes and to result in a
general decondensation of chromatin structure (28) that is
reflected by increased chromosome breakage and a low mitotic index
(11). Thus, E(z) appears to be a pleiotropic
Pc-G gene with functions in epigenetic gene regulation, chromosome
architecture, and growth control.
Mammalian homologues of E(z) have been isolated and
characterized and are encoded by two loci in the mouse, Ezh1
and Ezh2 (Enhancer of zeste homologues)
(15, 22). Alignments of Ezh1 and Ezh2 proteins with
Drosophila E(Z) reveal four conserved regions of homology,
including domain I, domain II, and a cysteine-rich amino acid stretch
which precedes the C-terminal SET domain (22). The
primary difference between the Ezh loci appears to lie
in their expression profiles, with Ezh2 being predominantly
expressed during embryonic development, whereas Ezh1 is more
abundant in adult tissues (22).
Genetic and biochemical evidence from both Drosophila and
the mouse support the existence of two distinct Pc-G complexes, with
eed-Ezh2 defining one complex (33, 37) and Pc-G
proteins such as Bmi-1 and Polycomb constituting the other
(2). eed is the murine homologue of the
Drosophila Pc-G gene esc (32) and is
required for gastrulation in the mouse (8). By contrast, gene targeting approaches in the mouse revealed later developmental functions for members of the Bmi-1-Polycomb complex (reviewed in
reference 36). Another Pc-G protein, YY1, was identified through homology to pleiohomeotic (3) and was subsequently shown to reside within the Bmi-1-Polycomb complex (10)
and also to interact with eed (31). YY1 is unique among
the Pc-G proteins in that it displays sequence-specific DNA binding
activity (3), with disruption of YY1 resulting
in peri-implantation lethality (7).
E(Z) is an original member of Drosophila chromatin
regulators which defined the evolutionarily conserved SET domain
(reviewed in reference 18). The SET domain of mammalian
SU(VAR)3-9 homologues has recently been shown to harbor an intrinsic
histone methyltransferase activity (29). Although
E(Z)-related proteins have not so far been associated with histone
methyltransferase activity, histone deacetylases (HDACs) 1 and 2 are
present in the eed-Ezh2 complex (35), and transcriptional
repression of this complex is mediated via histone deacetylation
(35). Ezh proteins interact with eed through domain II
(33, 37) but do not by themselves recruit HDACs
(35). The SET domain of E(Z)-related proteins has been shown to be a target for mammalian Sbf-1 (5), a SET domain binding antiphosphatase, providing a link for Ezh in the control of
proliferation and differentiation (5). In addition, domain II of Ezh2 interacts with the product of the proto-oncogene
Vav (14), suggesting that Ezh2 may also be
involved in signal-dependent T-cell activation. A potential role for
Ezh2 in B- and T-cell development and proliferation
is complemented by its abundant expression in the thymus and in organs
of the developing immune system (15, 22).
With strong evidence linking Ezh2 to conserved mechanisms of
eukaryotic gene silencing (22) and a possible role for
Ezh2 in mouse development, growth control, and B- and T-cell
proliferation, we decided to address the in vivo function of
Ezh2 by generating an Ezh2 null mutation in the
mouse germ line.
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MATERIALS AND METHODS |
Generation and genotyping of Ezh2 mutant
mice.
Ezh2 maps to mouse chromosome 6 (23). A partial genomic clone of Ezh2
(23) was used to generate short (1.4 kb) and long (4.2 kb)
arms of homology, in a strategy to produce an in-frame fusion of the
first 200 amino acids of Ezh2 with 
-galactosidase (LacZ) modified
with a nuclear localization signal. The diphtheria toxin A
(DTA) gene under the control of the MCI promoter (provided by T. Kallunki and M. Karin, San Diego, Calif.) was used to select against random integration of the targeting construct and was inserted
3' of the long arm of homology. A pGNA-derivative targeting construct
comprising a neomycin gene for positive selection and two
polyadenylation sites (see Fig. 1A) was linearized with NotI and electroporated into feeder-independent CCE and feeder-dependent embryonic day 14.1 (E14.1) embryonic stem (ES) cells.
