Histone H3K27 methyltransferase Ezh2 represses Wnt
genes to facilitate adipogenesis
Lifeng Wanga, Qihuang Jina, Ji-Eun Leea, I-hsin Sub,1, and Kai Gea,2
aNuclear Receptor Biology Section, Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of
Health, Bethesda, MD 20892; andbLaboratory of Lymphocyte Signaling, The Rockefeller University, New York, NY 10065
Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved March 12, 2010 (received for review January 4, 2010)
Wnt/β-catenin signaling inhibits adipogenesis. Genome-wide
profiling studies have revealed the enrichment of histone H3K27
methyltransferase Ezh2 on Wnt genes. However, the functional
significance of such a direct link between the two types of devel-
opmental regulators in mammalian cells, and the role of Ezh2 in
adipogenesis, remain unclear. Here we show Ezh2 and its H3K27
methyltransferase activity are required for adipogenesis. Ezh2
directly represses Wnt1, -6, -10a, and -10b genes in preadipocytes
and during adipogenesis. Deletion of Ezh2 eliminates H3K27me3
on Wnt promoters and derepresses Wnt expression, which leads
to activation of Wnt/β-catenin signaling and inhibition of adipo-
genesis. Ectopic expression of the wild-type (WT) Ezh2, but not the
enzymatically inactive F667I mutant, prevents
H3K27me3 and the defects in adipogenesis in Ezh2−/−preadipo-
cytes. The adipogenesis defects in Ezh2−/−cells can be rescued by
expression of adipogenic transcription factors PPARγ, C/EBPα, or
inhibitors of Wnt/β-catenin signaling. Interestingly, Ezh2−/−cells
show marked increase of H3K27 acetylation globally as well as on
Wnt promoters. These results indicate that H3K27 methyltransfer-
ase Ezh2 directly represses Wnt genes to facilitate adipogenesis
and suggest that acetylation and trimethylation on H3K27 play
opposing roles in regulating Wnt expression.
embryonic development and adult tissue homeostasis (1). In the
canonical Wnt signaling pathway, also know as the Wnt/
β-catenin signaling pathway, Wnt binding to cell surface recep-
tors leads to the stabilization and accumulation of free β-catenin
in the cytoplasm. The accumulated cytosolic β-catenin trans-
locates to the nucleus, where it binds to sequence-specific tran-
scription factors LEF/TCF and functions as a transcriptional
activates expression of Wnt target genes that regulate various
developmental processes. However, how Wnt genes are regulated
remains poorly understood.
Wnt/β-catenin signaling inhibits adipogenesis (2). Activation
of Wnt/β-catenin signaling by expression of Wnt1 or Wnt10b, or
by chemicals that stabilize cytosolic free β-catenin, blocks adi-
pogenesis (3). Wnt/β-catenin signaling prevents the induction of
peroxisome proliferator-activated receptor-γ (PPARγ) and
CCAAT/enhancer binding protein α (C/EBPα), the two principal
adipogenic transcription factors that cooperate to control pre-
adipocyte differentiation (adipogenesis). In addition, β-catenin
inhibits the transcriptional activity of PPARγ (4). Conversely,
inhibition of Wnt/β-catenin signaling by expressing Axin1 or
dominant-negative TCF4 (dnTCF4) promotes adipogenesis (3).
Polycomb group proteins are transcriptional repressors that
help maintain the cell identity during development through
chromatin modification (5). Mammalian polycomb group pro-
teins form two multisubunit complexes, polycomb repressive
complexes 1 and 2 (PRC1 and PRC2), respectively (5, 6). PRC2
contains three core subunits: Ezh2, Suz12, and EED. Through its
he Wnt genes encode an evolutionarily conserved family of
secreted proteins that play critical roles in regulating
histone methyltransferase subunit Ezh2, PRC2 methylates his-
tone H3 on lysine 27 (H3K27). The resulting H3K27 trimethy-
lation is specifically recognized and bound by the PRC1 complex
to facilitate transcriptional repression (6). PRC2 and PRC1 are
localized on a large number of developmental genes in embry-
onic stem (ES) cells. Disruption of PRC2 by deletion of Ezh2,
Suz12, or EED in ES cells markedly decreases the global levels
of H3K27 di- and trimethylation (H3K27me2 and H3K27me3)
and derepresses many polycomb target genes (7–12).
