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Lysine Methylation of the NF-Κb Subunit Rela by SETD6 Couples Activity of the Histone Methyltransferase GLP at Chromatin To

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Lysine Methylation of the NF-Κb Subunit Rela by SETD6 Couples Activity of the Histone Methyltransferase GLP at Chromatin To ARTICLES Lysine methylation of the NF-kB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-kB signaling Dan Levy1, Alex J Kuo1, Yanqi Chang2, Uwe Schaefer3, Christopher Kitson4, Peggie Cheung1, Alexsandra Espejo5, Barry M Zee6, Chih Long Liu1,7, Stephanie Tangsombatvisit7, Ruth I Tennen8, Andrew Y Kuo1, Song Tanjing9, Regina Cheung7, Katrin F Chua8,10, Paul J Utz7, Xiaobing Shi9, Rab K Prinjha4, Kevin Lee4, Benjamin A Garcia6, Mark T Bedford5, Alexander Tarakhovsky3, Xiaodong Cheng2 & Or Gozani1 Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-kB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation–linked phosphorylation of RelA at Ser311 by protein kinase C-z (PKC-z) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs. Chromatin dynamics regulate key cellular functions that influence proteins4,5. Stimulation of cells with NF­κB­activating ligands such survival, growth and proliferation, and disruption of chromatin homeo­ as the cytokine tumor necrosis factor (TNF) results in degradation stasis has been linked to diverse pathologic processes1. Methylation of of these inhibitors of NF­κB and translocation of the released NF­κB Nature America, Inc. All rights reserved. All rights Inc. America, Nature 5,6 1 lysine residues of histone, which is catalyzed by protein lysine methyl­ to the nucleus, where it directs various transcriptional programs . 1 transferases (PKMTs), is a principal chromatin­regulatory mecha­ In addition to that canonical pathway, there are several additional nism involved in directing fundamental DNA­templated processes mechanisms that regulate and fine­tune NF­κB signaling and activa­ © 20 such as transcription and DNA repair1. Histone methylation plays a tion of target genes7. For example, various post­translational modifi­ central part in orchestrating proper programming of the genome in cations of RelA influence the specificity, transcriptional activity and response to various stimuli, and aberrant signaling via lysine methyl­ activation kinetics of RelA target genes. Furthermore, even in resting ation has been linked to the initiation and progression of many human conditions, a population of RelA is present in the nucleus, bound at diseases2. Many non­histone proteins are also regulated by lysine chromatin; however, the functional relevance of this constitutively methylation, which indicates that this modification is probably a nuclear population is not known. common mechanism for the modulation of protein­protein inter­ Deregulation of NF­κB signaling is linked to many human diseases, actions and signaling pathways3. including cancer and autoimmune disorders8. Thus, understanding NF­κB is a transcription factor and key inducer of inflammatory the full range of molecular mechanisms that modulate this factor in responses4,5. One of the principal subunits of NF­κB is RelA (p65 response to diverse conditions has important biological and clinical (A001645)), which forms either a homodimer or a heterodimer implications. Here we screened over 40 known and candidate human with the structurally related p50 protein (A002937). Under basal PKMTs for methylation of RelA in vitro. We identify SETD6 (SET conditions, most RelA is sequestered in the cytoplasm because of domain–containing protein 6) as a PKMT that monomethylated its association with members of the inhibitor of NF­κB family of RelA at Lys310 (RelAK310me1). The ankryin repeat of the PKMT 1Department of Biology, Stanford University, Stanford, California, USA. 2Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA. 3Laboratory of Lymphocyte Signaling, The Rockefeller University, New York, New York, USA. 4EpiNova DPU, Immuno-Inflammation group, GlaxoSmithKline, Stevenage, UK. 5Department of Carcinogenesis, M.D. Anderson Cancer Center, Smithville, Texas, USA. 6Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA. 7Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, California, USA. 8Department of Endocrinology, Gerontology, and Metabolism Medicine, Stanford University School of Medicine, Stanford, California, USA. 9Center for Cancer Epigenetics, University