ES cells were put under G418 selection, and homologous recombination
was screened by PCR, using a nested reaction with primers external to
the short arm (Ezh2 PCR1, 5'-GTTCTGGATCAGGATGGCAC; Ezh2
PCR2n, 5'-GTTGCCATGACAGTGGCAGCTC) and primers in the
lacZ gene (lacZ PCR1,
5'-AACCCGTCGGATTCTCCGTGGGAAC; lacZ PCR2,
5'-CTCAGGAAGATCGCACTCCAGCC). Targeting was confirmed by
Southern blot analysis of EcoRI-digested ES cell DNA with a
360-bp internal probe, generated with the primers Ezh2 probe 4f
(5'-TCTGTTTATGGGGCATGTGC) and Ezh2 probe 4r
(5'-ATCTGGAAGAATAGTGAAGGC). This probe detects a 3.4-kb
fragment from the wild-type allele and a 1.7-kb fragment from the
targeted allele (see Fig. 1A and B).
E14.1 targeted ES cells were used to generate chimeric mice.
Heterozygous mice were interbred to obtain
Ezh2-deficient
embryos,
which were genotyped by PCR using the following primers: g33
(5'-GTGCTGATAGGCCTTCAC)
and lacZ PCR1 (above) to amplify a
580-bp fragment from the targeted
allele, and Ezh2 900r
(5'-CAGAGCACCTGGGAGCTGCTG) and g3
(5'-GAGGTTGATTCTTGTTTCCTATGC)
to amplify a 240-bp fragment
from the wild-type allele (see Fig.
1A and
C).
Embryological and histological techniques.
Basic
embryological and histological methods were performed as described
elsewhere (16). Pregnant mice at defined times postcoitus
were sacrificed by cervical dislocation, the deciduae were isolated,
and the embryos were dissected.
Whole-mount RNA in situ hybridization analysis.
A 275-bp
fragment of the EZH2 cDNA (22), encoding amino
acids 330 to 455, was cloned into the pGEM-3Zf vector (Promega) to
derive sense and antisense riboprobes. This human EZH2 probe contains 11 base pair mismatches (95% identity) with the corresponding portion of the murine Ezh2 gene and can be used to detect
Ezh2 transcripts. Whole-mount RNA in situ hybridization of
embryos was performed as described elsewhere (4).
Blastocyst outgrowth and ES cell derivation.
Blastocyst
outgrowth and ES cell derivation procedures were performed as described
elsewhere (1).
RT-PCR to detect Ezh1 and Ezh2
transcripts.
Total RNA was prepared from pools of oocytes,
fertilized oocytes, morulae, and blastocysts using glycogen as a
carrier. Reverse transcription (RT) was performed with random hexamers
(G. Schaffner, IMP) on the above-described RNA preparations and on 1 µg of total spleen RNA. Primer pairs that are specific for
Ezh1 (E1f, 5'-TGAAATCTGAGTATATGCGGC; E1r,
5'-AGATATCCTGGCTGTCGAAC) or Ezh2 (E2f,
5'-GCCAGACTGGGAAGAAATCTG; E2r,
5'-TGTGCTGGAAAATCCAAGTCA) were used in a subsequent PCR to generate a 236-bp product for Ezh1 and a 270-bp product for
Ezh2. The mouse Gapdh (30) primers
(G1, 5'-CGGAGTCAACGGATTTGGTCGTAT; G2,
5'-AGCCTTCTCCATGGTGGTGAAGAC) were used to control
for the quality and amount of the reverse-transcribed RNA.
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RESULTS |
Targeting the Ezh2 locus.
A conventional
targeting approach was used to inactivate the Ezh2 locus on
mouse chromosome 6 (23). The Ezh2 gene was
disrupted by homologous recombination in ES cells, replacing parts of
exons five and six with a lacZ gene and a neo
gene (Fig. 1A). This targeting strategy
produces an in-frame fusion of the first 200 amino acids of Ezh2 with
LacZ. Domain I, the eed interaction region (33, 37), is
present in the fusion protein, but domain II and the C-terminal SET
domain will not be expressed upon targeting (Fig. 1D). The Ezh2-LacZ
fusion protein could not be used as a marker of Ezh2
expression because it did not retain -galactosidase activity (data
not shown). The frequency of homologous recombination in feeder-dependent ES cells was 11%. Injection of three independently targeted clones produced several high chimeras from two clones. Chimeras derived from one targeted clone passed the mutation through the germ line. EcoRI-restricted DNA from heterozygous mice
produces a 3.4-kb fragment from the wild-type locus and a diagnostic
1.7-kb fragment from the targeted allele (Fig. 1B). Homozygous mutation of the Ezh2 gene results in early embryonic lethality (see
below). In order to genotype Ezh2 mutant embryos, a
PCR-based approach to detect both the wild-type and mutant alleles was
developed. The locations of the respective primer pairs are indicated
in Fig. 1A. PCR amplification produces a fragment of 240 bp for the endogenous allele and a fragment of 580 bp for the targeted allele (Fig. 1C). To demonstrate the generation of a loss-of-function allele,
RNA in situ hybridization was performed on wild-type and Ezh2-deficient day 7.5 embryos, using an
Ezh2-specific antisense probe; Ezh2 transcripts
hybridizing to this antisense probe were not detectable in mutant
embryos (Fig. 1D).