Genomewide profiling studies have revealed the enrichment
of H3K27 methyltransferase Ezh2 and associated H3K27me3 on
Wnt genes in Drosophila and mammalian cells (7, 13, 14). How-
ever, the functional significance of such a direct link between the
two types of developmental regulators in mammalian cell differ-
entiation has not been shown. In addition, the role of Ezh2 in
adipogenesis remains unclear. Using Ezh2 conditional knockout
cells,here weshow Ezh2and itsH3K27 methyltransferase activity
are required for adipogenesis. Ezh2 directly represses multiple
Wnt genes to facilitate adipogenesis. We also provide evidence
to suggest that acetylation and trimethylation on H3K27 play
opposing roles in regulating Wnt expression.
Severe Adipogenesis Defects in Ezh2−/−Primary Preadipocytes. To
investigate the role of H3K27 methyltransferase Ezh2 in adipo-
genesis, we isolated primary white preadipocytes from Ezh2
conditional knockout Ezh2flox/floxmice (15). Cells were infected
with adenovirus expressing Cre (Ad-Cre) to acutely delete the
Ezh2 gene. Deletion of Ezh2 was confirmed by quantitative
reverse-transcriptase PCR (qRT-PCR) (Fig. S1A). Gene expres-
sion analysis revealed increased expression of known Ezh2 target
genes including Hox, p16Ink4a, and p19Arfin Ezh2−/−primary
preadipocytes (Fig. S1B).
Two days after cells reached confluence, preadipocytes were
induced to undergo adipogenesis. Deletion of Ezh2 resulted in
a severe adipogenesis defect in primary white preadipocytes (Fig.
S1C). Consistent with the morphology, Ezh2 deletion blocked
expression of adipogenesis markers PPARγ, C/EBPα, and aP2
(Fig. S1D). Similarly, deletion of Ezh2 in primary brown pre-
adipocytes resulted in increased expression of known Ezh2 target
genes and severe defects in adipogenesis and associated
expression of markers for brown adipocytes (Fig. S1 E–H).
Author contributions: L.W. and K.G. designed research; L.W., Q.J., J.-E.L., and K.G. per-
formed research; I.-h.S. contributed new reagents/analytic tools; L.W., Q.J., J.-E.L., and
K.G. analyzed data; and L.W. and K.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: Microarray data have been deposited in NCBI GEO database (accession
1Present address: Division of Genomics and Genetics, School of Biological Sciences,
Nanyang Technological University, Singapore 639798.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
| April 20, 2010
| vol. 107
| no. 16
Because primary preadipocytes had a limited growth potential
of only several passages in culture, it was difficult to obtain
sufficient cells for mechanistic studies. Further, it was unclear
whether the observed adipogenesis failure in Ezh2−/−primary
preadipocytes was due to a differentiation defect or a potential
growth defect caused by derepression of tumor suppressor genes
p16Ink4aand p19Arf. To distinguish the role of Ezh2 in differ-
entiation from its role in cell proliferation, we immortalized
primary Ezh2flox/floxbrown preadipocytes with SV40 large T
antigen (SV40T) (16). The immortalized cells were infected with
retrovirus expressing Cre to generate Ezh2−/−brown pre-
adipocytes (Fig. 1A). Deletion of Ezh2 destabilized PRC2, as
shown by the reduced protein level of the Suz12 subunit, but had
no marked effects on the morphology or the growth rate of the
did not change the expression of Ezh2 paralog Ezh1, which has
been shown to display partial functional redundancy with Ezh2 in
ES cells (Fig. S1 B and F) (10, 14). Consistent with a previous
report on Ezh2−/−ES cells (10), Ezh2−/−brown preadipocytes
showed dramatic reduction of H3K27me2 and H3K27me3, but
retained robust H3K27 monomethylation (H3K27me1). Inter-
estingly, among the histone methylation and acetylation marks
that we examined, H3K27 acetylation (H3K27Ac) increased
markedly in Ezh2−/−brown preadipocytes (Fig. 1E). The decrease
of H3K27me2/me3, the increase of H3K27Ac, and the destabili-
zation of Suz12 in Ezh2−/−cells could be reversed by ectopic
expression of Ezh2 (Fig. S2A).
Preadipocytes. Ezh2 Is Required for Adipogenesis. The SV40T-immortalized
Ezh2flox/floxbrownpreadipocytesmaintainedthe full adipogenesis
potential, with over 90% of cells in the population differentiating
into adipocytes within 6 days following induction of adipogenesis
(Fig. 2A). In contrast, Ezh2−/−brown preadipocytes showed
ectopic Ezh2 (Fig. 2A and Fig. S2B). Deletion of Ezh2 in pre-
adipocytes did not significantly change the basal-level expression
adipocyte markers such as aP2, adiponectin, and the brown adi-
pocyte marker PRDM16 (17). However, the induction of these
genesduring adipogenesis wasseverely impaired (Fig. 2 Band C).
