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Histone H3K27 Methyltransferase Ezh2 Represses Wnt Genes to Facilitate Adipogenesis

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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 October 1, 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
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