Histone H3K27 Methyltransferase Ezh2 Represses Wnt Genes to Facilitate Adipogenesis

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; and bLaboratory 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 histone methyltransferase subunit Ezh2, PRC2 methylates his- profiling studies have revealed the enrichment of histone H3K27 tone H3 on lysine 27 (H3K27). The resulting H3K27 trimethy- methyltransferase Ezh2 on Wnt genes. However, the functional lation is specifically recognized and bound by the PRC1 complex significance of such a direct link between the two types of devel- to facilitate transcriptional repression (6). PRC2 and PRC1 are opmental regulators in mammalian cells, and the role of Ezh2 in localized on a large number of developmental genes in embry- adipogenesis, remain unclear. Here we show Ezh2 and its H3K27 onic stem (ES) cells. Disruption of PRC2 by deletion of Ezh2, methyltransferase activity are required for adipogenesis. Ezh2 Suz12, or EED in ES cells markedly decreases the global levels directly represses Wnt1,-6,-10a, and -10b genes in preadipocytes of H3K27 di- and trimethylation (H3K27me2 and H3K27me3) and during adipogenesis. Deletion of Ezh2 eliminates H3K27me3 and derepresses many polycomb target genes (7–12). on Wnt promoters and derepresses Wnt expression, which leads Genomewide profiling studies have revealed the enrichment β to activation of Wnt/ -catenin signaling and inhibition of adipo- of H3K27 methyltransferase Ezh2 and associated H3K27me3 on genesis. Ectopic expression of the wild-type (WT) Ezh2, but not the Wnt genes in Drosophila and mammalian cells (7, 13, 14). How- enzymatically inactive F667I mutant, prevents the loss of − − ever, the functional significance of such a direct link between the H3K27me3 and the defects in adipogenesis in Ezh2 / preadipo- Ezh2−/− two types of developmental regulators in mammalian cell differ- cytes. The adipogenesis defects in cells can be rescued by entiation has not been shown. In addition, the role of Ezh2 in expression of adipogenic transcription factors PPARγ, C/EBPα,or − − adipogenesis remains unclear. Using Ezh2 conditional knockout inhibitors of Wnt/β-catenin signaling. Interestingly, Ezh2 / cells cells, here we show Ezh2 and its H3K27 methyltransferase activity show marked increase of H3K27 acetylation globally as well as on Wnt are required for adipogenesis. Ezh2 directly represses multiple promoters. These results indicate that H3K27 methyltransfer- Wnt ase Ezh2 directly represses Wnt genes to facilitate adipogenesis genes to facilitate adipogenesis. We also provide evidence to suggest that acetylation and trimethylation on H3K27 play and suggest that acetylation and trimethylation on H3K27 play Wnt opposing roles in regulating Wnt expression. opposing roles in regulating expression. Results epigenetics | histone methylation | polycomb | PRC2 Severe Adipogenesis Defects in Ezh2−/− Primary Preadipocytes. To Wnt investigate the role of H3K27 methyltransferase Ezh2 in adipo- he genes encode an evolutionarily conserved family of genesis, we isolated primary white preadipocytes from Ezh2 fl fl Tsecreted proteins that play critical roles in regulating conditional knockout Ezh2 ox/ ox mice (15). Cells were infected embryonic development and adult tissue homeostasis (1). In the with adenovirus expressing Cre (Ad-Cre) to acutely delete the canonical Wnt signaling pathway, also know as the Wnt/ Ezh2 gene. Deletion of Ezh2 was confirmed by quantitative β-catenin signaling pathway, Wnt binding to cell surface recep- reverse-transcriptase PCR (qRT-PCR) (Fig. S1A). Gene expres- tors leads to the stabilization and accumulation of free β-catenin sion analysis revealed increased expression of known Ezh2 target in the cytoplasm. The accumulated cytosolic β-catenin trans- Ink4a Arf − − genes including Hox, p16 , and p19 in Ezh2 / primary locates to the nucleus, where it binds to sequence-specific tran- preadipocytes (Fig. S1B). scription factors LEF/TCF and functions as a transcriptional Two days after cells reached confluence, preadipocytes were coactivator to promote expression of Wnt target genes. Numerous induced to undergo adipogenesis. Deletion of Ezh2 resulted in studies have pinpointed the details of how Wnt/β-catenin signaling a severe adipogenesis defect in primary white preadipocytes (Fig. activates expression of Wnt target genes that regulate various S1C). Consistent with the morphology, Ezh2 deletion blocked developmental processes. However, how Wnt genes are regulated expression of adipogenesis markers PPARγ, C/EBPα, and aP2 remains poorly understood.