Interestingly, Ezh2 deletion in preadipocytes had no effect on
induction of the adipogenic transcription factor C/EBPβ, which
works upstream of PPARγ and C/EBPα (Fig. 2C) (4). Next we
infected Ezh2−/−brown preadipocytes with retroviruses express-
ing either PPARγ or C/EBPα (Fig. S3A). Ectopic expression of
either PPARγ or C/EBPα fully rescued adipogenesis in Ezh2−/−
preadipocytes (Fig. 2D). These results indicate that Ezh2 is
required for adipogenesis and suggest that Ezh2 functions in
the early phase of adipogenesis before the induction of PPARγ
Deletion of Ezh2 Derepresses Wnt Expression and Activates Wnt/
β-Catenin Signaling. Because Ezh2 is a transcriptional repressor,
we hypothesized that Ezh2 represses expression of adipogenesis
inhibitor(s) to facilitate adipogenesis. Because Wnt/β-catenin
signaling inhibits adipogenesis, we examined expression of Wnt/
β-catenin signaling pathway components by qRT-PCR. Increased
expression of Wnt1, -6, -10a, and -10b but not β-catenin were
observed in both immortalized and primary Ezh2−/−pre-
adipocytes (Fig. 3A and Figs. S3B and S4). Consistent with the
increased Wnt expression, cytosolic β-catenin protein accumu-
lated in Ezh2−/−preadipocytes (Fig. 3B). Further, expression of
Axin2, a direct target gene of β-catenin and an indicator of acti-
vation of Wnt/β-catenin signaling, increased markedly in Ezh2−/−
white and brown preadipocytes (Fig. 3C and Fig. S4). Deletion of
Ezh2 also increased levels of Wnt genes but not β-catenin during
adipogenesis (Fig. S5A). These results indicate that Ezh2
represses Wnt genes in preadipocytes and during adipogenesis
andthat deletionofEzh2derepresses Wntexpression andleadsto
activation of Wnt/β-catenin signaling.
By chromatin immunoprecipitation (ChIP) assays, we observed
Ezh2-dependent enrichment of Suz12 subunit of PRC2 on the
proximal promoters of Wnt1, -6, -10a, and -10b but not β-catenin
or GAPDH in brown preadipocytes (Fig. 3 D andE). Ezh2,Suz12,
and H3K27me3 were also enriched on these Wnt promoters in
3T3-L1 white preadipocytes (Fig. S6). Consistent with the
decreased H3K27me3 and the increased H3K27Ac in Ezh2−/−
nuclear extracts (Fig. 1E), deletion of Ezh2 led to marked
decrease of H3K27me3 concomitant with marked increase of
H3K27Ac on the promoters of Wnt1, -6, -10a, and -10b but not
β-catenin or GAPDH (Fig. 3 F and G). We also observed
decreased H3K27me3 and increased H3K27Ac on the proximal
promoters of the majority of reported Ezh2 target genes that we
have examined in Ezh2−/−preadipocytes (Fig. S7 B–D). The
increase of H3K27Ac correlated generally not only with the
increased Pol II recruitment on the promoters of these genes in
Ezh2−/−preadipocytes (Fig. 3I and Fig. S7 A and F). Deletion of
Ezh2 reduced binding of the Bmi-1 subunit of PRC1 to the pro-
moters of Wnt1, -6, -10a, and -10b, which was consistent with the
reduced H3K27me3 on these promoters (Fig. S8). These results
indicate that Wnt1, -6, -10a, and -10b are direct and functional
Ezh2 target genes in preadipocytes and suggest that H3K27Ac
may promote expression of Wnts and other Ezh2 target genes.
adipocytes. SV40T-immortalized Ezh2flox/floxbrown preadipocytes were
infected with retroviruses MSCVhygro expressing Cre (MSCVhygro-Cre) or
vector (Vec) alone. After selection with 150 μg/mL hygromycin for 2 weeks,
cells were maintained at subconfluence. (A) Confirmation of Cre-mediated
deletion of Ezh2 gene by qRT-PCR. (B) Western blot of Ezh2 and Suz12. (C)
Cell morphology under the microscope. (D) To analyze the short-term cell
growth rates, 1 × 105cells were plated at day 0 and the cumulative cell
numbers were determined every day for 5 days. (E) Western blot analysis of
histone methylation and acetylation in nuclear extracts. me1, me2, and me3
refer to mono-, di-, and trimethylation, respectively. Ac, acetylation. Quan-
titative PCR data in all figures are presented as means ± SD.
