Hypoxia-induced methylation of a pontin chromatin remodeling factor Jason S. Leea,1, Yunho Kima,1, Jinhyuk Bhinb, Hi-Jai R. Shina, Hye Jin Nama, Seung Hoon Leec, Jong-Bok Yoonc, Olivier Bindad, Or Gozanid, Daehee Hwangb,e, and Sung Hee Baeka,2 aDepartment of Biological Sciences, Creative Research Initiative Center for Chromatin Dynamics, Seoul National University, Seoul 151-742, South Korea; bDepartment of Chemical Engineering, Pohang University of Science and Technology (POSTECH), Pohang 790-784, South Korea; cDepartment of Biochemistry and Translational Research Center for Protein Function Control, Yonsei University, Seoul 120-749, South Korea; dDepartment of Biological Sciences, Stanford University, Stanford, CA 94305; and eSchool of Interdisciplinary Biosciences and Bioengineering, POSTECH, Pohang 790-784, South Korea Edited by Gregg L. Semenza, Johns Hopkins University School of Medicine, Baltimore, MD, and approved July 12, 2011 (received for review April 19, 2011) Pontin is a chromatin remodeling factor that possesses both ATPase such as ATP-dependent SWI/SNF proteins as well as histone and DNA helicase activities. Although Pontin is frequently overex- modifying enzymes. Numerous enzymatic activities are associated pressed in human cancers of various types and implicated in onco- with coregulator functions and the activities are regulated by genic functions, the upstream signaling network leading to the posttranslational modifications, including methylation, acetyla- regulation of Pontin that in turn affects transcription of down- tion, phosphorylation, ubiquitylation, and small ubiquitin-like stream target genes has not been extensively studied. Here, we modifier (SUMO)-ylation (13–16). SUMOylation of Reptin has identify Pontin is methylated by G9a/GLP methyltransferases in been shown to be crucial for transcriptional repression of a KAI1 hypoxic condition and potentiates HIF-1α-mediated activation by metastasis suppressor gene (17, 18), whereas SUMOylation of increasing the recruitment of p300 coactivator to a subset of Pontin has been shown to function as a transcriptional coactivator HIF-1α target promoters. Intriguingly, Pontin methylation results of androgen receptor-mediated transcription in prostate cancer in the increased invasive and migratory properties by activating (19). Recently, we reported that Reptin chromatin-remodeling downstream target gene, Ets1. In contrast, inhibition of Pontin factor negatively regulates a subset of hypoxia-responsive genes methylation results in the suppression of tumorigenic and meta- (20). Biochemical purification of Reptin-binding proteins identi- BIOCHEMISTRY static properties. Together, our data provide new approaches by fied G9a, and hypoxia-induced Reptin methylation turned out to targeting Pontin methylation and its downstream targets for the participate in downregulating a subset of hypoxia target genes development of therapeutic agents for human cancers. involved in metabolism and tumor development using a gen- ome-wide analysis approach. In this manuscript, we provide evi- ∣ ∣ epigenetics transcriptional regulation covalent nonhistone modification dence that Pontin chromatin-remodeling factor is methylated by G9a and GLP in hypoxic condition. We address a detailed mo- efining the molecular mechanisms that coordinate specific lecular mechanism by which Pontin methylation mediates and Dupstream signal to diverse transcriptional responses remains elaborates the transcriptional regulation, thereby strongly activat- an important goal in biology. The main downstream effect of ing a subset of hypoxia target genes differentially regulated by signaling cascades is the modulation of transcription factors and Pontin compared to those of Reptin. coregulators functioning in the nucleus in response to specific upstream signals (1–4). Oxygen deficiency affects not only phy- Results siological processes such as those involved in embryonic develop- Pontin Is Methylated by G9a and GLP Methyltransferases. To screen ment, wound healing, and inflammation, but also in pathological for enzymes responsible for Pontin methylation, we performed conditions such as tumor progression, ischemic disease, and in vitro methyltransferase assays and found that out of sixteen atherosclerosis (5, 6). Many hypoxic responses are mediated by histone methyltransferases (HMTs), G9a was the only enzyme hypoxia inducible factor 1 (HIF-1), a heterodimeric transcription A α able to methylate Pontin (Fig. 1 ). Because G9a often functions factor that is comprised of an oxygen-regulated subunit (HIF- together with GLP (21, 22), we examined whether G9a and/or 1α or HIF-2α) and a constitutively expressed β subunit (HIF-1β) GLP are responsible for Pontin methylation. Further in vitro (7, 8). Under normoxic conditions, HIF-1α is unstable and subject HMT assays using G9a and GLP revealed that Pontin is methy- to degradation mediated by the von Hippel–Lindau E3 ligase. lated by both enzymes (Fig. 1 B and C). To determine whether However, under hypoxic conditions, HIF-1α is stabilized and it G9a/GLP can methylate Pontin in vivo, we examined Pontin