of Texas M.D. Anderson, Houston, Texas, USA. 10Geriatric Research, Education, and Clinical Center, VA Palo Alto Health Care System, Palo Alto, California, USA. Correspondence should be addressed to O.G. ([email protected]). Received 15 October; accepted 9 November; published online 5 December 2010; doi:10.1038/ni.1968 NATURE IMMUNOLOGY VOLUME 12 NUMBER 1 JANUARY 2011 29 ARTICLES a b RelA(1–431) RelA(1–431) SETD6 (kDa) SETD2(SET)SETD4SETD5(SET)SETD6SETD8PRDM2(SET)PRDM4(SET)SETDB1SETDB2GLP(SET)G9a(SET)SETMAR(SET)ASH1(SET)SUV39H2 250 – WT K122RK123RK218RK221RK310R 150 RelA(1–431) 3H – SETD2(SET)SETD4SETD5(SET)SETD6SETD8PRDM2(SET)PRDM4(SET)– SETDB1SETDB2GLP(SET)G9a(SET)SETMAR(SET)ASH1(SET)SUV39H2 100 75 SETD6 3H Coomassie 50 RelA RelA 37 RelA(300–320) RelA(300–320) + SETD6 c 100 555.896 100 555.896 d e f 556.096 556.096 SETD6 – + + + RelA(1–431) 80 80 SETD6 ––+ + RelA WT – – + – 555.696 555.695 RelA(1–431) WT K310R RelA(K310R) – – – + 60 60 556.296 556.296 RelAK310me1 RelAK310me1 – SETD6SETD6(Y285A) WT 40 40 SETD6 RelA 3 556.497 556.497 + me1 H Abundance (%) SETD6 Abundance (%) RelA 20 20 558.699 SETD6 556.697 558.498 559.098 556.697 559.297 Tubulin Coomassie RelA 0 0 554 555 556 557 558 559 560 554 555 556 557 558 559 560 m/z m/z 293T U2OS g SETD6 WT – + – h SETD6 siRNA: C 1 2 i293T j k Cyt ChromCyt Chrom SETD6(Y285A) –– + RelAK310me1 ChIP IP: anti-RelA TNF ––+ + – +– + RelAK310me1 Cyt Nuc Chrom IP: anti-RelA RelA RelAK310me1 RelAK310me1 RelA Input lgG RelA RelA SETD6 H3 Tubulin SETD6 Tubulin WCE WCE RelA RelA RelA H3 H3 Tubulin Figure 1 SETD6 monomethylates RelA at Lys310. (a) Methylation reactions (3H autoradiogram; left) with recombinant RelA(1–431) as substrate and recombinant PKMT enzymes (full-length or SET domains only; above lanes) and Coomassie staining (right) of recombinant enzymes (left margin, molecular size in kilodaltons (kDa)). Top band, automethylated SETD6. (b) SETD6-catalyzed methylation assay (autoradiogram; top) with wild-type or mutant RelA(1–431) (above lanes), and Coomassie staining (below) of proteins used. (c) Mass spectrometry analysis of methylation assays of RelA peptide (amino acids 300–320; RelA(300–320)) with (right) or without (left) SETD6; results are presented relative to those of the most abundant ion, set as 100%. Numbers above peaks indicate the mass/charge (m/z) ratio. (d) Immunoblot analysis of methylation reactions with (+) or without (−) SETD6 on wild- type (WT) RelA(1–431) or RelA(1–431) with the K310R substitution. (e) Immunoblot analysis of whole-cell extracts (WCE; 5% of total) of 293T cells transfected with Flag-tagged SETD6 and either RelA or RelA(K310R), probed with antibodies to various molecules (left margin). (f) In vitro methylation reaction (top) with wild-type SETD6 or SETD6(Y285A) on RelA(1–431) and Coomassie staining (below) of recombinant proteins used. (g) Immunoblot analysis of immunoprecipitated RelA (IP) or WCE (5% of total) from 293T cells transfected with wild-type SETD6 or SETD6(Y285A). (h) Immunoblot analysis (as in g) of 293T cells treated with control (C) or SETD6-specific siRNA (two independent siRNAs: 1 and 2). (i) Immunoblot analysis of RelA or control immunoglobulin G (IgG) protein-protein ChIP, probed with antibodies to various molecules (left margin). Input, 5% of total. (j) Immunoblot analysis Nature America, Inc. All rights reserved. All rights Inc. America, Nature of 293T cells biochemically separated into cytoplasmic (Cyt), nucleoplasmic (Nuc) or chromatin-enriched (Chrom) fractions; tubulin and H3 signals serve 1 1 as controls for fractionation integrity. (k) Immunoblot analysis of cytoplasmic and chromatin-enriched fractions (as in j) from 293T and U2OS cells with (+) or without (−) treatment with TNF (10 ng/ml for 1 h). Data are representative of three (a,b,e,g,h) or two (c,d,f,i–k) independent experiments. © 20 GLP (G9A­like protein) functioned as a recognition module for catalytic activity on various histone and non­histone candidate RelAK310me1, which links this mark to localized methylation of substrates (Supplementary Table 1 and data not shown). SETD6, histone H3 Lys9 (H3K9) and repressed chromatin at RelAK310me1­ a previously uncharacterized PKMT, methylated an N­terminal occupied genes9,10. The SETD6­initiated lysine­methylation signaling RelA polypeptide encompassing amino acids 1­431 (RelA(1–431)) cascade acted to restrain activation of NF­κB­mediated
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