G9a-mediated Lysine Methylation Alters the Function of CCAAT/Enhancer-binding Protein-β

Abstract

The functional capacity of the transcriptional regulatory CCAAT/enhancer-binding protein-β (C/EBPβ) is governed by protein interactions and post-translational protein modifications. In a proteome-wide interaction screen, the histone-lysine N-methyltransferase, H3 lysine 9-specific 3 (G9a), was found to directly interact with the C/EBPβ transactivation domain (TAD). Binding between G9a and C/EBPβ was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation. Metabolic labeling showed that C/EBPβ is post-translationally modified by methylation in vivo. A conserved lysine residue in the C/EBPβ TAD served as a substrate for G9a-mediated methylation. G9a, but not a methyltransferase-defective G9a mutant, abrogated the transactivation potential of wild type C/EBPβ. A C/EBPβ TAD mutant that contained a lysine-to-alanine exchange was resistant to G9a-mediated inhibition. Moreover, the same mutation conferred super-activation of a chromatin-embedded, endogenous C/EBPβ target gene. Our data identify C/EBPβ as a direct substrate of G9a-mediated post-translational modification that alters the functional properties of C/EBPβ during gene regulation. The functional capacity of the transcriptional regulatory CCAAT/enhancer-binding protein-β (C/EBPβ) is governed by protein interactions and post-translational protein modifications. In a proteome-wide interaction screen, the histone-lysine N-methyltransferase, H3 lysine 9-specific 3 (G9a), was found to directly interact with the C/EBPβ transactivation domain (TAD). Binding between G9a and C/EBPβ was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation. Metabolic labeling showed that C/EBPβ is post-translationally modified by methylation in vivo. A conserved lysine residue in the C/EBPβ TAD served as a substrate for G9a-mediated methylation. G9a, but not a methyltransferase-defective G9a mutant, abrogated the transactivation potential of wild type C/EBPβ. A C/EBPβ TAD mutant that contained a lysine-to-alanine exchange was resistant to G9a-mediated inhibition. Moreover, the same mutation conferred super-activation of a chromatin-embedded, endogenous C/EBPβ target gene. Our data identify C/EBPβ as a direct substrate of G9a-mediated post-translational modification that alters the functional properties of C/EBPβ during gene regulation. CCAAT/enhancer-binding protein-β (C/EBPβ) 2The abbreviations used are: C/EBPβCCAAT/enhancer-binding protein-βGSTglutathione S-transferasePCAFp300/CREB-binding protein associated factorAAamino acid(s)PMAphorbol 12-myristate 13-acetateGFPgreen fluorescent proteinIPimmunoprecipitationMES4-morpholineethanesulfonic acidTADtransactivation domainCRconserved regionPKAprotein kinase A. 2The abbreviations used are: C/EBPβCCAAT/enhancer-binding protein-βGSTglutathione S-transferasePCAFp300/CREB-binding protein associated factorAAamino acid(s)PMAphorbol 12-myristate 13-acetateGFPgreen fluorescent proteinIPimmunoprecipitationMES4-morpholineethanesulfonic acidTADtransactivation domainCRconserved regionPKAprotein kinase A. is a basic leucine zipper transcription factor that regulates tissue-specific gene expression, proliferation, and differentiation in a variety of cell types. C/EBPβ is an important factor for female reproduction (1Sterneck E. Tessarollo L. Johnson P.F. Genes Dev. 1997; 11: 2153-2162Crossref PubMed Scopus (342) Google Scholar), liver function and regeneration (2Greenbaum L.E. Li W. Cressman D.E. Peng Y. Ciliberto G. Poli V. Taub R. J. Clin. Investig. 1998; 102: 996-1007Crossref PubMed Scopus (232) Google Scholar), acute phase response (3Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar), as well as innate and adopted immune functions (4Tanaka T. Akira S. Yoshida K. Umemoto M. Yoneda Y. Shirafuji N. Fujiwara H. Suematsu S. Yoshida N. Kishimoto T. Cell. 1995; 80: 353-361Abstract Full Text PDF PubMed Scopus (466) Google Scholar). CCAAT/enhancer-binding protein-β glutathione S-transferase p300/CREB-binding protein associated factor amino acid(s) phorbol 12-myristate 13-acetate green fluorescent protein immunoprecipitation 4-morpholineethanesulfonic acid transactivation domain conserved region protein kinase A. CCAAT/enhancer-binding protein-β glutathione S-transferase p300/CREB-binding protein associated factor amino acid(s) phorbol 12-myristate 13-acetate green fluorescent protein immunoprecipitation 4-morpholineethanesulfonic acid transactivation domain conserved region protein kinase A. Although the locus encoding C/EBPβ lacks any intron, three protein isoforms are generated from a single mRNA transcript by alternative translation initiation, resulting in full-length and N-terminally truncated proteins termed LAP*, LAP, and LIP proteins, respectively (5Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (857) Google Scholar, 