1. Characterization of SV40T-immortalized Ezh2−/−
| www.pnas.org/cgi/doi/10.1073/pnas.1000031107 Wang et al.
Ezh2 Methyltransferase Activity Is Required for Adipogenesis.
Because Ezh2 and PRC2 may also function beyond H3K27
methylation (5), it was necessary to determine whether the Ezh2
methyltransferase activity was required for adipogenesis and for
repression of Wnt genes. To address this issue, we generated a
mutant form of mouse Ezh2 (F667I). F667I corresponds to the
F681I mutation in E(Z), which is the Drosophila ortholog of
mammalian Ezh2 and the enzymatic subunit of Drosophila PRC2.
The F681I mutation has been shown to eliminate the H3K27
methyltransferase activity of E(Z) with little effects on the
integrity of Drosophila PRC2 (18). The immortalized Ezh2flox/flox
brown preadipocytes were infected with retroviruses expressing
FLAG-tagged Ezh2, either wild-type (WT) or F667I mutant,
followed by infection with retroviruses expressing Cre to delete
endogenous Ezh2. Deletion of the endogenous Ezh2 gene was
confirmed by quantitative PCR of genomic DNA (Fig. 4A). As
shown in Fig. 4 B and C, deletion of endogenous Ezh2 led to
markedly decreased H3K27me3 levels globally as well as on Wnt
promoters, both of which could be prevented by ectopic expres-
sion of wild-type Ezh2, but not the F667I mutant, indicating that
the F667I mutation inactivated the H3K27 methyltransferase
Ezh2. Gene expression analysis revealed that deletion of endo-
genous Ezh2 derepressed Wnt genes and activated Wnt/β-catenin
signaling, which could be prevented by ectopic expression of wild-
type but not the mutant Ezh2 (Fig. 4D). Consistent with these
results, ectopic expression of wild-type Ezh2, but not the F667I
mutant, prevented the adipogenesis defects in Ezh2-deficient
preadipocytes (Fig. 4E). These results demonstrate that the
H3K27 methyltransferase activity of Ezh2 is required for adipo-
genesis and for repression of Wnt genes in preadipocytes.
Blocking Wnt/β-Catenin Signaling Rescues Adipogenesis in Ezh2−/−
Preadipocytes. To find out whether Ezh2 represses expression
of other adipogenesis regulators, we performed microarray
analysis in the immortalized Ezh2flox/floxbrown preadipocytes
infected with retroviruses expressing Cre or vector alone. Gene
ontology (GO) analysis of genes with over 2.5-fold increase in
Ezh2-deficient cells revealed a remarkable enrichment of genes
involved in developmental regulation (Fig. S9). These results are
consistent with previous reports that Ezh2 and PRC2 repress
developmental regulators in ES cells (7, 8).
Microarray analysis followed by qRT-PCR confirmation
revealed that deletion of Ezh2 in preadipocytes also led to
increased expression of GATA3 (Fig. S7A), Pref-1 (Dlk1), BMP4,
KLF5, and IGF1 (Fig. S10A). GATA3 and Pref-1 are negative
regulators of adipogenesis whereas BMP4, KLF5, and IGF1
have been implicated in the positive regulation of adipogenesis
(2, 4). By ChIP assays, we observed Ezh2-dependent enrichment
of Ezh2, Suz12, and H3K27me3 on the promoters of GATA3,
Pref- 1, and BMP4 but not KLF5 and IGF1 (Figs. S7 B–D and S10
B–D). These results indicate that GATA3, Pref-1, and BMP4 are
direct targets of Ezh2 and that the up-regulation of KLF5 and
IGF1 is a secondary effect. The increase of BMP4 expression in
Ezh2−/−preadipocytes was largely due to the activation of Wnt/
β-catenin signaling, as inhibitors of Wnt/β-catenin signaling
blocked the up-regulation of BMP4 (see below). Nevertheless,
the identification of GATA3 and Pref-1 as direct Ezh2 targets
raised the question on the role of Ezh2-mediated repression of
Wnt genes in adipogenesis.
