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• W2024634513 abstract "Posttranslational modification of transcription factors by the small ubiquitin-related modifier SUMO is associated with transcriptional repression, but the underlying mechanisms remain incompletely described. We have identified binding of the LSD1/CoREST1/HDAC corepressor complex to SUMO-2. Here we show that CoREST1 binds directly and noncovalently to SUMO-2, but not SUMO-1, and CoREST1 bridges binding of the histone demethylase LSD1 to SUMO-2. Depletion of SUMO-2/3 conjugates led to transcriptional derepression, reduced occupancy of CoREST1 and LSD1, and changes in histone methylation and acetylation at some, but not all, LSD1/CoREST1/HDAC target genes. We have identified a nonconsensus SUMO-interaction motif (SIM) in CoREST1 required for SUMO-2 binding, and we show that mutation of the CoREST1 SIM disrupted SUMO-2 binding and transcriptional repression of some neuronal-specific genes in nonneuronal cells. Our results reveal that direct interactions between CoREST1 and SUMO-2 mediate SUMO-dependent changes in chromatin structure and transcription that are important for cell-type-specific gene expression. Posttranslational modification of transcription factors by the small ubiquitin-related modifier SUMO is associated with transcriptional repression, but the underlying mechanisms remain incompletely described. We have identified binding of the LSD1/CoREST1/HDAC corepressor complex to SUMO-2. Here we show that CoREST1 binds directly and noncovalently to SUMO-2, but not SUMO-1, and CoREST1 bridges binding of the histone demethylase LSD1 to SUMO-2. Depletion of SUMO-2/3 conjugates led to transcriptional derepression, reduced occupancy of CoREST1 and LSD1, and changes in histone methylation and acetylation at some, but not all, LSD1/CoREST1/HDAC target genes. We have identified a nonconsensus SUMO-interaction motif (SIM) in CoREST1 required for SUMO-2 binding, and we show that mutation of the CoREST1 SIM disrupted SUMO-2 binding and transcriptional repression of some neuronal-specific genes in nonneuronal cells. Our results reveal that direct interactions between CoREST1 and SUMO-2 mediate SUMO-dependent changes in chromatin structure and transcription that are important for cell-type-specific gene expression. Transcription repression mechanisms contribute to cell-type-specific gene expression programs that are important for cell fate determination. Posttranslational modification of histones plays a central role in regulating chromatin structure and gene transcription (Jenuwein and Allis, 2001Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7211) Google Scholar). The first histone lysine-specific demethylase identified, LSD1 (KDM1), regulates both gene activation and repression programs important for mammalian organogenesis (Shi et al., 2004Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Shi Y. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (2863) Google Scholar, Wang et al., 2007Wang J. Scully K. Zhu X. Cai L. Zhang J. Prefontaine G.G. Krones A. Ohgi K.A. Zhu P. Garcia-Bassets I. et al.Opposing LSD1 complexes function in developmental gene activation and repression programmes.Nature. 2007; 446: 882-887Crossref PubMed Scopus (391) Google Scholar). Transcriptional repression by LSD1 has been correlated with enzymatic removal of mono- and dimethyl groups from histone H3 Lys4 (Shi et al., 2004Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Shi Y. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (2863) Google Scholar). LSD1 is a component of several multiprotein complexes, where it has been found tightly associated with CoREST1 and histone deacetylases (HDAC) 1 and 2 (Hakimi et al., 2003Hakimi M.A. Dong Y. Lane W.S. Speicher D.W. Shiekhattar R. A candidate X-linked mental retardation gene is a component of a new family of histone deacetylase-containing complexes.J. Biol. Chem. 2003; 278: 7234-7239Crossref PubMed Scopus (133) Google Scholar, Humphrey et al., 2001Humphrey G.W. Wang Y. Russanova V.R. Hirai T. Qin J. Nakatani Y. Howard B.H. Stable histone deacetylase complexes distinguished by the presence of SANT domain proteins CoREST/kiaa0071 and Mta-L1.J. Biol. Chem. 2001; 276: 6817-6824Crossref PubMed Scopus (253) Google Scholar, Shi et al., 2003Shi Y. Sawada J. Sui G. Affar el B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Shi Y. Coordinated histone modifications mediated by a CtBP co-repressor complex.Nature. 