D CELL BIOLOGY Wnt/β-catenin signaling inhibits adipogenesis (2). Activation (Fig. S1 ). Similarly, deletion of Ezh2 in primary brown pre- of Wnt/β-catenin signaling by expression of Wnt1 or Wnt10b, or adipocytes resulted in increased expression of known Ezh2 target β genes and severe defects in adipogenesis and associated by chemicals that stabilize cytosolic free -catenin, blocks adi- E–H pogenesis (3). Wnt/β-catenin signaling prevents the induction of expression of markers for brown adipocytes (Fig. S1 ). 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- Author contributions: L.W. and K.G. designed research; L.W., Q.J., J.-E.L., and K.G. per- β formed research; I.-h.S. contributed new reagents/analytic tools; L.W., Q.J., J.-E.L., and adipocyte differentiation (adipogenesis). In addition, -catenin K.G. analyzed data; and L.W. and K.G. wrote the paper. γ inhibits the transcriptional activity of PPAR (4). Conversely, The authors declare no conﬂict of interest. β inhibition of Wnt/ -catenin signaling by expressing Axin1 or This article is a PNAS Direct Submission. dominant-negative TCF4 (dnTCF4) promotes adipogenesis (3). Data deposition: Microarray data have been deposited in NCBI GEO database (accession Polycomb group proteins are transcriptional repressors that number GSE20054). help maintain the cell identity during development through 1Present address: Division of Genomics and Genetics, School of Biological Sciences, chromatin modiﬁcation (5). Mammalian polycomb group pro- Nanyang Technological University, Singapore 639798. teins form two multisubunit complexes, polycomb repressive 2To whom correspondence should be addressed. E-mail: [email protected]. complexes 1 and 2 (PRC1 and PRC2), respectively (5, 6). PRC2 This article contains supporting information online at www.pnas.org/cgi/content/full/ contains three core subunits: Ezh2, Suz12, and EED. Through its 1000031107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1000031107 PNAS | April 20, 2010 | vol. 107 | no. 16 | 7317–7322 Downloaded by guest on September 25, 2021 − − Characterization of SV40T-Immortalized Ezh2 / Preadipocytes. Ezh2 Is Required for Adipogenesis. The SV40T-immortalized fl fl Because primary preadipocytes had a limited growth potential Ezh2 ox/ ox brown preadipocytes maintained the full adipogenesis of only several passages in culture, it was difficult to obtain potential, with over 90% of cells in the population differentiating sufficient cells for mechanistic studies. Further, it was unclear into adipocytes within 6 days following induction of adipogenesis − − −/− whether the observed adipogenesis failure in Ezh2 / primary (Fig. 2A). In contrast, Ezh2 brown preadipocytes showed preadipocytes was due to a differentiation defect or a potential severe defects in adipogenesis, which could be partially rescued by growth defect caused by derepression of tumor suppressor genes ectopic Ezh2 (Fig. 2A and Fig. S2B). Deletion of Ezh2 in pre- Ink4a Arf fi p16 and p19 . To distinguish the role of Ezh2 in differ- adipocytes did not signi cantly change the basal-level expression γ α entiation from its role in cell proliferation, we immortalized of adipogenic transcription factors PPAR and C/EBP and other fl fl primary Ezh2 ox/ ox brown preadipocytes with SV40 large T adipocyte markers such as aP2, adiponectin, and the brown adi- antigen (SV40T) (16). The immortalized cells were infected with pocyte marker PRDM16 (17). However, the induction of these − − B C retrovirus expressing Cre to generate Ezh2 / brown pre- genes during adipogenesis was severely impaired (Fig. 2 and ). adipocytes (Fig. 1A). Deletion of Ezh2 destabilized PRC2, as