PR-Set7/Set8/KMT5a is a chromatin-modifying enzyme that specifically monomethylates lysine 20 of histone H4 (H4K20me1). In this study we attempted to identify PR-Set7-interacting proteins reasoning that these proteins would provide important insights into the role of PR-Set7 in transcriptional regulation. Using an unbiased yeast two-hybrid approach, we discovered that PR-Set7 interacts with the UBC9 E2 SUMO conjugating enzyme. This interaction was confirmed in human cells and we demonstrated that PR-Set7 was preferentially modified with SUMO1 in vivo. Further in vitro studies revealed that UBC9 directly binds PR-Set7 proximal to the catalytic SET domain. Two putative SUMO consensus sites were identified in this region and both were capable of being SUMOylated in vitro. The absence of either or both SUMO sites did not perturb nuclear localization of PR-Set7. By employing whole genome expression arrays, we identified a panel of genes whose expression was significantly altered in the absence of PR-Set7. The vast majority of these genes displayed increased expression strongly suggesting that PR-Set7 predominantly functions as a transcriptional repressor. Importantly, the reduction of UBC9 resulted in the consistent derepression of several of these newly identified genes regulated by PR-Set7. Our findings indicate that direct interaction with UBC9 facilitates the repressive effects of PR-Set7 at specific target genes, most likely by SUMOylating PR-Set7.
References
[1]
Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389: 251–260.
[2]
Strahl BD, Allis CD (2000) The language of covalent histone modifications. Nature 403: 41–45.
[3]
Couture JF, Collazo E, Brunzelle JS, Trievel RC (2005) Structural and functional analysis of SET8, a histone H4 Lys-20 methyltransferase. Genes Dev 19: 1455–1465.
[4]
Fang J, Feng Q, Ketel CS, Wang H, Cao R, et al. (2002) Purification and functional characterization of SET8, a nucleosomal histone H4-lysine 20-specific methyltransferase. Curr Biol 12: 1086–1099.
[5]
Nishioka K, Rice JC, Sarma K, Erdjument-Bromage H, Werner J, et al. (2002) PR-Set7 is a nucleosome-specific methyltransferase that modifies lysine 20 of histone H4 and is associated with silent chromatin. Mol Cell 9: 1201–1213.
[6]
Xiao B, Jing C, Kelly G, Walker PA, Muskett FW, et al. (2005) Specificity and mechanism of the histone methyltransferase Pr-Set7. Genes Dev 19: 1444–1454.
[7]
Houston SI, McManus KJ, Adams MM, Sims JK, Carpenter PB, et al. (2008) Catalytic function of the PR-Set7 histone H4 lysine 20 monomethyltransferase is essential for mitotic entry and genomic stability. J Biol Chem 283: 19478–19488.
[8]
Huen MS, Sy SM, van Deursen JM, Chen J (2008) Direct interaction between SET8 and proliferating cell nuclear antigen couples H4-K20 methylation with DNA replication. J Biol Chem 283: 11073–11077.
[9]
Oda H, Okamoto I, Murphy N, Chu J, Price SM, et al. (2009) Monomethylation of histone H4-lysine 20 is involved in chromosome structure and stability and is essential for mouse development. Mol Cell Biol 29: 2278–2295.
[10]
Sakaguchi A, Steward R (2007) Aberrant monomethylation of histone H4 lysine 20 activates the DNA damage checkpoint in Drosophila melanogaster. J Cell Biol 176: 155–162.
[11]
Abbas T, Shibata E, Park J, Jha S, Karnani N, et al. (2010) CRL4(Cdt2) regulates cell proliferation and histone gene expression by targeting PR-Set7/Set8 for degradation. Mol Cell 40: 9–21.
[12]
Centore RC, Havens CG, Manning AL, Li JM, Flynn RL, et al. (2010) CRL4(Cdt2)-mediated destruction of the histone methyltransferase Set8 prevents premature chromatin compaction in S phase. Mol Cell 40: 22–33.
[13]
Oda H, Hubner MR, Beck DB, Vermeulen M, Hurwitz J, et al. (2010) Regulation of the histone H4 monomethylase PR-Set7 by CRL4(Cdt2)-mediated PCNA-dependent degradation during DNA damage. Mol Cell 40: 364–376.
[14]
Tardat M, Brustel J, Kirsh O, Lefevbre C, Callanan M, et al. (2010) The histone H4 Lys 20 methyltransferase PR-Set7 regulates replication origins in mammalian cells. Nat Cell Biol 12: 1086–1093.
[15]
Wu S, Wang W, Kong X, Congdon LM, Yokomori K, et al. (2010) Dynamic regulation of the PR-Set7 histone methyltransferase is required for normal cell cycle progression. Genes Dev 24: 2531–2542.
[16]
Sims JK, Houston SI, Magazinnik T, Rice JC (2006) A trans-tail histone code defined by monomethylated H4 Lys-20 and H3 Lys-9 demarcates distinct regions of silent chromatin. J Biol Chem 281: 12760–12766.
