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Lysine methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling

Abstract

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-κB 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-ζ (PKC-ζ) 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.

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Figure 1: SETD6 monomethylates RelA at Lys310.
Figure 2: Monomethylation of RelA by SETD6 inhibits the transactivation activity of RelA.
Figure 3: SETD6 attenuates RelA-driven cell proliferation.
Figure 4: SETD6 attenuates RelA-driven inflammatory responses.
Figure 5: GLP(ANK) binds specifically to RelAK310me1.
Figure 6: Phosphorylation of RelA at S311 by PKC-ζ blocks GLP recognition of RelAK310me1.
Figure 7: RelA methylation-phosphorylation switch at chromatin regulates NF-κB signaling.

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References

  1. Kouzarides, T. Chromatin modifications and their function. Cell 128, 693–705 (2007).

    Article  CAS  Google Scholar 

  2. Albert, M. & Helin, K. Histone methyltransferases in cancer. Semin. Cell Dev. Biol. 2, 209–220 (2009).

    Google Scholar 

  3. Huang, J. & Berger, S.L. The emerging field of dynamic lysine methylation of non-histone proteins. Curr. Opin. Genet. Dev. 18, 152–158 (2008).

    Article  CAS  Google Scholar 

  4. Hoffmann, A., Natoli, G. & Ghosh, G. Transcriptional regulation via the NF-κB signaling module. Oncogene 25, 6706–6716 (2006).

    Article  CAS  Google Scholar 

  5. Natoli, G. Control of NF-κB-dependent transcriptional responses by chromatin organization. Cold Spring Harb Perspect Biol 1, a000224 (2009).

    Article  Google Scholar 

  6. Ghosh, S. & Hayden, M.S. New regulators of NF-κB in inflammation. Nat. Rev. Immunol. 8, 837–848 (2008).

    Article  CAS  Google Scholar 

  7. Perkins, N.D. Post-translational modifications regulating the activity and function of the nuclear factor κB pathway. Oncogene 25, 6717–6730 (2006).

    Article  CAS  Google Scholar 

  8. Grivennikov, S.I., Greten, F.R. & Karin, M. Immunity, inflammation, and cancer. Cell 140, 883–899 (2010).

    Article  CAS  Google Scholar 

  9. Tachibana, M. et al. Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3–K9. Genes Dev. 19, 815–826 (2005).

    Article  CAS  Google Scholar 

  10. Saccani, S. & Natoli, G. Dynamic changes in histone H3 Lys 9 methylation occurring at tightly regulated inducible inflammatory genes. Genes Dev. 16, 2219–2224 (2002).

    Article  CAS  Google Scholar 

  11. Duran, A., Diaz-Meco, M.T. & Moscat, J. Essential role of RelA Ser311 phosphorylation by ζPKC in NF-κB transcriptional activation. EMBO J. 22, 3910–3918 (2003).

    Article  CAS  Google Scholar 

  12. Ea, C.K. & Baltimore, D. Regulation of NF-κB activity through lysine monomethylation of p65. Proc. Natl. Acad. Sci. USA 106, 18972–18977 (2009).

    Article  CAS  Google Scholar 

  13. Yang, X.D. et al. Negative regulation of NF-κB action by Set9-mediated lysine methylation of the RelA subunit. EMBO J. 28, 1055–1066 (2009).

    Article  CAS  Google Scholar 

  14. Trievel, R.C., Flynn, E.M., Houtz, R.L. & Hurley, J.H. Mechanism of multiple lysine methylation by the SET domain enzyme Rubisco LSMT. Nat. Struct. Biol. 10, 545–552 (2003).

    Article  CAS  Google Scholar 

  15. Dong, J., Jimi, E., Zeiss, C., Hayden, M.S. & Ghosh, S. Constitutively active NF-κB triggers systemic TNFα-dependent inflammation and localized TNFα-independent inflammatory disease. Genes Dev. 24, 1709–1717 (2010).

    Article  CAS  Google Scholar 

  16. Chen, L., Fischle, W., Verdin, E. & Greene, W.C. Duration of nuclear NF-κB action regulated by reversible acetylation. Science 293, 1653–1657 (2001).

    Article  CAS  Google Scholar 

  17. Tachibana, M., Sugimoto, K., Fukushima, T. & Shinkai, Y. Set domain-containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3. J. Biol. Chem. 276, 25309–25317 (2001).