Interestingly, Ezh2 deletion in preadipocytes had no effect on induction of the adipogenic transcription factor C/EBPβ, which shown by the reduced protein level of the Suz12 subunit, but had works upstream of PPARγ and C/EBPα (Fig. 2C) (4). Next we no marked effects on the morphology or the growth rate of the − − infected Ezh2 / brown preadipocytes with retroviruses express- immortalized cells (Fig. 1 B–D). Deletion of Ezh2 in preadipocytes ing either PPARγ or C/EBPα (Fig. S3A). Ectopic expression of did not change the expression of Ezh2 paralog Ezh1, which has − − either PPARγ or C/EBPα fully rescued adipogenesis in Ezh2 / been shown to display partial functional redundancy with Ezh2 in D B F preadipocytes (Fig. 2 ). These results indicate that Ezh2 is ES cells (Fig. S1 and ) (10, 14). Consistent with a previous required for adipogenesis and suggest that Ezh2 functions in Ezh2−/− Ezh2−/− report on ES cells (10), brown preadipocytes the early phase of adipogenesis before the induction of PPARγ showed dramatic reduction of H3K27me2 and H3K27me3, but and C/EBPα. retained robust H3K27 monomethylation (H3K27me1). Inter- estingly, among the histone methylation and acetylation marks Deletion of Ezh2 Derepresses Wnt Expression and Activates Wnt/ that we examined, H3K27 acetylation (H3K27Ac) increased β-Catenin Signaling. Because Ezh2 is a transcriptional repressor, − − markedly in Ezh2 / brown preadipocytes (Fig. 1E). The decrease we hypothesized that Ezh2 represses expression of adipogenesis of H3K27me2/me3, the increase of H3K27Ac, and the destabili- inhibitor(s) to facilitate adipogenesis. Because Wnt/β-catenin − − zation of Suz12 in Ezh2 / cells could be reversed by ectopic signaling inhibits adipogenesis, we examined expression of Wnt/ expression of Ezh2 (Fig. S2A). 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 / preadipocytes (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 and that deletion of Ezh2 derepresses Wnt expression and leads to 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 and E). 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 −/− − − Ezh2 B–D Fig. 1. Characterization of SV40T-immortalized Ezh2 / brown pre- have examined in preadipocytes (Fig. S7 ). The fl fl adipocytes. SV40T-immortalized Ezh2 ox/ ox brown preadipocytes were increase of H3K27Ac correlated generally not only with the infected with retroviruses MSCVhygro expressing Cre (MSCVhygro-Cre) or derepression of Wnt and other target Ezh2 genes, but also with the vector (Vec) alone. After selection with 150 μg/mL hygromycin for 2 weeks, increased Pol II recruitment on the promoters of these genes in − − cells were maintained at subconfluence. (A) Confirmation of Cre-mediated Ezh2 / preadipocytes (Fig. 3I and Fig. S7 A and F). Deletion of deletion of Ezh2 gene by qRT-PCR. (B) Western blot of Ezh2 and Suz12. (C) Ezh2 reduced binding of the Bmi-1 subunit of PRC1 to the pro- D Cell morphology under the microscope. ( ) To analyze the short-term cell moters of Wnt1,-6,-10a, and -10b, which was consistent with the growth rates, 1 × 105 cells were plated at day 0 and the cumulative cell E reduced H3K27me3 on these promoters (Fig. S8). These results numbers were determined every day for 5 days. ( ) Western blot analysis of Wnt1 6 10a 10b histone methylation and acetylation in nuclear extracts. me1, me2, and me3 indicate that ,- ,- , and - are direct and functional refer to mono-, di-, and trimethylation, respectively. Ac, acetylation. Quan- Ezh2 target genes in preadipocytes and suggest that H3K27Ac titative PCR data in all figures are presented as means ± SD. may promote expression of Wnts and other Ezh2 target genes.