To address this issue, we investigated whether blocking Wnt/
β-catenin signaling could rescue adipogenesis in Ezh2−/−pre-
adipocytes. Axin1 associates directly with β-catenin and is impli-
cated in down-regulating Wnt/β-catenin signaling. Overexpressed
Axin1 inhibits Wnt/β-catenin signaling through destabilization
of β-catenin in the cytoplasm (1). Wnt/β-catenin signaling can
also be blocked by expression of the dominant-negative form
of transcription factor TCF4 (dnTCF4), which can bind to the
consensus LEF/TCF binding sites on Wnt target genes but cannot
be activated by β-catenin (3). We found that ectopic expression
of Axin1 or dnTCF4 in Ezh2−/−preadipocytes partially blocked
expression of Wnt target genes such as cyclin D1, BMP4, and
Axin2 (Fig. 5 A and B) and partially rescued adipogenesis and
associated expression of adipogenesis markers PPARγ and C/
EBPα (Fig. 5 C and D). These results indicate that derepression
of Wnt genes is responsible, at least in part, for the adipogenesis
immortalized Ezh2−/−brown preadipocytes. SV40T-immortalized Ezh2flox/flox
brown preadipocytes were infected with MSCVhygro-Cre, followed by adipo-
genesis assay. Adipogenesis was induced at day 0. Whole cell extracts for
Western blot and RNA samples for qRT-PCR were prepared at indicated time
points. (A) Morphological differentiation at day 6. Cells were stained with Oil
the p85α subunit ofPI3-kinaseisused asa loading control.(C) qRT-PCRanalysis
of expression of adipogenesis makers during adipogenesis. (D) Ectopic PPARγ
and C/EBPα can fully rescue adipogenesis in Ezh2−/−brown preadipocytes.
Ezh2−/−brown preadipocytes were infected with retroviruses WZLneo
expressingPPARγ2orC/EBPα. Afterselectionwith 500μg/mLG418for2 weeks,
cells were induced to undergo adipogenesis. Shown are Oil Red O-stained
dishes and cells under the microscope.
Wang et al. PNAS
| April 20, 2010
| vol. 107
| no. 16
defects in Ezh2−/−preadipocytes. These data also suggest that the
increased expression of GATA3 and Pref-1 likely contributes to
the adipogenesis defects in Ezh2−/−cells.
Ezh2 and Wnts are both important regulators of development. In
this paper, we demonstrate a direct, functional link between
these two types of developmental regulators and show that Ezh2
and its H3K27 methyltransferase activity are required for adipo-
genesis. Ezh2 directly represses Wnt genes to facilitate adipo-
genesis, a function that is independent of the well-established role
of Ezh2 in regulating cell proliferation. Finally, we provide evi-
dence to suggest that acetylation and trimethylation on H3K27
play opposing roles in regulating Wnt expression.
Separation of Ezh2 Functions in Cell Proliferation and Differentiation.
Ezh2 is dispensable for the self-renewal of ES cells but appears to
be required for the proliferation of fibroblasts, pancreatic islet β
cells, epidermal progenitors, and cancer cells. Ezh2 directly
represses the Ink4a-Arf locus. Deletion of Ezh2 derepresses the
Ink4a-Arf locus and increases levels of p16Ink4aand p19Arf, which
inhibitcell proliferation (19). Consistently, weobserved increased
expression of both p16Ink4aand p19Arfin Ezh2−/−primary pre-
adipocytes. It is possible that the increased expression of p16Ink4a
and p19Arfcontributes to the observed adipogenesis defects in
primary Ezh2−/−preadipocytes. To separate Ezh2 function in cell
differentiation from its role in cell proliferation, we established
SV40T-immortalized Ezh2flox/floxbrown preadipocytes. Although
SV40T inhibits adipogenesis of white preadipocytes, it does not
interfere with differentiation of brown preadipocytes toward
mature adipocytes that express markers of brown adipose tissue
(16). p16Ink4aand p19Arfinhibit cell proliferation through activa-
tion of tumor suppressors RB and p53. SV40T directly interacts
with and inactivates RB and p53 and thus functionally inactivates
the potential growth defects in Ezh2−/−preadipocytes, which
makes it possible to study the roles of Ezh2 in regulating pre-
adipocyte differentiation (adipogenesis).
Wnt Genes as Functional Ezh2 Targets. Previous genomewide anal-
yses in human cancer cell lines, ES cells, and embryonic fibro-
blasts have revealed the enrichment of Ezh2 and H3K27me3 on
Wnt promoters (7, 13, 14). However, it was unclear from these
MSCVhygro-Cre or Vec were maintained at subconfluence condition. (A) qRT-
PCR of Wnt and β-catenin expression. (B) Western blot analysis of β-catenin
levels in the cytosolic fractions and the whole cell extracts. GAPDH serves as
theloading control. (C) qRT-PCR of Axin2 expression. (D–I) ChIP assays of Ezh2
(D), Suz12 (E), H3K27me3 (F), H3K27Ac (G), H3K4me3 (H), and RNA poly-
merase II (Pol II) (I) on Wnt, β-catenin, and GAPDH proximal promoters.