[17]
Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, et al. (2007) High-resolution profiling of histone methylations in the human genome. Cell 129: 823–837.
[18]
Talasz H, Lindner HH, Sarg B, Helliger W (2005) Histone H4-lysine 20 monomethylation is increased in promoter and coding regions of active genes and correlates with hyperacetylation. J Biol Chem 280: 38814–38822.
[19]
Vakoc CR, Sachdeva MM, Wang H, Blobel GA (2006) Profile of histone lysine methylation across transcribed mammalian chromatin. Mol Cell Biol 26: 9185–9195.
[20]
Gareau JR, Lima CD (2010) The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nat Rev Mol Cell Biol 11: 861–871.
[21]
Lin D, Tatham MH, Yu B, Kim S, Hay RT, et al. (2002) Identification of a substrate recognition site on Ubc9. J Biol Chem 277: 21740–21748.
[22]
Yunus AA, Lima CD (2006) Lysine activation and functional analysis of E2-mediated conjugation in the SUMO pathway. Nat Struct Mol Biol 13: 491–499.
[23]
Sampson DA, Wang M, Matunis MJ (2001) The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification. J Biol Chem 276: 21664–21669.
[24]
Fernandez-Miranda G, de Castro IP, Carmena M, Aguirre-Portoles C, Ruchaud S, et al. (2010) SUMOylation modulates the function of Aurora-B kinase. J Cell Sci 123: 2823–2833.
[25]
Kirsh O, Seeler JS, Pichler A, Gast A, Muller S, et al. (2002) The SUMO E3 ligase RanBP2 promotes modification of the HDAC4 deacetylase. EMBO J 21: 2682–2691.
[26]
Congdon LM, Houston SI, Veerappan CS, Spektor TM, Rice JC (2010) PR-Set7-mediated monomethylation of histone H4 lysine 20 at specific genomic regions induces transcriptional repression. J Cell Biochem 110: 609–619.
[27]
Sims JK, Rice JC (2008) PR-Set7 establishes a repressive trans-tail histone code that regulates differentiation. Mol Cell Biol 28: 4459–4468.
[28]
Zeng L, Yap KL, Ivanov AV, Wang X, Mujtaba S, et al. (2008) Structural insights into human KAP1 PHD finger-bromodomain and its role in gene silencing. Nat Struct Mol Biol 15: 626–633.
[29]
Kim JH, Kim B, Cai L, Choi HJ, Ohgi KA, et al. (2005) Transcriptional regulation of a metastasis suppressor gene by Tip60 and beta-catenin complexes. Nature 434: 921–926.
[30]
Bernier-Villamor V, Sampson DA, Matunis MJ, Lima CD (2002) Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell 108: 345–356.
[31]
Tatham MH, Chen Y, Hay RT (2003) Role of two residues proximal to the active site of Ubc9 in substrate recognition by the Ubc9.SUMO-1 thiolester complex. Biochemistry 42: 3168–3179.
[32]
Yang M, Hsu CT, Ting CY, Liu LF, Hwang J (2006) Assembly of a polymeric chain of SUMO1 on human topoisomerase I in vitro. J Biol Chem 281: 8264–8274.
[33]
Pichler A, Gast A, Seeler JS, Dejean A, Melchior F (2002) The nucleoporin RanBP2 has SUMO1 E3 ligase activity. Cell 108: 109–120.
[34]
Shin JA, Choi ES, Kim HS, Ho JC, Watts FZ, et al. (2005) SUMO modification is involved in the maintenance of heterochromatin stability in fission yeast. Mol Cell 19: 817–828.
[35]
Kalakonda N, Fischle W, Boccuni P, Gurvich N, Hoya-Arias R, et al. (2008) Histone H4 lysine 20 monomethylation promotes transcriptional repression by L3MBTL1. Oncogene 27: 4293–4304.
[36]
Girdwood D, Bumpass D, Vaughan OA, Thain A, Anderson LA, et al. (2003) P300 transcriptional repression is mediated by SUMO modification. Mol Cell 11: 1043–1054.
[37]
Kim JH, Choi HJ, Kim B, Kim MH, Lee JM, et al. (2006) Roles of sumoylation of a reptin chromatin-remodelling complex in cancer metastasis. Nat Cell Biol 8: 631–639.
[38]
Yang SH, Sharrocks AD (2004) SUMO promotes HDAC-mediated transcriptional repression. Mol Cell 13: 611–617.
[39]
Rice JC, Briggs SD, Ueberheide B, Barber CM, Shabanowitz J, et al. (2003) Histone methyltransferases direct different degrees of methylation to define distinct chromatin domains. Mol Cell 12: 1591–1598.
[40]
Ren J, Gao X, Jin C, Zhu M, Wang X, et al. (2009) Systematic study of protein sumoylation: Development of a site-specific predictor of SUMOsp 2.0. Proteomics 9: 3409–3412.