7318 | www.pnas.org/cgi/doi/10.1073/pnas.1000031107 Wang et al. Downloaded by guest on September 25, 2021 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 fl fl integrity of Drosophila PRC2 (18). The immortalized Ezh2 ox/ ox 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 expression 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 endogenous 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 adipogenesis 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 fl fl analysis in the immortalized Ezh2 ox/ ox brown 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,andIGF1 (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,andBMP4 but not KLF5 and IGF1 (Figs. S7 B–D and S10 B–D). These results indicate that GATA3, Pref-1,andBMP4 are Fig. 2. Ezh2 is required for adipogenesis. (A–C) Severe adipogenesis defects in direct targets of Ezh2 and that the up-regulation of KLF5 and Ezh2−/− Ezh2flox/flox immortalized brown preadipocytes. SV40T-immortalized IGF1 is a secondary effect. The increase of BMP4 expression in − − brown preadipocytes were infected with MSCVhygro-Cre, followed by adipo- Ezh2 / preadipocytes was largely due to the activation of Wnt/ genesis assay. Adipogenesis was induced at day 0. Whole cell extracts for β-catenin signaling, as inhibitors of Wnt/β-catenin signaling Western blot and RNA samples for qRT-PCR were prepared at indicated time BMP4 points. (A) Morphological differentiation at day 6. Cells were stained with Oil blocked the up-regulation of (see below). Nevertheless, Red O. Top panels, stained dishes; Lower panels, representative fields under the the identification of GATA3 and Pref-1 as direct Ezh2 targets B

microscope. ( ) Western blot analysis of expression of adipogenesis makers and raised the question on the role of Ezh2-mediated repression of CELL BIOLOGY Ezh2 during adipogenesis. The 54.5-kDa PPARγ1 and 57.5-kDa PPARγ2 bands as Wnt genes in adipogenesis. well as the 30-kDa and 42-kDa C/EBPα isoforms are indicated. The expression of To address this issue, we investigated whether blocking Wnt/ − − the p85α subunit of PI3-kinase is used as a loading control. (C) qRT-PCR analysis β-catenin signaling could rescue adipogenesis in Ezh2 / pre- D γ of expression of adipogenesis makers during adipogenesis. ( ) Ectopic PPAR adipocytes. Axin1 associates directly with β-catenin and is impli- and C/EBPα can fully rescue adipogenesis in Ezh2−/− brown preadipocytes. − − cated in down-regulating Wnt/β-catenin signaling. Overexpressed Ezh2 / brown preadipocytes were infected with retroviruses WZLneo γ α μ Axin1 inhibits Wnt/β-catenin signaling through destabilization expressing PPAR 2 or C/EBP . After selection with 500 g/mL G418 for 2 weeks, β β cells were induced to undergo adipogenesis. Shown are Oil Red O-stained of -catenin in the cytoplasm (1). Wnt/ -catenin signaling can dishes and cells under the microscope. 