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FIG. 1.
Targeting and genotyping of
Ezh2-deficient mice. (A) Diagrammatic representation of
the Ezh2 genomic locus, the replacement vector, and the
targeted Ezh2 allele. Exons are indicated by black boxes
with numbers referring to the starting amino acid positions of the
respective exons (23). Also shown are the diagnostic
EcoRI restriction sites and the internal probe used for
Southern blot analysis, as are the primer pairs (arrowheads) for PCR
genotyping. pA indicates polyadenylation signals. (B) Southern blot
analysis of EcoRI-restricted DNA isolated from offspring
of heterozygous (het) intercrosses. wt, wild type. (C) PCR genotyping
of genomic DNA isolated from embryos of heterozygous intercrosses. (D)
Whole-mount RNA in situ hybridization with day 7.5 wild-type and
Ezh2 null embryos with an Ezh2-specific
antisense probe.
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Ezh2 is required for mouse development.
Genotyping of offspring by Southern blot analysis from Ezh2
heterozygous intercrosses revealed the absence of mice homozygous for
the Ezh2 mutation (Table 1),
indicating that Ezh2 is required for embryonic development.
In order to determine when the Ezh2 mutation produces a
lethal phenotype, timed matings were set up and embryos were obtained
at day 7.5 and day 10.5. Genotyping of day 10.5 embryos by Southern
blot analysis revealed that no Ezh2 mutant mice were
identified (Table 1). We next analyzed day 7.5 embryos. Genotyping by
PCR indicated the presence of Ezh2 mutants, albeit just
under Mendelian ratios. All mutant embryos identified by PCR were
significantly smaller than littermates and fell into two distinct
categories. The first category comprised embryos which were extremely
growth retarded and resembled in size a day 5.5 embryo. The second
category of embryos comprised those that were growth retarded and
displayed increased amounts of extraembryonic tissue. Indeed, the ratio
between embryonic and extraembryonic tissues was visibly skewed, with
only a small part of the embryo consisting of embryonic regions.
Sub-Mendelian numbers of Ezh2 heterozygous animals were
observed at birth (Table 1). Genotyping of day 10.5 embryos derived
from either heterozygous intercrosses or wild-type-to-heterozygous
crosses revealed the presence of Mendelian ratios of heterozygous
animals (Table 1 and data not shown). This suggests that a fraction of
Ezh2 heterozygous mice develop problems during mid- to late
gestation. However, the nature of these heterozygous defects was not
further investigated.
Arrested development and gastrulation failure in embryos from
Ezh2 heterozygous intercrosses.
We then examined
day 7.0 to day 8.5 embryos derived from heterozygous intercrosses
histologically. Entire deciduae were isolated, fixed, and stained with
hematoxylin-eosin. Preparations were sectioned, and embryos were scored
for being either of wild-type (Fig. 2A) morphology, abnormal, or resorbed. At day 8.5, no abnormal embryos were
identified, although a significant number of resorptions were observed
(Table 2). By contrast, at day 7.0 to day
7.5, 11 abnormal embryos and 3 resorptions were detected. These 11 presumptive Ezh2-deficient mice fell into two distinct
categories, similar to the analysis with whole-mount preparations (see
above). The most severely growth-retarded embryos ceased developing
shortly after implantation and resembled a day 5.5 embryo (Fig. 2B).
This category of mutants accounted for 45% (5 of 11) of the abnormal embryos. The other mutants appeared to initiate but not complete gastrulation (Fig. 2C and D). The initiation of gastrulation was confirmed by the presence of mesoderm cells and verified by RNA in situ
analysis of the mesoderm marker brachyury (13)
in whole-mount null embryos (data not shown). However, the majority of
the mesoderm-like cells appear to migrate into extraembryonic regions
and form a bulge of cells which pushes on the embryonic portion of the
embryo (Fig. 2C). The embryo illustrated in Fig. 2D consists almost
completely of extraembryonic tissues and represents a mutant which
continued the aberrant cell migration shown in Fig. 2C. The mutant
embryos represented in Fig. 2C and D each accounted for 27% (3 of 11) of abnormal embryos. This later-onset phenotype resembles that of the
eed mutation, where embryos fail to complete gastrulation due to defects in morphogenetic movements (9).