2003; 422: 735-738Crossref PubMed Scopus (598) Google Scholar, Shi et al., 2005Shi Y.J. Matson C. Lan F. Iwase S. Baba T. Shi Y. Regulation of LSD1 histone demethylase activity by its associated factors.Mol. Cell. 2005; 19: 857-864Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, You et al., 2001You A. Tong J.K. Grozinger C.M. Schreiber S.L. CoREST is an integral component of the CoREST- human histone deacetylase complex.Proc. Natl. Acad. Sci. USA. 2001; 98: 1454-1458Crossref PubMed Scopus (368) Google Scholar). CoREST1 binds directly to LSD1 and is required for LSD1-mediated demethylation of nucleosomal substrates (Lee et al., 2005Lee M.G. Wynder C. Cooch N. Shiekhattar R. An essential role for CoREST in nucleosomal histone 3 lysine 4 demethylation.Nature. 2005; 437: 432-435Crossref PubMed Scopus (561) Google Scholar, Shi et al., 2005Shi Y.J. Matson C. Lan F. Iwase S. Baba T. Shi Y. Regulation of LSD1 histone demethylase activity by its associated factors.Mol. Cell. 2005; 19: 857-864Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, Yang et al., 2006Yang M. Gocke C.B. Luo X. Borek D. Tomchick D.R. Machius M. Otwinowski Z. Yu H. Structural basis for CoREST-dependent demethylation of nucleosomes by the human LSD1 histone demethylase.Mol. Cell. 2006; 23: 377-387Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). The presence of LSD1 and HDACs in a common complex supports coordinated deacetylation and demethylation of histone tails to generate a repressive chromatin structure (Lan et al., 2008Lan F. Nottke A.C. Shi Y. Mechanisms involved in the regulation of histone lysine demethylases.Curr. Opin. Cell Biol. 2008; 20: 316-325Crossref PubMed Scopus (181) Google Scholar, Shi et al., 2003Shi Y. Sawada J. Sui G. Affar el B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Shi Y. Coordinated histone modifications mediated by a CtBP co-repressor complex.Nature. 2003; 422: 735-738Crossref PubMed Scopus (598) Google Scholar). In addition to modulating LSD1 enzymatic activity, CoREST1 and other components of the LSD1 complex are key regulators of LSD1 recruitment to specific genes. CoREST1 was initially identified as a corepressor for the DNA-binding protein REST (also known as NRSF); binding of CoREST1 to REST recruits the CoREST1/LSD1 complex to silence neuronal genes in nonneuronal cells (Ballas et al., 2001Ballas N. Battaglioli E. Atouf F. Andres M.E. Chenoweth J. Anderson M.E. Burger C. Moniwa M. Davie J.R. Bowers W.J. et al.Regulation of neuronal traits by a novel transcriptional complex.Neuron. 2001; 31: 353-365Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, Ballas et al., 2005Ballas N. Grunseich C. Lu D.D. Speh J.C. Mandel G. REST and its corepressors mediate plasticity of neuronal gene chromatin throughout neurogenesis.Cell. 2005; 121: 645-657Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, Hakimi et al., 2002Hakimi M.A. Bochar D.A. Chenoweth J. Lane W.S. Mandel G. Shiekhattar R. A core-BRAF35 complex containing histone deacetylase mediates repression of neuronal-specific genes.Proc. Natl. Acad. Sci. USA. 2002; 99: 7420-7425Crossref PubMed Scopus (223) Google Scholar, Shi et al., 2004Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Shi Y. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (2863) Google Scholar, Shi et al., 2005Shi Y.J. Matson C. Lan F. Iwase S. Baba T. Shi Y. Regulation of LSD1 histone demethylase activity by its associated factors.Mol. Cell. 2005; 19: 857-864Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). The LSD1/CoREST1/HDAC core complex is also associated with the corepressor CtBP, which interacts directly with many DNA-binding transcription factors (Chinnadurai, 2002Chinnadurai G. CtBP, an unconventional transcriptional corepressor in development and oncogenesis.Mol. Cell. 2002; 9: 213-224Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). CoREST1 binds directly to CtBP, and this interaction may contribute to repression of some CtBP target genes (Cowger et al., 2007Cowger J.J. Zhao Q. Isovic M. Torchia J. Biochemical characterization of the zinc-finger protein 217 transcriptional repressor complex: identification of a ZNF217 consensus recognition sequence.Oncogene. 