Deletion of Ezh2 derepresses Wnt expression and activates Wnt/
brown preadipocytes were infected with MSCVhygro expressing FLAG-tagged
WT or F667I mutant Ezh2, followed by infection with retrovirus WZLneo-Cre.
Experiments in (A–D) were done before differentiation. (A) Confirmation of
Cre-mediated deletion of endogenous Ezh2 gene by quantitative genomic
PCR. (B) Western blot analysis in nuclear extracts. (C) ChIP assays of H3K27me3
on Wnt, β-catenin, and GAPDH proximal promoters. (D) qRT-PCR of Wnt
by staining with Oil Red O.
| www.pnas.org/cgi/doi/10.1073/pnas.1000031107Wang et al.
studies whether Ezh2 represses Wnt expression, as knockdown of
Ezh2 in human embryonic fibroblasts failed to increase Wnt
expression, which could be due to insufficient knockdown and/or
the functional redundancy between Ezh1 and Ezh2 (10, 14).
Deletion of Ezh2 in ES cells led to derepression of many Ezh2
target genes. However, its effect on Wnt expression was unclear
(7–11). We show derepression of known Ezh2 target genes in
Ezh2−/−white and brown preadipocytes, which suggests a func-
tional conservation of the Ezh2-mediated gene repression across
different cell types. Further, Ezh2 directly represses expression
of Wnt1, -6, -10a, and -10b but not β-catenin in preadipocytes.
Furthermore, Ezh2 requires its H3K27 methyltransferase activity
to repress Wnt expression. Finally, we demonstrate that der-
epression of Wnt genes in Ezh2−/−preadipocytes has a functional
consequence—inhibition of adipogenesis. The identification of
Wnt1, -6, -10a, and -10b as functional Ezh2 target genes in white
and brown preadipocytes thus provides a direct, functional link
between these two important types of developmental regulators.
The Wnt10b level is high in 3T3-L1 white preadipocytes but
declines rapidly after induction of adipogenesis (3). During
adipogenesis of wild-type brown preadipocytes, the Ezh2 protein
level in nuclear extracts and the H3K27me3 levels on Wnt pro-
moters show little changes (Fig. 2B and Fig. S5B). However,
Wnt1 and -10b levels decrease markedly during adipogenesis of
both wild-type and Ezh2−/−brown preadipocytes. Deletion of
Ezh2 derepresses Wnt expression not only in preadipocytes but
also during adipogenesis (Fig. S5A). These results suggest that
Ezh2 constitutively represses Wnt expression and that the de-
creased Wnt1 and -10b expression during adipogenesis is due to
transcriptional repressor(s) other than Ezh2.
Regulation of Ezh2 Target Genes by H3K27me3 and H3K27Ac. In
preadipocytes, deletion of Ezh2 leads to a marked increase of
H3K27Ac concomitant with a marked decrease of H3K27me3
not only globally but also on the promoters of Wnt and other
target genes of Ezh2. As H3K27Ac associates with active genes
whereas H3K27me3 associates with repressed genes (21), these
results are consistent with the derepression of Wnt and other
Ezh2 target genes in Ezh2−/−cells. It was shown recently that
knockdown of E(Z), the Drosophila ortholog of Ezh2, resulted in
increased H3K27Ac concomitant with decreased H3K27me3,
although it was unclear whether knockdown of E(Z) was suffi-
cient to increase expression of E(Z) target genes (22). Never-
theless, the inverse changes of H3K27Ac and H3K27me3 caused
by depletion of Ezh2 appears to be conserved between Droso-
phila and mammalian cells. Interestingly, deletion of Ezh2 spe-
cifically increases the global H3K27Ac level, suggesting that the
increase of H3K27Ac in Ezh2−/−cells is not secondary to gene
derepression, and that H3K27Ac plays a specific role in activa-
tion of Wnt and other Ezh2 target genes. Future work will be
needed to find out the exact role of H3K27Ac in activation of
Ezh2 target genes. The robust H3K27me1 in Ezh2−/−cells is also
interesting. Because a single lysine residue cannot be acetylated
and methylated simultaneously, this suggests that the bulk of
H3K27me1 occurs on distinct nucleosomes, and perhaps at dis-
tinct genetic loci, as H3K27me2 and H3K27me3.
Trimethylation on histone H3 lysine 4 (H3K4me3) is an epi-
genetic mark associated with gene activation and has been sug-
gested to antagonize PRC2- and H3K27me3-mediated gene
repression (5). Ezh2 deletion significantly increased H3K4me3
signal on the promoters of Wnt6 and Wnt10a genes, which are
localized adjacently on mouse chromosome 1, but had no effect
on H3K4me3 signal on the promoters of Wnt1 and Wnt10b genes,
which are localized adjacently on mouse chromosome 15 (Fig.