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FIG. 2.
Arrested development and gastrulation failure in
presumptive Ezh2-deficient embryos. Deciduae at day 7.0 to day 7.5 were isolated, sectioned at 5-µm thickness, stained with
hematoxylin-eosin, and scored for being normal or abnormal based on
size and morphology. (A) An almost-sagittal section through a wild-type
embryo at day 7.5. (B to D) Sections of presumptive Ezh2
mutants isolated at days 7.0 to 7.5. The arrow in panel C highlights
excessive amounts of mesoderm aberrantly accumulating onto the abnormal
embryo. The different magnifications of each photograph are indicated.
Abbreviations: A, anterior; Al, allantois; Am, amnion; E, embryonic;
EE, embryonic ectoderm; Ec, ectoplacental cone; Ex, extraembryonic;
ExE, extraembryonic ectoderm; P, posterior; Ch, chorion; VE, visceral
endoderm.
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We investigated the spatial expression profiles of
Ezh2 by
whole-mount RNA in situ hybridizations with day 6.5 to day 9.5
mouse
embryos. Whereas no staining was observed with the control
sense probe
(Fig.
3D), the
Ezh2 antisense
probe revealed broad
expression of
Ezh2 throughout the
gastrulating mouse embryo (day
6.5; Fig.
3A). This expression profile
indicates a role for
Ezh2 during gastrulation and would be
consistent with the defects of
the presumptive
Ezh2-deficient embryos described above. However,
Ezh2 was also expressed broadly at later stages of mouse
development
(E7.5 to E9.5; Fig.
3B and C), suggesting additional
functions
for
Ezh2 in postgastrulation development.
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FIG. 3.
Ezh2 is broadly expressed during early
mouse embryogenesis. Whole-mount RNA in situ hybridization analysis of
wild-type day 6.5 (A), day 7.5 (B), and day 9.5 (C) embryos with an
Ezh2-specific antisense probe or, as a control, with an
Ezh2-specific sense probe (D) is shown. Abbreviations:
A, anterior; Al, allantois; Am, amnion; Ch, chorion; EE, embryonic
ectoderm; M, mesoderm; P, posterior.
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Ezh2 is required for ES cell derivation.
To
further analyze the Ezh2 null phenotype, we decided to
generate Ezh2-deficient ES cells, which would allow us to
gain insight into Ezh2 function through cellular assays and
chimera studies. We derived ES cells from blastocysts obtained from
heterozygous intercrosses. The inner cell mass (ICM) of expanded
blastocysts was disaggregated and seeded under ES cell conditions,
while the trophoblast layer was used for PCR genotyping. From 62 blastocysts processed for ES cell derivation, 33 ES cell lines were
established, and none of these lines was homozygous for the
Ezh2 mutation (Table 3). A
representative wild-type ES cell clone is shown in Fig. 4A. Attempts to derive ES cells from null
blastocysts resulted in non-ES-like cells that failed to proliferate,
acquired large vacuoles, and subsequently died (Fig. 4B). ES cell lines
were established from both wild-type and heterozygous blastocysts with an approximate efficiency of 62% (Table 3). With this degree of
efficiency, we would have expected 6 null ES cell lines from 10 null
blastocysts; we therefore conclude that Ezh2 is required for
the derivation of ES cells.
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FIG. 4.
Impaired ES cell derivation and outgrowth potential of
Ezh2-deficient blastocysts. Blastocysts were isolated
from heterozygous intercrosses at day 3.5 and processed for either ES
cell derivation (A and B) or outgrowth experiments (C to F). In panel
A, a wild-type (wt) ES cell colony is shown, whereas panel B depicts
the highly vacuoled and short-lived mutant cells derived from an
Ezh2 null blastocyst. (C to F) Blastocysts were cultured
in vitro for 7 days and photographed. Panels C and D show successful
outgrowths from a wild-type blastocyst and an Ezh2 null
blastocyst. Arrows point to the ICM and to the trophoblast giant cells
(GC). Each percentage indicates the proportion of blastocysts
(n = 68) displaying the depicted phenotype (see
Table 4).