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 Ezh2 Methyltransferase Activity Is Required for Adipogenesis. be activated by β-catenin (3). We found that ectopic expression − − Because Ezh2 and PRC2 may also function beyond H3K27 of Axin1 or dnTCF4 in Ezh2 / preadipocytes partially blocked methylation (5), it was necessary to determine whether the Ezh2 expression of Wnt target genes such as cyclin D1, BMP4, and methyltransferase activity was required for adipogenesis and for Axin2 (Fig. 5 A and B) and partially rescued adipogenesis and repression of Wnt genes. To address this issue, we generated a associated expression of adipogenesis markers PPARγ and C/ mutant form of mouse Ezh2 (F667I). F667I corresponds to the EBPα (Fig. 5 C and D). These results indicate that derepression F681I mutation in E(Z), which is the Drosophila ortholog of of Wnt genes is responsible, at least in part, for the adipogenesis

Wang et al. PNAS | April 20, 2010 | vol. 107 | no. 16 | 7319 Downloaded by guest on September 25, 2021 Fig. 4. Ezh2 methyltransferase activity is required for adipogenesis. Ezh2flox/flox 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,andGAPDH proximal promoters. (D)qRT-PCRofWnt Wnt Fig. 3. Deletion of Ezh2 derepresses expression and activates Wnt/ and Axin2 expression. (E) Cells were induced to undergo adipogenesis, followed β Ezh2flox/flox -catenin signaling. brown preadipocytes infected with by staining with Oil Red O. 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 represses the Ink4a-Arf locus. Deletion of Ezh2 derepresses the C Axin2 D–I the loading control. ( ) qRT-PCR of expression. ( ) ChIP assays of Ezh2 Ink4a-Arf locus and increases levels of p16Ink4a and p19Arf, which (D), Suz12 (E), H3K27me3 (F), H3K27Ac (G), H3K4me3 (H), and RNA poly- I Wnt β-catenin GAPDH inhibit cell proliferation (19). Consistently, we observed increased merase II (Pol II) ( )on , , and proximal promoters. Ink4a Arf − − expression of both p16 and p19 in Ezh2 / primary preadipocytes. It is possible that the increased expression of p16Ink4a − − defects in Ezh2 / preadipocytes. These data also suggest that the and p19Arf contributes to the observed adipogenesis defects in − − increased expression of GATA3 and Pref-1 likely contributes to primary Ezh2 / preadipocytes. To separate Ezh2 function in cell − − the adipogenesis defects in Ezh2 / cells. differentiation from its role in cell proliferation, we established fl fl SV40T-immortalized Ezh2 ox/ ox brown preadipocytes. Although Discussion SV40T inhibits adipogenesis of white preadipocytes, it does not Ezh2 and Wnts are both important regulators of development. In interfere with differentiation of brown preadipocytes toward this paper, we demonstrate a direct, functional link between mature adipocytes that express markers of brown adipose tissue these two types of developmental regulators and show that Ezh2 (16). p16Ink4a and p19Arf inhibit cell proliferation through activa- and its H3K27 methyltransferase activity are required for adipo- tion of tumor suppressors RB and p53. SV40T directly interacts genesis. Ezh2 directly represses Wnt genes to facilitate adipo- with and inactivates RB and p53 and thus functionally inactivates genesis, a function that is independent of the well-established role both p16Ink4a and p19Arf (20). Immortalization by SV40T prevents − − of Ezh2 in regulating cell proliferation. Finally, we provide evi- the potential growth defects in Ezh2 / preadipocytes, which dence to suggest that acetylation and trimethylation on H3K27 makes it possible to study the roles of Ezh2 in regulating pre- play opposing roles in regulating Wnt expression. adipocyte differentiation (adipogenesis).