    Article  CAS  Google Scholar 

  18. Buerki, C. et al. Functional relevance of novel p300-mediated lysine 314 and 315 acetylation of RelA/p65. Nucleic Acids Res. 36, 1665–1680 (2008).

    Article  CAS  Google Scholar 

  19. Kim, J. et al. Tudor, MBT and chromo domains gauge the degree of lysine methylation. EMBO Rep. 7, 397–403 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Collins, R.E. et al. The ankyrin repeats of G9a and GLP histone methyltransferases are mono- and dimethyllysine binding modules. Nat. Struct. Mol. Biol. 15, 245–250 (2008).

    Article  CAS  Google Scholar 

  21. Tachibana, M., Matsumura, Y., Fukuda, M., Kimura, H. & Shinkai, Y. G9a/GLP complexes independently mediate H3K9 and DNA methylation to silence transcription. EMBO J. 27, 2681–2690 (2008).

    Article  CAS  Google Scholar 

  22. Smith, L. et al. Activation of atypical protein kinase C ζ by caspase processing and degradation by the ubiquitin-proteasome system. J. Biol. Chem. 275, 40620–40627 (2000).

    Article  CAS  Google Scholar 

  23. Leitges, M. et al. Targeted disruption of the zetaPKC gene results in the impairment of the NF-κB pathway. Mol. Cell 8, 771–780 (2001).

    Article  CAS  Google Scholar 

  24. Su, I.H. & Tarakhovsky, A. Lysine methylation and 'signaling memory'. Curr. Opin. Immunol. 18, 152–157 (2006).

    Article  CAS  Google Scholar 

  25. Hirota, T., Lipp, J.J., Toh, B.H. & Peters, J.M. Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin. Nature 438, 1176–1180 (2005).

    Article  CAS  Google Scholar 

  26. Fischle, W. et al. Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation. Nature 438, 1116–1122 (2005).

    Article  CAS  Google Scholar 

  27. Zhang, K. et al. The Set1 methyltransferase opposes Ipl1 aurora kinase functions in chromosome segregation. Cell 122, 723–734 (2005).

    Article  CAS  Google Scholar 

  28. Toney, L.M. et al. BCL-6 regulates chemokine gene transcription in macrophages. Nat. Immunol. 1, 214–220 (2000).

    Article  CAS  Google Scholar 

  29. Yasuda, K. et al. Murine dendritic cell type I IFN production induced by human IgG-RNA immune complexes is IFN regulatory factor (IRF)5 and IRF7 dependent and is required for IL-6 production. J. Immunol. 178, 6876–6885 (2007).

    Article  CAS  Google Scholar 

  30. Michishita, E. et al. SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin. Nature 452, 492–496 (2008).

    Article  CAS  Google Scholar 

  31. Kattah, M.G., Coller, J., Cheung, R.K., Oshidary, N. & Utz, P.J. HIT: a versatile proteomics platform for multianalyte phenotyping of cytokines, intracellular proteins and surface molecules. Nat. Med. 14, 1284–1289 (2008).

    Article  CAS  Google Scholar 

  32. van der Pouw Kraan, T.C. et al. Rheumatoid arthritis subtypes identified by genomic profiling of peripheral blood cells: assignment of a type I interferon signature in a subpopulation of patients. Ann. Rheum. Dis. 66, 1008–1014 (2007).

    Article  CAS  Google Scholar 

  33. Julia, A. et al. An eight-gene blood expression profile predicts the response to infliximab in rheumatoid arthritis. PLoS One 4, e7556 (2009).

    Article  Google Scholar 

  34. Barnes, M.G. et al. Subtype-specific peripheral blood gene expression profiles in recent-onset juvenile idiopathic arthritis. Arthritis Rheum. 60, 2102–2112 (2009).

    Article  CAS  Google Scholar 

  35. Wong, H.R. et al. Genome-level expression profiles in pediatric septic shock indicate a role for altered zinc homeostasis in poor outcome. Physiol. Genomics 30, 146–155 (2007).

    Article  CAS  Google Scholar 

  36. Demeter, J. et al. The Stanford Microarray Database: implementation of new analysis tools and open source release of software. Nucleic Acids Res. 35, D766–D770 (2007).