2007; 26: 3378-3386Crossref PubMed Scopus (71) Google Scholar, Kuppuswamy et al., 2008Kuppuswamy M. Vijayalingam S. Zhao L.J. Zhou Y. Subramanian T. Ryerse J. Chinnadurai G. Role of the PLDLS-binding cleft region of CtBP1 in recruitment of core and auxiliary components of the corepressor complex.Mol. Cell. Biol. 2008; 28: 269-281Crossref PubMed Scopus (83) Google Scholar). Recent studies of promoter-specific repressors such as Gfi-1 and Gfi-1b, as well as genome-wide analyses, have led to the identification of a large number of genes regulated by LSD1 (Garcia-Bassets et al., 2007Garcia-Bassets I. Kwon Y.S. Telese F. Prefontaine G.G. Hutt K.R. Cheng C.S. Ju B.G. Ohgi K.A. Wang J. Escoubet-Lozach L. et al.Histone methylation-dependent mechanisms impose ligand dependency for gene activation by nuclear receptors.Cell. 2007; 128: 505-518Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, Saleque et al., 2007Saleque S. Kim J. Rooke H.M. Orkin S.H. Epigenetic regulation of hematopoietic differentiation by Gfi-1 and Gfi-1b is mediated by the cofactors CoREST and LSD1.Mol. Cell. 2007; 27: 562-572Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). In most cases, however, the protein-protein interactions that mediate recruitment of LSD1 to specific promoters have not been described. Posttranslational modification by the small ubiquitin-related modifier SUMO regulates diverse cellular processes, including cell-cycle progression, genomic stability, intracellular trafficking, and transcription (Geiss-Friedlander and Melchior, 2007Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1248) Google Scholar, Gill, 2004Gill G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms?.Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (587) Google Scholar, Hay, 2005Hay R.T. SUMO: a history of modification.Mol. Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar, Johnson, 2004Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1322) Google Scholar). In most cases, SUMO conjugation alters localization and/or activity of the substrate by providing a new protein-protein interaction interface. SUMO interaction motifs (SIMs) that mediate noncovalent binding to SUMO have been described. SIMs in several proteins, such as the DNA repair enzyme TDG and the tumor suppressor PML, have been shown to be important for biological activity (Baba et al., 2005Baba D. Maita N. Jee J.G. Uchimura Y. Saitoh H. Sugasawa K. Hanaoka F. Tochio H. Hiroaki H. Shirakawa M. Crystal structure of thymine DNA glycosylase conjugated to SUMO-1.Nature. 2005; 435: 979-982Crossref PubMed Scopus (177) Google Scholar, Shen et al., 2006Shen T.H. Lin H.K. Scaglioni P.P. Yung T.M. Pandolfi P.P. The mechanisms of PML-nuclear body formation.Mol. Cell. 2006; 24: 331-339Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, Takahashi et al., 2005Takahashi H. Hatakeyama S. Saitoh H. Nakayama K.I. Noncovalent SUMO-1 binding activity of thymine DNA glycosylase (TDG) is required for its SUMO-1 modification and colocalization with the promyelocytic leukemia protein.J. Biol. Chem. 2005; 280: 5611-5621Crossref PubMed Scopus (92) Google Scholar). In mammals, three SUMO paralogs are widely expressed: SUMO-2 and SUMO-3, which are 96% identical, and SUMO-1, which is 45% identical to SUMO-2. Growing evidence suggests that SUMO-2/3 and SUMO-1 have some unique biological functions (Ayaydin and Dasso, 2004Ayaydin F. Dasso M. Distinct in vivo dynamics of vertebrate SUMO paralogues.Mol. Biol. Cell. 2004; 15: 5208-5218Crossref PubMed Scopus (155) Google Scholar, Saitoh and Hinchey, 2000Saitoh H. Hinchey J. Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3.J. Biol. Chem. 2000; 275: 6252-6258Crossref PubMed Scopus (652) Google Scholar, Vertegaal et al., 2006Vertegaal A.C. Andersen J.S. Ogg S.C. Hay R.T. Mann M. Lamond A.I. Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics.Mol. Cell. Proteomics. 2006; 5: 2298-2310Crossref PubMed Scopus (234) Google Scholar). Although proteins that bind preferentially to SUMO-2/3 or SUMO-1 may contribute to distinct functions of the SUMO paralogs, with only a single type of SIM described to date, it is not clear how paralog-specific interactions are determined. SUMO modification of transcription factors and cofactors has generally been correlated with transcriptional repression (Geiss-Friedlander and Melchior, 2007Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1248) Google Scholar, Gill, 2004Gill G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms?.Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (587) Google Scholar, Hay, 2005Hay R.T. SUMO: a history of modification.Mol. Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar). Investigations of the molecular mechanisms underlying SUMO-dependent repression largely support the hypothesis that covalent attachment of SUMO provides a new interaction interface that mediates recruitment of transcriptional corepressors (Ivanov et al., 2007Ivanov A.V. Peng H. Yurchenko V. Yap K.L. Negorev D.G. Schultz D.C. Psulkowski E. Fredericks W.J. White D.E. Maul G.G. et al.PHD domain-mediated E3 ligase activity directs intramolecular sumoylation of an adjacent bromodomain required for gene silencing.Mol. Cell. 2007; 28: 823-837Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, Lin et al., 2006Lin D.Y. Huang Y.S. Jeng J.C. Kuo H.Y. Chang C.C. Chao T.T. Ho C.C. Chen Y.C. Lin T.P. Fang H.I. et al.Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors.Mol. Cell. 2006; 24: 341-354Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, Ross et al., 2002Ross S. Best J.L. Zon L.I. Gill G. SUMO-1 modification represses Sp3 transcriptional activation and modulates its subnuclear localization.Mol. Cell. 2002; 10: 831-842Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, Yang and Sharrocks, 2004Yang S.H. Sharrocks A.D. SUMO promotes HDAC-mediated transcriptional repression.Mol. Cell. 2004; 13: 611-617Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Several chromatin-modifying enzymes and chromatin-binding proteins have been implicated as effectors of SUMO-mediated repression. For example, SUMO modification of the transcription factor Elk-1 promotes recruitment of HDAC2, associated with histone deacetylation and transcriptional repression of the c-fos promoter (Yang and Sharrocks, 2004Yang S.H. Sharrocks A.D. SUMO promotes HDAC-mediated transcriptional repression.Mol. Cell. 2004; 13: 611-617Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Previous affinity chromatography studies revealed an association between LSD1 and SUMO-2 (Rosendorff et al., 2006Rosendorff A. Sakakibara S. Lu S. Kieff E. Xuan Y. DiBacco A. Shi Y. Shi Y. Gill G. NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression.Proc. Natl. Acad. Sci. USA. 2006; 103: 5308-5313Crossref PubMed Scopus (86) Google Scholar). In that study, although repression by Gal4-SUMO-2 correlated with demethylation of histone H3K4, LSD1 was not essential for repression of the synthetic reporter gene analyzed. Thus, the molecular basis for LSD1 association with SUMO-2 and the functional significance of this association for LSD1 and/or SUMO-dependent repression of endogenous genes has not been described. Here we show that CoREST1 binds directly and noncovalently to SUMO-2, but not SUMO-1, and bridges LSD1 binding to SUMO-2 in vitro. We identify a nonconsensus SIM required for SUMO-2 binding by CoREST1 and establish that this motif also mediates SUMO-2-specific binding in at least two other proteins. We found that deconjugation of SUMO-2/3 led to derepression, loss of promoter occupancy by CoREST1 and LSD1, increased H3K4 methylation, and increased H3 acetylation of the SCN1A and SCN3A genes but did not affect another CoREST1/LSD1 target gene, SCN2A2. Furthermore, we show that mutation of the CoREST1 SIM disrupted binding of SUMO-2/3 and led to loss of CoREST1 occupancy and transcriptional repression of specific LSD1/CoREST1 target genes. Our findings reveal a