Separation of Ezh2 Functions in Cell Proliferation and Differentiation. Wnt Genes as Functional Ezh2 Targets. Previous genomewide anal- Ezh2 is dispensable for the self-renewal of ES cells but appears to yses in human cancer cell lines, ES cells, and embryonic ﬁbro- be required for the proliferation of ﬁbroblasts, pancreatic islet β blasts have revealed the enrichment of Ezh2 and H3K27me3 on cells, epidermal progenitors, and cancer cells. Ezh2 directly Wnt promoters (7, 13, 14). However, it was unclear from these

7320 | www.pnas.org/cgi/doi/10.1073/pnas.1000031107 Wang et al. Downloaded by guest on September 25, 2021 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 decreased 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 sufficient 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 specifically 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 activation of Wnt and other Ezh2 target genes. Future work will be fi β needed to nd out the exact role of H3K27Ac in activation of Fig. 5. Blocking Wnt/ -catenin signaling partially rescues adipogenesis −/− − − fl fl Ezh2 in Ezh2 / preadipocytes. Ezh2 ox/ ox brown preadipocytes were infected Ezh2 target genes. The robust H3K27me1 in cells is also with MSCVhygro expressing FLAG-tagged dominant negative form of TCF4 interesting. Because a single lysine residue cannot be acetylated (F-dnTCN4) or FLAG-tagged Axin1 (F-Axin1), followed by infection with and methylated simultaneously, this suggests that the bulk of WZLneo-Cre. (A) Western blot analysis of F-dnTCN4 and F-Axin1 expression H3K27me1 occurs on distinct nucleosomes, and perhaps at dis- before differentiation. (B) Ectopic expression of dnTCF4 and Axin1 partially tinct genetic loci, as H3K27me2 and H3K27me3. −/− blocks up-regulation of Cyclin D1, BMP4, and Axin2 in Ezh2 preadipocytes. Trimethylation on histone H3 lysine 4 (H3K4me3) is an epi- Gene expression was analyzed by qRT-PCR. (C and D) Ectopic expression of Ezh2−/− genetic mark associated with gene activation and has been sug- dnTCF4 and Axin1 partially rescues adipogenesis in preadipocytes. gested to antagonize PRC2- and H3K27me3-mediated gene Cells were stained with Oil Red O (C) or subjected to Western blot analysis of γ α D repression (5). Ezh2 deletion significantly increased H3K4me3 expression of adipogenesis markers PPAR and C/EBP ( ) 6 days after Wnt6 Wnt10a induction of adipogenesis. (E) Model for how H3K27 methyltransferase Ezh2 signal on the promoters of and genes, which are facilitates adipogenesis. Expression of multiple Wnt genes leads to stabili- localized adjacently on mouse chromosome 1, but had no effect zation of β-catenin protein, which inhibits adipogenesis. H3K27 methyl- on H3K4me3 signal on the promoters of Wnt1 and Wnt10b genes, transferase Ezh2 represses Wnt gene expression to facilitate adipogenesis. which are localized adjacently on mouse chromosome 15 (Fig. Derepression of Pref-1 and GATA3 likely contributes to the adipogenesis 3H). Similarly, the increase of H3K4me3 was only found on a − − defect in Ezh2-deficient cells. subset of reported Ezh2 target gene promoters in Ezh2 / preadipocytes (Fig. S7E). Compared with H3K4me3, the increase of Wnt studies whether Ezh2 represses Wnt expression, as knockdown of H3K27Ac correlates better with both the derepression of and Ezh2 in human embryonic fibroblasts failed to increase Wnt other Ezh2 target genes and the increased recruitment of Pol II Ezh2−/− expression, which could be due to insufficient knockdown and/or on the promoters of these genes in preadipocytes (Fig. 3 the functional redundancy between Ezh1 and Ezh2 (10, 14). and Fig. S7). Deletion of Ezh2 in ES cells led to derepression of many Ezh2 Regulation of Adipogenesis by Ezh2. The adipogenesis defects in target genes. However, its effect on Wnt expression was unclear fi