translocates into the nucleus and binds to HIF-1β. The HIF-1α/β heterodimer is then able to bind to the hypoxia response element methylation by performing immunoprecipitation using anti- (HRE) that contains ACGTG as a core sequence. methyl lysine antibody by introducing either G9a or GLP. Pontin Although HIF-1α and HIF-2α share some common targets appears to have very low basal level of methylation, whereas its VEGF GLUT1 methylation status can be dramatically induced by G9a/GLP including and that are involved in regulating D angiogenesis and glycolytic pathway, HIF-2α appears to have its (Fig. 1 ). This was supported by coimmunoprecipitation assays unique targets during embryonic development by regulating factors such as Oct4 (9) and antioxidant enzymes, such as SOD2 Author contributions: J.S.L., Y.K., and S.H.B. designed research; J.S.L., Y.K., J.B., H.-J.R.S., (10). Both isoforms of HIFα interact with Sirt1 and the transcrip- H.J.N., S.H.L., and O.B. performed research; J.S.L., J.B., J.-B.Y., O.G., D.H., and S.H.B. tional activity of HIF-2α is enhanced upon deacetylation by Sirt1 analyzed data; and J.S.L., Y.K., and S.H.B. wrote the paper. whereas the transcriptional activity of HIF-1α is repressed by The authors declare no conflict of interest. deacetylation (11, 12). Therefore, although the two isoforms of This article is a PNAS Direct Submission. HIFα both execute hypoxic response, it appears that they have Data deposition: The data reported in this paper have been deposited in the Gene Expression distinct functions through differential regulatory mechanisms. Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE27813). Gene expression is not only influenced by presence of tran- 1J.S.L. and Y.K. contributed equally to this work. scription factors, but also by chromatin structure regulated by 2To whom correspondence should be addressed: E-mail: [email protected]. chromatin modifiers. The transcription of most genes is regulated This article contains supporting information online at www.pnas.org/lookup/suppl/ by the coordinate action of chromatin-remodeling complexes doi:10.1073/pnas.1106106108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1106106108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 28, 2021 A Autoradiogram of in vitro HMTase assay histone H3K9 suggesting that there exist a specificity amongst several HMTs in response to hypoxia in upregulating their activity D2 (Fig. 2A). We then examined whether Pontin methylation is HMTs: ET Mr(K) G9a SET8 EZH2 ASH1 NSD3 NSD1(SET) SETD5 S SETD4 PRDM2 PRDM4 ZNF298 SETDB2 PRMD14 SUV420H2 SUV39H2 100 induced in hypoxic condition, and found that Pontin methylation Pontin-me * 75 continued to increase with hypoxic exposure up to at least 12 h B C with corresponding accumulation of G9a and GLP in MCF7 breast cancer cells and HEK293 cells (Fig. 2B and Fig. S1). M (K) M (K) To further establish that the increase in Pontin methylation is r r directly caused by the increase in G9a and GLP enzymatic activ- 91 91 Pontin-me Pontin-me # ity, we exposed cells to hypoxia in the presence and absence of * * 39 39 G9a and GLP inhibitor, BIX-01294 (23, 24). BIX-01294 signifi- Coomassie Autorad. Coomassie Autorad. cantly reduced hypoxia-mediated Pontin methylation further P G a demonstrating that the methyltransferase activity of G9a/GLP D E g I F GL Pontin G9 IgG Mock GLP α IP: α C α IP: α is required for Pontin methylation (Fig. 2 ). Dependency of Pon- α α IP: Kme Pontin-me αG9a Pontin tin methylation on the expression of G9a was also demonstrated Pontin α αG9a Pontin in G9a-deficient mouse embryonic fibroblasts (MEFs) (Fig. 2D). WCL WCL WCL GLP α αGLP G9a Pontin These data strongly suggest that G9a and GLP are required for hypoxia-mediated Pontin methylation. To identify specific methy- Fig. 1. G9a and GLP methylate Pontin. (A) Histone lysine methyltransferases lation sites on Pontin, we performed in vivo methyltransferase were prepared as GST-fusion proteins and incubated with GST-Pontin in the 3 assays on Pontin deletion mutants and found that a region con- presence of S-adenosyl-L-[methyl- H] methionine (SAM). Reaction products taining the amino acid residues between 251 and 350, was methy- were analyzed by autoradiogram. (B and C) In vitro methylation assay was E performed using GST-G9a SET domain (B) or GST-GLP SET domain (C) with lated (Fig. 2 ). Within this region, there were six lysine residues, Coomassie brilliant blue staining (Left). Asterisks (*) indicate nonspecific and we mutated each one to alanine, however, these single bands, and the hash mark (#) indicates automethylation. (D) HEK293 cells mutants (K265A, K267A, K268A, K274A, K281A, and K285A) were transfected with empty vector or with expression vector encoding G9a still retained some methylation (Fig. S2). Therefore we eventually or GLP. Cell lysates were immunoprecipitated with antibody against anti- had to mutate all 6 lysine residues to alanine to completely elim- methyl-lysine antibody followed by immunoblot analysis with anti-Pontin inate the residual methylation. We found that Pontin mutant antibody to detect methylated Pontin.