role for SUMO-2/3 in gene-specific recruitment and activity of the LSD1/CoREST1/HDAC corepressor complex, supporting a role for SUMOylation in regulation of histone modifications and transcriptional silencing of some neuronal-specific genes in nonneuronal cells. A prevailing hypothesis to explain SUMO-mediated transcriptional repression is that SUMO provides a protein-protein interaction interface that recruits transcriptional corepressors. We have therefore used an affinity purification strategy to identify nuclear SUMO-binding proteins. We mixed soluble HeLa nuclear extracts with a nonconjugatable form of GST-SUMO-2 (GST-SUMO-2-GA) or control GST affinity resins and identified the bound proteins by mass spectrometry. Among the proteins retained specifically by GST-SUMO-2 (see Table S1 available online), we have been particularly interested in a group of proteins, including LSD1, CoREST1, HDAC1, and HDAC2, that were previously reported as components of the LSD1/CoREST1/HDAC corepressor complex (Hakimi et al., 2003Hakimi M.A. Dong Y. Lane W.S. Speicher D.W. Shiekhattar R. A candidate X-linked mental retardation gene is a component of a new family of histone deacetylase-containing complexes.J. Biol. Chem. 2003; 278: 7234-7239Crossref PubMed Scopus (133) Google Scholar, Humphrey et al., 2001Humphrey G.W. Wang Y. Russanova V.R. Hirai T. Qin J. Nakatani Y. Howard B.H. Stable histone deacetylase complexes distinguished by the presence of SANT domain proteins CoREST/kiaa0071 and Mta-L1.J. Biol. Chem. 2001; 276: 6817-6824Crossref PubMed Scopus (253) Google Scholar, Shi et al., 2003Shi Y. Sawada J. Sui G. Affar el B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Shi Y. Coordinated histone modifications mediated by a CtBP co-repressor complex.Nature. 2003; 422: 735-738Crossref PubMed Scopus (598) Google Scholar, Shi et al., 2005Shi Y.J. Matson C. Lan F. Iwase S. Baba T. Shi Y. Regulation of LSD1 histone demethylase activity by its associated factors.Mol. Cell. 2005; 19: 857-864Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, You et al., 2001You A. Tong J.K. Grozinger C.M. Schreiber S.L. CoREST is an integral component of the CoREST- human histone deacetylase complex.Proc. Natl. Acad. Sci. USA. 2001; 98: 1454-1458Crossref PubMed Scopus (368) Google Scholar). Previous studies using whole-cell extracts also revealed LSD1 association with GST-SUMO-2 (Rosendorff et al., 2006Rosendorff A. Sakakibara S. Lu S. Kieff E. Xuan Y. DiBacco A. Shi Y. Shi Y. Gill G. NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression.Proc. Natl. Acad. Sci. USA. 2006; 103: 5308-5313Crossref PubMed Scopus (86) Google Scholar). Identification of multiple LSD1/CoREST1/HDAC corepressor complex subunits suggested that a multiprotein complex was retained on the GST-SUMO-2 affinity resin. We therefore investigated which subunits bound directly to SUMO-2. Using purified recombinant proteins, we found that (His)6-CoREST1 bound efficiently to GST-SUMO-2 (Figure 1A). SUMO-2 possesses stronger intrinsic transcriptional repression activity than SUMO-1 in reporter gene assays, and residues K33 and K35 contribute to the transcriptional repression activity of SUMO-2 (Chupreta et al., 2005Chupreta S. Holmstrom S. Subramanian L. Iniguez-Lluhi J.A. A small conserved surface in SUMO is the critical structural determinant of its transcriptional inhibitory properties.Mol. Cell. Biol. 2005; 25: 4272-4282Crossref PubMed Scopus (60) Google Scholar, Rosendorff et al., 2006Rosendorff A. Sakakibara S. Lu S. Kieff E. Xuan Y. DiBacco A. Shi Y. Shi Y. Gill G. NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression.Proc. Natl. Acad. Sci. USA. 2006; 103: 5308-5313Crossref PubMed Scopus (86) Google Scholar). As shown in Figure 1A, we found that (His)6-CoREST1 bound to GST-SUMO-2, but almost no binding was observed to GST-SUMO-1 or GST-SUMO-2-K33,35A. Mutation of SUMO-2 K33, K35 did not affect binding to UBC9 (Rosendorff et al., 2006Rosendorff A. Sakakibara S. Lu S. Kieff E. Xuan Y. DiBacco A. Shi Y. Shi Y. Gill G. NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression.Proc. Natl. Acad. Sci. USA. 2006; 103: 5308-5313Crossref PubMed