Ezh2-de cient preadipocytes are consistent with the well-established CELL BIOLOGY (7–11). We show derepression of known Ezh2 target genes in β Ezh2−/− inhibitory role of Wnt/ -catenin signaling in adipogenesis. Deletion white and brown preadipocytes, which suggests a func- of Ezh2 in preadipocytes does not change expression of the β-catenin − − tional conservation of the Ezh2-mediated gene repression across gene. Rather, the increased Wnt expression in Ezh2 / cellsleadsto different cell types. Further, Ezh2 directly represses expression stabilization of the cytosolic β-catenin protein, which inhibits the Wnt1 6 10a 10b β-catenin of ,-,- , and - but not in preadipocytes. activity of the master adipogenic transcription factor PPARγ (4). In Furthermore, Ezh2 requires its H3K27 methyltransferase activity addition to Wnt genes, Ezh2 directly represses GATA3 and Pref-1, Wnt to repress expression. Finally, we demonstrate that der- which are known inhibitors of adipogenesis (2, 4). The increased −/− epression of Wnt genes in Ezh2 preadipocytes has a functional expression of Pref-1, GATA3, or other inhibitors of adipogenesis − − consequence—inhibition of adipogenesis. The identiﬁcation of likely contributes to the adipogenesis defects in Ezh2 / preadipo- Wnt1,-6,-10a, and -10b as functional Ezh2 target genes in white cytes. However, our results that blocking Wnt/β-catenin signaling and brown preadipocytes thus provides a direct, functional link pathway by expression of dnTCF4 or Axin1 can partially rescue adi- − − between these two important types of developmental regulators. pogenesis in Ezh2 / cells indicate that derepression of Wnt genes is The Wnt10b level is high in 3T3-L1 white preadipocytes but responsible, at least in part, for the observed adipogenesis defects. declines rapidly after induction of adipogenesis (3). During The partial rescue of adipogenesis could be due to incomplete adipogenesis of wild-type brown preadipocytes, the Ezh2 protein inhibition of Wnt/β-catenin signaling and/or derepression of GATA3 level in nuclear extracts and the H3K27me3 levels on Wnt pro- and Pref-1. Nevertheless, both the derepression of Wnt genes and − − moters show little changes (Fig. 2B and Fig. S5B). However, the adipogenesis defects in Ezh2 / preadipocytes are prevented by

Wang et al. PNAS | April 20, 2010 | vol. 107 | no. 16 | 7321 Downloaded by guest on September 25, 2021 ectopic expression of wild-type but not the enzymatically inactive virus pBabepuro expressing SV40 large T antigen following a published pro- Ezh2, indicating that the H3K27 methyltransferase activity of Ezh2 tocol (16). Retroviral infection was done as described (17). Adenoviral is required for adipogenesis. Taken together, our results suggest a infection of preadipocytes was done at 100 moi as described (17). model in which H3K27 methyltransferase Ezh2 represses Wnt genes, Adipogenesis of primary white preadipocytes was carried out as described as well as Pref-1 and GATA3, to facilitate adipogenesis (Fig. 5E). (17). For adipogenesis of brown preadipocytes, cells were plated at a density of × 5 μ Misregulation of Wnt/β-catenin signaling leads to devel- 5 10 per 10-cm dish in differentiation medium (DMEM plus 10% FBS, 0.1 M opmental defects and diseases (1). Because Ezh2-mediated gene 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- repression appears to be conserved across different cell types, it mented with 0.5 mM 3-isobutyl-1-methyl-xanthine (IBMX), 2 μg/mL dex- is possible that derepression of Wnt genes may contribute to the fi amethasone, and 0.125 mM indomethacin. Two days later, cells were changed developmental defects observed in other Ezh2-de cient cells and to the differentiation medium. The medium was replenished at 2-day intervals. tissues. Consistent with this possibility, it has been shown that At day 3–4, small lipid droplets appeared in differentiating cells. At day 6–8 Ezh2 inhibits, whereas Wnt/β-catenin signaling promotes, myo- postinduction, fully differentiated cells were either stained with Oil Red O or genesis (23, 24). It will be interesting to determine whether