6Calkhoven C.F. Muller C. Leutz A. Genes Dev. 2000; 14: 1920-1932PubMed Google Scholar). The long C/EBPβ isoforms LAP* and LAP are transactivator proteins, and the small LIP isoform is thought of as a transcriptional dominant-negative inhibitor, although LAP may also function as a latent transcriptional repressor dependent on post-translational modifications (7Screpanti I. Musiani P. Bellavia D. Cappelletti M. Aiello F.B. Maroder M. Frati L. Modesti A. Gulino A. Poli V. J. Exp. Med. 1996; 184: 1561-1566Crossref PubMed Scopus (57) Google Scholar, 8Lamb J. Ramaswamy S. Ford H.L. Contreras B. Martinez R.V. Kittrell F.S. Zahnow C.A. Patterson N. Golub T.R. Ewen M.E. Cell. 2003; 114: 323-334Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). A number of post-translational modifications have been detected on C/EBPβ, including phosphorylation, acetylation, and SUMOylation. Of particular interest is a phosphoacceptor site in the central, regulatory part of the protein that is conserved throughout evolution (Thr-235 in human (9Nakajima T. Kinoshita S. Sasagawa T. 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Chem. 2002; 277: 38037-38044Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) occurs at a highly conserved lysine residue in the regulatory domain. Mutation of the lysine acceptor site increases the activity of the respective transcription factor, suggesting that SUMOylation is involved in repressive C/EBP functions. In addition, C/EBPβ can be acetylated at multiple lysine residues (26Cesena T.I. Cardinaux J.R. Kwok R. Schwartz J. J. Biol. Chem. 2007; 282: 956-967Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 27Joo M. Park G.Y. Wright J.G. Blackwell T.S. Atchison M.L. Christman J.W. J. Biol. Chem. 2004; 279: 6658-6665Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 28Xu M. Nie L. Kim S.H. Sun X.H. EMBO J. 2003; 22: 893-904Crossref PubMed Scopus (130) Google Scholar). The acetyltransferases p300 and p300/CREB-binding protein-associated factor (PCAF) catalyze acetylation of lysine 39, and mutation of lysine 39 has been shown to affect C/EBPβ-dependent reporter activity (26Cesena T.I. Cardinaux J.R. Kwok R. Schwartz J. J. Biol. Chem. 2007; 282: 956-967Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Acetylation is predominantly observed on N-terminal histone tails (29Allfrey V.G. Faulkner R. Mirsky A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Scopus (1727) Google Scholar) and accompanied by methylation of lysine side chains (30Comb D.G. Sarkar N. Pinzino C.J. J. Biol. Chem. 1966; 241: 1857-1862Abstract Full Text PDF PubMed Google Scholar). Methylation is involved in gene activation or gene repression depending on the state of methylation (mono-, di-, or trimethylation) and on its position on the histone tail (31Jenuwein T. Allis C.D. 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G9a can methylate C/EBPβ. G9a or the mutant was as proteins in and from cell G9a proteins were with or in the of shown in C/EBPβ was by G9a but not by of G9a served as an for the activity of the M. J. D. S. 2007; PubMed Scopus (87) Google Scholar). of the amino acid of the C/EBPβ TAD between and a single conserved lysine residue in and in in the region between and Methylation with were with the C/EBPβ TAD to a G9a methylation target and served as a A that C/EBPβ other that may or may not lysine residues and The data that G9a that the conserved in methylation with proteins and G9a were was in wild type was by data that of C/EBPβ as a target site for G9a methylation. The G9a for of C/EBPβ functional of the interaction between C/EBPβ and G9a was by reporter gene G9a, but not the transactivation potential of C/EBPβ in a on the reporter A and A with the C/EBPβ TAD and the domain of (11Kowenz-Leutz E. Twamley G. Ansieau S. Leutz A. 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Kurash J.K. Wilson J.R. Xiao B. Justin N. Ivanov G.S. McKinney K. Tempst P. Prives C. Gamblin S.J. Barlev N.A. Reinberg D. Nature. 2004; 432: 353-360Crossref PubMed Scopus (626) Google Scholar, 33Huang J. Perez-Burgos L. Placek B.J. Sengupta R. Richter M. Dorsey J.A. Kubicek S. Opravil S. Jenuwein T. Berger S.L. Nature. 2006; 444: 629-632Crossref PubMed Scopus (479) Google Scholar, J. Sengupta R. M.G. Dorsey J.A. Richter M. Opravil S. R. Jenuwein T. Berger S.L. Nature. 2007; PubMed Scopus Google Scholar). Methylation of p53 may or transcription in a target by its activity J. Sengupta R. M.G. Dorsey J.A. Richter M. Opravil S. R. Jenuwein T. Berger S.L. Nature. 2007; PubMed Scopus Google Scholar). also at a single lysine This modification leads to a of the protein factor with the A. E. A. L. I. Mol. Cell. 2004; 14: Full Text Full Text PDF PubMed Scopus Google Scholar). The acid was also found to be in its domain. This modification to the of acid to interact with Mol. 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