Scopus (86) Google Scholar) or SENP5 (J.O. and G.G., unpublished data), indicating that these mutations do not disturb the overall conformation of SUMO-2. Thus, CoREST1 binds directly and noncovalently to SUMO-2 in vitro and this binding correlates with transcriptional repression. We then examined CoREST1 association with SUMO-2/3 in vivo. As shown in Figure 1B, 3xFLAG-CoREST1 coimmunoprecipitated endogenous SUMO-2/3 conjugates from cell lysates. Notably, almost no SUMO-1 conjugates coimmunoprecipitated with CoREST1, consistent with the preference for SUMO-2 binding observed in vitro. The SUMO-2/3 conjugates coimmunoprecipitated with CoREST1 are not SUMO-2/3-modified forms of CoREST1 because they were not immunoreactive to anti-FLAG or anti-CoREST1 antibody (data not shown). In reciprocal assays, CoREST1, as well as LSD1 and HDACs 1 and 2, coimmunoprecipitated with HA-SUMO-2-GG in vivo (Figure S1), further confirming that the LSD1/CoREST1/HDAC corepressor core complex associates with SUMO-2/3 conjugates in cells. In contrast to our findings with CoREST1, we did not observe binding of purified LSD1 to SUMO-2 (Figure 2). We therefore reasoned that LSD1 was retained on the GST-SUMO-2 affinity resin through an indirect mechanism. Since CoREST1 binds directly to both LSD1 and SUMO-2, we asked if CoREST1 can bridge recruitment of LSD1 by SUMO-2. To this end, we mixed purified recombinant (His)6-LSD1 with purified GST-SUMO-2 in the absence or presence of purified (His)6-CoREST1. As shown in Figure 2, LSD1 did not bind to SUMO-2 directly. However, LSD1 was retained by the GST-SUMO-2 resin in the presence of CoREST1. These data suggest that SUMO-2 binding by CoREST1 could be the mechanism underlying the observed association of LSD1 with SUMO-2 in vivo (Figure S1). In order to map the SUMO-2 binding domain in CoREST1, we generated a series of CoREST1 truncation mutants (Figure 3A). Results from in vitro binding assays performed with recombinant (His)6-tagged CoREST1 deletions and GST-SUMO-2 indicated that the region between amino acids 241–300 of CoREST1, which includes part of the first coiled-coil region, is important for binding to SUMO-2 (Figures 3B and S2). As shown in Figure 3B, an amino-terminal fragment of CoREST1, residues 1–300, bound SUMO-2 as well as wild-type (WT) CoREST1, whereas truncations 1–240 or 301–482 did not bind to GST-SUMO-2, although the C-terminal fragment 301–482 bound to GST-LSD1 perfectly well. As summarized in Figure 3A, all of the truncated CoREST1 derivatives lacking amino acids 241–300 failed to bind to GST-SUMO-2 in vitro. A small fragment encompassing this region, 237–300, was not sufficient to bind SUMO-2 under these conditions. Truncated forms of CoREST1 that failed to bind to GST-SUMO-2 in vitro also had decreased binding to SUMO-2/3 conjugates in vivo (data not shown). Based on these results, we conclude that CoREST1 residues 241–300 are necessary, but not sufficient, for binding SUMO-2. To further localize the SUMO-2 interaction motif in CoREST1, we made internal deletions of CoREST1 within the region essential for binding, 241–300. The consensus SUMO interaction motif (SIM) has key hydrophobic residues often flanked by acidic residues (Hecker et al., 2006Hecker C.M. Rabiller M. Haglund K. Bayer P. Dikic I. Specification of SUMO1- and SUMO2-interacting motifs.J. Biol. Chem. 2006; 281: 16117-16127Crossref PubMed Scopus (384) Google Scholar, Song et al., 2004Song J. Durrin L.K. Wilkinson T.A. Krontiris T.G. Chen Y. Identification of a SUMO-binding motif that recognizes SUMO-modified proteins.Proc. Natl. Acad. Sci. USA. 2004; 101: 14373-14378Crossref PubMed Scopus (440) Google Scholar). Although there are predicted consensus SIMs in the C terminus of CoREST1, our deletion analyses revealed that these are not important for SUMO-2 binding. Nonetheless, guided by previous studies, we made small deletions within the region 241–300, including deletions of an acidic stretch (aa 255–263), a hydrophobic patch (aa 270–275), and the sequence in between (aa 264–270) (Figure 4B). Individual deletion of these small regions significantly reduced but did not eliminate CoREST1 