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Impaired outgrowth potential of Ezh2-deficient
blastocysts.
We next examined the outgrowth potential of
blastocysts. Blastocysts were cultured under conditions where the ICM
should expand and outgrow, while the trophoblast layer should become
adherent and differentiate into giant cells (Fig. 4C). Cells were kept in culture for 7 days after seeding, photographed for documentation, and subsequently genotyped by PCR. Seventy-five percent of wild-type blastocysts and 51% of heterozygous blastocysts underwent normal outgrowth, compared to only 18% of null blastocysts (Table
4; Fig. 4D). The null blastocysts, which
failed to expand normally, displayed three main phenotypes. One subset
of null blastocysts did not show growth of the ICM, while the
trophoblast layer attached and differentiated into giant cells (termed
"no ICM" in Table 4; Fig. 4E). Another group of null blastocysts
did not show any growth of the ICM or of the trophoblast (termed "no
outgrowth" in Table 4; Fig. 4F). The final group of null blastocysts
was nonviable and died after a brief time in culture. These data
demonstrate that blastocyst outgrowth in vitro is impaired in the
absence of Ezh2, which appears to be primarily attributable
to the failure of the growth of the ICM, since the trophoblast layer of
Ezh2-deficient blastocysts has the potential to adhere and
differentiate into giant cells.
Ezh2 is up-regulated in fertilized oocytes and
preimplantation embryos.
Finally, we determined the
preimplantation expression profile of Ezh2 by RT-PCR on
total RNA prepared from oocytes, fertilized oocytes, and
preimplantation embryos. To control for the relative abundance of
Ezh2 transcripts, we included RT-PCR analyses for the second
murine Ezh gene, Ezh1 (22), and for
the ubiquitously expressed Gapdh gene. Whereas
Ezh1 is expressed in fertilized oocytes, Ezh1
transcripts are not detectable in morulae or blastocysts and are also
not present in unfertilized oocytes (Fig.
5, upper panel). By contrast,
Ezh2 is also up-regulated upon fertilization but remains
abundantly expressed at all stages of preimplantation (Fig. 5, middle
panel). Splenic RNA was used as a positive control for the RT-PCR,
since both Ezh loci are similarly expressed in this tissue
(22). These data indicate persistent expression of
Ezh2, but not of Ezh1, in mouse preimplantation
embryos.
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FIG. 5.
Preimplantation expression of Ezh1 and
Ezh2 in the early mouse embryo. RT-PCR was used to
detect Ezh1, Ezh2, and
Gapdh transcripts in total RNA preparations from
wild-type oocytes, fertilized oocytes, morulae, and blastocysts. As a
control, RT-PCR was also performed on total RNA from spleen tissue.
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DISCUSSION |
Ezh2 is an early-acting Pc-G gene during mouse
preimplantation.
Based on the above analysis, Ezh2
joins YY1 as the only other described Pc-G gene
(7) with expression at preimplantation stages of mouse
development (Fig. 5). By contrast, although eed and Ezh2 physically
interact (33, 37), zygotic eed expression does
not appear to start until shortly after implantation at day 5.5 (32). These data suggest additional and earlier roles for Ezh2 outside the eed-Ezh2 complex. Ezh2 could function
during preimplantation development to prepare the embryo for rapid cell proliferations and the initiation and maintenance of complex
gene expression programs. Since Ezh2 is a SET domain-containing
protein, it is intriguing to infer that some of these early functions
may impinge on a more regional modification of the underlying chromatin structure, presumably by enhancing or interfering with the methylation of a yet-unknown substrate.
Alternatively, Ezh2 may be recruited to sites in the genome of
the preimplantation embryo which require heritable regulation
upon
implantation. During implantation and concomitant with the
onset of
eed expression, Ezh2 would interact with eed to target
HDACs
(
35) to these genomic sites. Indeed, this hierarchy is
supported by this and other (G. Lagger, D. O'Carroll, T. Jenuwein,
and
C. Seiser, unpublished data) genetic studies with the mouse.
Ezh2-deficient blastocysts display an impaired potential for
outgrowth
(Table
4 and Fig.
4D to F), and a subset of
Ezh2
mutants (45%)
cease to progress after implantation (Fig.
2B). The
later-developing
Ezh2 mutants bear a striking resemblance to
those with the
eed phenotype, which is required for
morphogenetic movements during
gastrulation, with
eed
mutants dying around day 8.5 (
8,
9).