3H). Similarly, the increase of H3K4me3 was only found on a
subset of reported Ezh2 target gene promoters in Ezh2−/−pre-
adipocytes (Fig. S7E). Compared with H3K4me3, the increase of
H3K27Accorrelates better with both thederepression of Wntand
other Ezh2 target genes and the increased recruitment of Pol II
on the promoters of these genes in Ezh2−/−preadipocytes (Fig. 3
and Fig. S7).
Regulation of Adipogenesis by Ezh2. The adipogenesis defects in
inhibitory role of Wnt/β-catenin signaling in adipogenesis. Deletion
gene. Rather, the increased Wnt expression in Ezh2−/−cells leads to
stabilization of the cytosolic β-catenin protein, which inhibits the
activity of the master adipogenic transcription factor PPARγ (4). In
addition to Wnt genes, Ezh2 directly represses GATA3 and Pref-1,
which are known inhibitors of adipogenesis (2, 4). The increased
expression of Pref-1, GATA3, or other inhibitors of adipogenesis
likely contributes to the adipogenesis defects in Ezh2−/−preadipo-
cytes. However, our results that blocking Wnt/β-catenin signaling
pathway by expression of dnTCF4 or Axin1 can partially rescue adi-
pogenesis in Ezh2−/−cells indicate that derepression of Wnt genes is
responsible, at least in part, for the observed adipogenesis defects.
The partial rescue of adipogenesis could be due to incomplete
inhibitionof Wnt/β-catenin signaling and/or derepressionof GATA3
and Pref-1. Nevertheless, both the derepression of Wnt genes and
the adipogenesis defects in Ezh2−/−preadipocytes are prevented by
in Ezh2−/−preadipocytes. Ezh2flox/floxbrown preadipocytes were infected
with MSCVhygro expressing FLAG-tagged dominant negative form of TCF4
(F-dnTCN4) or FLAG-tagged Axin1 (F-Axin1), followed by infection with
WZLneo-Cre. (A) Western blot analysis of F-dnTCN4 and F-Axin1 expression
before differentiation. (B) Ectopic expression of dnTCF4 and Axin1 partially
blocks up-regulation of Cyclin D1, BMP4, and Axin2 in Ezh2−/−preadipocytes.
Gene expression was analyzed by qRT-PCR. (C and D) Ectopic expression of
dnTCF4 and Axin1 partially rescues adipogenesis in Ezh2−/−preadipocytes.
Cells were stained with Oil Red O (C) or subjected to Western blot analysis of
expression of adipogenesis markers PPARγ and C/EBPα (D) 6 days after
induction of adipogenesis. (E) Model for how H3K27 methyltransferase Ezh2
facilitates adipogenesis. Expression of multiple Wnt genes leads to stabili-
zation of β-catenin protein, which inhibits adipogenesis. H3K27 methyl-
transferase Ezh2 represses Wnt gene expression to facilitate adipogenesis.
Derepression of Pref-1 and GATA3 likely contributes to the adipogenesis
defect in Ezh2-deficient cells.
Blocking Wnt/β-catenin signaling partially rescues adipogenesis
Wang et al. PNAS
| April 20, 2010
| vol. 107
| no. 16
ectopic expression of wild-type but not the enzymatically inactive Download full-text
Ezh2, indicating that the H3K27 methyltransferase activity of Ezh2
is required for adipogenesis. Taken together, our results suggest a
as well as Pref-1 and GATA3, to facilitate adipogenesis (Fig. 5E).
Misregulation of Wnt/β-catenin signaling leads to devel-
opmental defects and diseases (1). Because Ezh2-mediated gene
repression appears to be conserved across different cell types, it
is possible that derepression of Wnt genes may contribute to the
developmental defects observed in other Ezh2-deficient cells and
tissues. Consistent with this possibility, it has been shown that
Ezh2 inhibits, whereas Wnt/β-catenin signaling promotes, myo-
genesis (23, 24). It will be interesting to determine whether
repression of Wnt genes by Ezh2 is a conserved mechanism in
regulation of cell differentiation and animal development.