subjected to gene expression analysis by qRT-PCR or Western blot. repression of Wnt genes by Ezh2 is a conserved mechanism in regulation of cell differentiation and animal development. Western Blot, Microarray, qRT-PCR, and ChIP. The cytosolic fraction of β-catenin was prepared as described (26). Western blot was done as described (25). Materials and Methods Microarray analysis in the SV40T-immortalized Ezh2flox/flox brown pre- Plasmids and Antibodies. The retroviral plasmids WZLneo-PPARγ2 and adipocytes was performed on Mouse Genome 430 2.0 Array (Affymetrix) as WZLneo-C/EBPα have been described (25). MSCVhygro-Cre and WZLneo-Cre described (17). Data are deposited in NCBI GEO database (accession no. were generated by cloning Cre cDNA into MSCVhygro (Clontech) and GSE20054). GO analysis of genes that show increased expression in Ezh2- WZLneo, respectively. The SV40T-expressing retroviral plasmid pBabepuro- deficient cells was done using the MGI GO_Slim Chart Tool. For qRT-PCR, largeTcDNA was from Addgene (no. 14088). Full-length mouse Ezh2 with N- purified total RNA was reverse transcribed using random hexamers and the terminal FLAG tag was amplified by PCR from Mammalian Gene Collection SuperScript First-Strand Synthesis system (Invitrogen). The resulting first- (MGC) clone BC016391 and cloned into MSCVhygro to generate MSCVhygro- strand cDNAs were quantified with Sybr-Green assays on PRISM 7900HT F-Ezh2. The F667I mutant Ezh2 was generated by PCR. A similar method was system using the standard curve and relative quantitation method with 18S used to construct MSCVhyg-F-dnTCF4 (pLXSN-dnTCF4E from Ormond rRNA as control (Applied Biosystems). The sequences of most SYBR Green McDougald as PCR template) and MSCVhyg-F-Axin1 (MGC clone BC113171 primers were obtained from the PrimerBank (http://pga.mgh.harvard.edu/ as PCR template). All plasmids were confirmed by DNA sequencing. primerbank/index.html) and are available upon request. qRT-PCR in Fig. S3B α γ α Anti-C/EBP (sc-61), anti-PPAR (sc-7273), and anti-p85 subunit of PI3- was done using predesigned Taqman gene expression assays from Applied kinase (sc-1637) antibodies were from Santa Cruz. Anti-FLAG (F3165) was Biosystems. Data are presented as means ± SD. β from Sigma. Anti-Ezh2 for Western blot (612666) and anti- -catenin (610153) ChIP was performed as described (17) except that protein A sepharose were from BD Biosciences. Anti-Ezh2 for ChIP (39103) was from Active Motif. CL-4B (GE Healthcare) was used for immunoprecipitation. PCR quantitation Anti-RNA Pol II (Ab5408) and anti-Suz12 (ab12073) were from Abcam. Anti- of precipitated genomic DNA relative to inputs was performed in duplicate GAPDH (mAb374) and Bmi-1 (05-637) were from Millipore. Histone meth- or triplicate using SYBR Green kit. SYBR Green primers were designed within ylation and acetylation antibodies have been described (21). All chemicals 500-bp distance to the transcription start sites. The sequences of primers are were from Sigma unless indicated otherwise. available upon request.

Isolation of Primary Preadipocytes, Virus Infection, and Adipogenesis Assay. ACKNOWLEDGMENTS. We thank A. Tarakhovsky for kindly providing Primary brown preadipocytes were cultured in DMEM plus 20% FBS. Immor- fl fl Ezh2 ox/ ox mice, O. MacDougald for dnTCF4 cDNA, Y. Tseng and Y.-W. Cho talized brown preadipocytes and all other cells were routinely cultured in for help on brown preadipocyte immortalization, and Z. Wang and K. Zhao DMEM plus 10% FBS. Primary white preadipocytes were isolated as described for validated histone modification antibodies. This work was supported by (17). Primary brown preadipocytes were isolated from interscapular brown the Intramural Research Program of the National Institute of Diabetes and adipose tissues of newborn Ezh2flox/flox pups and immortalized using retro- Digestive and Kidney Diseases, National Institutes of Health to K.G.

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