binding to SUMO-2 in vitro (Figure 3C). Strikingly, deletion of all three regions together (d255–275) led to complete abrogation of SUMO-2 binding activity of CoREST1, although binding to GST-LSD1 was not affected. Thus, deletion analysis reveals that CoREST1 amino acids 255–275 are required for binding to SUMO-2 (Figure 3C). A close homolog of CoREST1, CoREST3, is also a component of the LSD1/CoREST1/HDAC complex (Shi et al., 2005Shi Y.J. Matson C. Lan F. Iwase S. Baba T. Shi Y. Regulation of LSD1 histone demethylase activity by its associated factors.Mol. Cell. 2005; 19: 857-864Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar), and CoREST3 was associated with GST-SUMO-2 in our affinity chromatography assays (Table S1). Interestingly, we found that CoREST3 did not bind to SUMO-2 in vitro, although CoREST3 bound well to GST-LSD1 (Figure 4A). These findings raise the possibility that CoREST3, like CoREST1, may regulate LSD1 demethylase activity; differential binding to SUMO-2, however, supports distinct biological functions of CoREST1 and CoREST3. We compared the sequences of CoREST1 and CoREST3 in the region required for SUMO-2 binding of CoREST1 (amino acids 255–275). As shown in Figure 4B, the alignment reveals that CoREST3 lacks hydrophobic residues conserved in CoREST1. Structural studies have revealed that the hydrophobic core (I/V-X-I/V-I/V or I/V-I/V-X-I/V) in the consensus SIM makes direct contact with SUMO (Baba et al., 2005Baba D. Maita N. Jee J.G. Uchimura Y. Saitoh H. Sugasawa K. Hanaoka F. Tochio H. Hiroaki H. Shirakawa M. Crystal structure of thymine DNA glycosylase conjugated to SUMO-1.Nature. 2005; 435: 979-982Crossref PubMed Scopus (177) Google Scholar, Reverter and Lima, 2005Reverter D. Lima C.D. Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex.Nature. 2005; 435: 687-692Crossref PubMed Scopus (352) Google Scholar). Although the hydrophobic core in the CoREST1 SUMO-2 interaction motif is notably different from the consensus SIM, largely due to absence of a hydrophobic residue at position 4, we nonetheless reasoned that the hydrophobic residues could be important for direct binding of CoREST1 to SUMO-2. As shown in Figure 4C, mutation of three hydrophobic residues in CoREST1 (IIV) into alanines (AAA) resulted in very weak to no binding to SUMO-2 in vitro. Also, when the hydrophobic residues were replaced with the CoREST3 counterparts (NSY), binding to SUMO-2 was almost completely abolished. Neither the AAA nor NSY mutations affected binding to LSD1, consistent with the fact that the hydrophobic patch is distinct from the region of CoREST1 binding to LSD1 (Shi et al., 2005Shi Y.J. Matson C. Lan F. Iwase S. Baba T. Shi Y. Regulation of LSD1 histone demethylase activity by its associated factors.Mol. Cell. 2005; 19: 857-864Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, Yang et al., 2006Yang M. Gocke C.B. Luo X. Borek D. Tomchick D.R. Machius M. Otwinowski Z. Yu H. Structural basis for CoREST-dependent demethylation of nucleosomes by the human LSD1 histone demethylase.Mol. Cell. 2006; 23: 377-387Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). These studies define a hydrophobic core in the CoREST1 SIM that is required for high-affinity binding to SUMO-2. Since CoREST1 binds specifically to SUMO-2 dependent on a region of CoREST1 that does not match the current SIM consensus, we wondered if this unusual SIM contributes to SUMO-2-specific binding in other proteins. We searched for proteins containing a small sequence pattern: I/V/L-D/E-I/V/L-D/E-I/V/L with N-terminal acidic residues. We tested 11 out of approximately 50 matches for direct binding to SUMO-2 in vitro. Using this approach we have identified three proteins, including FIP1L1 and RBBP4, that bound directly to SU" @default.
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• W2024634513 title "Direct Binding of CoREST1 to SUMO-2/3 Contributes to Gene-Specific Repression by the LSD1/CoREST1/HDAC Complex" @default.
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• W2024634513 doi "https://doi.org/10.1016/j.molcel.2009.03.013" @default.
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