Similarly, a portion
of
Ezh2 mutants (55%) initiate but fail to
complete
gastrulation and also display an apparent defect in mesoderm
migration
(Fig.
2C and
D).
Ezh2: a regulator of proliferation?
The
Ezh2 null mutation results in early embryonic lethality, and
Ezh2-deficient mouse embryos die during the transition
from pre- to postimplantation development (Tables 1 and 2 and
Fig. 2). This transition is accompanied by elevated rates of cell
division, and around gastrulation these divisions are characterized by
very short cell cycles (4 to 6 h for some cell types)
(16). The broad expression profile of Ezh2
during pre- and postimplantation stages of mouse development (Fig. 5
and Fig. 3, respectively) would be consistent with Ezh2
having a more global role in the control and/or progression of
proliferation. Although Ezh2-deficient blastocysts maintain the potential to outgrow both the ICM and the trophoblast cells, most null blastocysts fail to expand their ICM in vitro (Tables
3 and 4 and Fig. 4). Similarly, Ezh2-deficient embryos show arrested development after implantation (Fig. 2).
There are many additional lines of evidence linking
Ezh2
function to proliferation in cell types that are more specialized
than
those present in the preimplantation mouse embryo. For example,
Ezh2
was also identified through an interaction with the gene
product of the
proto-oncogene
Vav (
14), which is required as
a
signaling molecule for stimulated lymphocyte proliferation
(
34).
Resting splenic B or T cells do not express
Ezh2, but upon mitogen
stimulation,
Ezh2 is
rapidly up-regulated (D. O'Carroll and T.
Jenuwein, unpublished data).
In addition, the transition of resting
mantle B cells to proliferating
follicular centroblasts coincides
with the appearance of both Ezh2 and
eed expression (
27). In
general, SET domain proteins
appear to be important chromatin-associated
targets for signaling
pathways involved in regulating growth control.
Forced expression of
Sbf-1, a SET domain binding factor resembling
an antiphosphatase
(
5), results in oncogenic transformation
of fibroblasts or
enhanced proliferation of primary B-cell cultures
(
6).
Sbf-1 was shown to interact with the SET domain of E(Z)-related
proteins (
5).
In summary, we have shown that the Pc-G gene
Ezh2 is
required for early mouse development. The severity of the phenotype
distinguishes
Ezh2 from most other Pc-G genes and defines
Ezh2 as an early-acting
gene along with
YY1 and
eed (
7,
32). Our genetic analysis
thus
indicates a functional overlap for these Pc-G genes during
mouse
preimplantation and extends the recent observation that
YY1 can
physically interact with the eed-Ezh2 protein complex
(
31). Although
Ezh2 represents one of the most
early-acting
Pc-G genes, it is also broadly expressed in the
postimplantation
embryo and in lymphoid organs (
15,
22).
Based on the data
presented here, we propose that
Ezh2 is an
important regulator
for growth control whose disruption at later stages
of mouse development
would severely impair the overall proliferation
potential of
Ezh2-mutated
cells.
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ACKNOWLEDGMENTS |
We thank Gotthold Schaffner for sequence analysis and
oligonucleotide synthesis, Hans-Christian Theussl for blastocyst
injection of ES cell clones, Terry Magnuson and Elizabeth M. Morin-Kensicki (Case Western Reserve University, Cleveland, Ohio) for
kind teaching of early embryological techniques, Annette Neubüser
for advice on Ezh2 mutant morphology, and Stephen Rea for
critical reading of the manuscript.
Research in T.J.'s laboratory is supported by the IMP through
Boehringer Ingelheim, the Austrian Research Promotion Fund, and the
Vienna Economy Promotion Fund. Research in M.A.S.'s laboratory is
supported by a Wellcome Trust Grant (036481), and S.E. holds a Ph.D.
scholarship from Boehringer Ingelheim.
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FOOTNOTES |
*
Corresponding author. Mailing address: Research
Institute for Molecular Pathology, Dr. Bohrgasse 7, A-1030 Vienna,
Austria. Phone: (43/1) 797-30-474. Fax: (43/1) 798-7153. E-mail:
jenuwein{at}nt.imp.univie.ac.at.
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Molecular and Cellular Biology, July 2001, p. 4330-4336, Vol. 21, No. 13
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.13.4330-4336.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Miller, T., Krogan, N. J., Dover, J., Erdjument-Bromage, H., Tempst, P., Johnston, M., Greenblatt, J. F., Shilatifard, A.
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[Abstract]
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Abstract
Introduction