Materials and Methods
Plasmids and Antibodies. The retroviral plasmids WZLneo-PPARγ2 and
WZLneo-C/EBPα have been described (25). MSCVhygro-Cre and WZLneo-Cre
were generated by cloning Cre cDNA into MSCVhygro (Clontech) and
WZLneo, respectively. The SV40T-expressing retroviral plasmid pBabepuro-
largeTcDNA was from Addgene (no. 14088). Full-length mouse Ezh2 with N-
terminal FLAG tag was amplified by PCR from Mammalian Gene Collection
(MGC) clone BC016391 and cloned into MSCVhygro to generate MSCVhygro-
F-Ezh2. The F667I mutant Ezh2 was generated by PCR. A similar method was
used to construct MSCVhyg-F-dnTCF4 (pLXSN-dnTCF4E from Ormond
McDougald as PCR template) and MSCVhyg-F-Axin1 (MGC clone BC113171
as PCR template). All plasmids were confirmed by DNA sequencing.
Anti-C/EBPα (sc-61), anti-PPARγ (sc-7273), and anti-p85α subunit of PI3-
kinase (sc-1637) antibodies were from Santa Cruz. Anti-FLAG (F3165) was
from Sigma. Anti-Ezh2 for Western blot (612666) and anti-β-catenin (610153)
were from BD Biosciences. Anti-Ezh2 for ChIP (39103) was from Active Motif.
Anti-RNA Pol II (Ab5408) and anti-Suz12 (ab12073) were from Abcam. Anti-
GAPDH (mAb374) and Bmi-1 (05-637) were from Millipore. Histone meth-
ylation and acetylation antibodies have been described (21). All chemicals
were from Sigma unless indicated otherwise.
Isolation of Primary Preadipocytes, Virus Infection, and Adipogenesis Assay.
Primary brown preadipocytes were cultured in DMEM plus 20% FBS. Immor-
talized brown preadipocytes and all other cells were routinely cultured in
DMEM plus 10% FBS. Primary white preadipocytes were isolated as described
(17). Primary brown preadipocytes were isolated from interscapular brown
adipose tissues of newborn Ezh2flox/floxpups and immortalized using retro-
virus pBabepuro expressing SV40 large T antigen following a published pro-
tocol (16). Retroviral infection was done as described (17). Adenoviral
infection of preadipocytes was done at 100 moi as described (17).
Adipogenesis of primary white preadipocytes was carried out as described
5 × 105per 10-cm dish in differentiation medium (DMEM plus 10% FBS, 0.1 μM
insulin, and 1 nM T3) 4 days before induction of adipogenesis. At day 0, cells
were fully confluent and were treated with differentiation medium supple-
mented with 0.5 mM 3-isobutyl-1-methyl-xanthine (IBMX), 2 μg/mL dex-
amethasone, and 0.125 mM indomethacin. Two days later, cells were changed
At day 3–4, small lipid droplets appeared in differentiating cells. At day 6–8
postinduction, fully differentiated cells were either stained with Oil Red O or
subjected to gene expression analysis by qRT-PCR or Western blot.
Western Blot, Microarray, qRT-PCR, and ChIP. The cytosolic fraction ofβ-catenin
was prepared as described (26). Western blot was done as described (25).
Microarray analysis in the SV40T-immortalized Ezh2flox/floxbrown pre-
adipocytes was performed on Mouse Genome 430 2.0 Array (Affymetrix) as
described (17). Data are deposited in NCBI GEO database (accession no.
GSE20054). GO analysis of genes that show increased expression in Ezh2-
deficient cells was done using the MGI GO_Slim Chart Tool. For qRT-PCR,
purified total RNA was reverse transcribed using random hexamers and the
SuperScript First-Strand Synthesis system (Invitrogen). The resulting first-
strand cDNAs were quantified with Sybr-Green assays on PRISM 7900HT
system using the standard curve and relative quantitation method with 18S
rRNA as control (Applied Biosystems). The sequences of most SYBR Green
primers were obtained from the PrimerBank (http://pga.mgh.harvard.edu/
primerbank/index.html) and are available upon request. qRT-PCR in Fig. S3B
was done using predesigned Taqman gene expression assays from Applied
Biosystems. Data are presented as means ± SD.
ChIP was performed as described (17) except that protein A sepharose
CL-4B (GE Healthcare) was used for immunoprecipitation. PCR quantitation
of precipitated genomic DNA relative to inputs was performed in duplicate
or triplicate using SYBR Green kit. SYBR Green primers were designed within
500-bp distance to the transcription start sites. The sequences of primers are
available upon request.
ACKNOWLEDGMENTS. We thank A. Tarakhovsky for kindly providing
Ezh2flox/floxmice, O. MacDougald for dnTCF4 cDNA, Y. Tseng and Y.-W. Cho
for help on brown preadipocyte immortalization, and Z. Wang and K. Zhao
for validated histone modification antibodies. This work was supported by
the Intramural Research Program of the National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health to K.G.
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