    Article  CAS  Google Scholar 

  37. Shanley, T.P. et al. Genome-level longitudinal expression of signaling pathways and gene networks in pediatric septic shock. Mol. Med. 13, 495–508 (2007).

    Article  CAS  Google Scholar 

  38. Shi, X. et al. ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression. Nature 442, 96–99 (2006).

    Article  CAS  Google Scholar 

  39. Schnitzler, G.R. in Current Protocols in Molecular Biology Ch 21, 21.5.1–21.5.12 (John Wiley & Sons, Hoboken, New Jersey, 2001).

  40. Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).

    Article  CAS  Google Scholar 

  41. Mendez, J. & Stillman, B. Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol. Cell. Biol. 20, 8602–8612 (2000).

    Article  CAS  Google Scholar 

  42. Michishita, E., Park, J.Y., Burneskis, J.M., Barrett, J.C. & Horikawa, I. Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins. Mol. Biol. Cell 16, 4623–4635 (2005).

    Article  CAS  Google Scholar 

  43. Ainbinder, E. et al. Mechanism of rapid transcriptional induction of tumor necrosis factor α-responsive genes by NF-κB. Mol. Cell. Biol. 22, 6354–6362 (2002).

    Article  CAS  Google Scholar 

  44. Nelson, J.D., Denisenko, O. & Bomsztyk, K. Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat. Protocols 1, 179–185 (2006).

    Article  CAS  Google Scholar 

  45. Kelley, L.A. & Sternberg, M.J. Protein structure prediction on the web: a case study using the Phyre server. Nat. Protocols 4, 363–371 (2009).

    Article  CAS  Google Scholar 

  46. Couture, J.F., Hauk, G., Thompson, M.J., Blackburn, G.M. & Trievel, R.C. Catalytic roles for carbon-oxygen hydrogen bonding in SET domain lysine methyltransferases. J. Biol. Chem. 281, 19280–19287 (2006).

    Article  CAS  Google Scholar 

  47. Bua, D.J. et al. Epigenome microarray platform for proteome-wide dissection of chromatin-signaling networks. PLoS One 4, e6789 (2009).

    Article  Google Scholar 

  48. Espejo, A., Cote, J., Bednarek, A., Richard, S. & Bedford, M.T. A protein-domain microarray identifies novel protein-protein interactions. Biochem. J. 367, 697–702 (2002).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank R. Kingston and M. Simon (Harvard Medical School) for recombinant nucleosomes; J. Smith (University of Alabama Birmingham) for the PKC-ζ(ca) plasmid; W.C. Greene (University of California San Francisco) for RelA(1–431) cDNA and the κB-Luc luciferase reporter plasmid; D. Reinberg (New York University) for the NSD1(SET) plasmid; M. Covert (Stanford University) and T.D. Gilmore (Boston University) for the wild-type and Rela−/− mouse 3T3 cells; J. Moscat (University of Cincinnati College of Medicine) for the wild-type and Prkcz−/− MEFs; E. Engleman (Stanford University) for FL-B16 cells; E. Green for critical reading of the manuscript; and A. Alizadeh for comments. Supported by the National Institutes of Health (DA025800 to O.G. and M.T.B.; GM068680 to X.C.; and F32AI080086 to C.L.L.), the American Society for Mass Spectrometry (B.A.G.), the National Heart, Lung and Blood Institute (HHSN-268201999934C to P.J.U.), the National Institute of Allergy and Infectious Diseases (U19-AI082719 to P.J.U.), the Floren Family Trust (P.J.U.), the Genentech Foundation (A.J.K.), the European Molecular Biology Organization (D.L.), the Human Frontier Science Program (D.L.), the Machiah Foundation (D.L.), the Georgia Research Alliance (X.C.) and the Ellison Medical Foundation (O.G.)

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Authors

Contributions

D.L. did most of the molecular biology and cellular studies; Y.C. did binding affinity studies and modeling; A.J.K., P.C. and X.S. generated the PKMT library; A.J.K. identified and initially characterized the activity of SETD6 on RelA Lys310; B.Z. did mass spectrometry analysis; U.S. and C.K. did the primary cells experiments; A.E. did CADOR array experiments; C.L.L. analyzed gene expression data sets; R.I.T., S.T., A.Y.K., R.C. and S.T. provided technical support; X.S., P.J.U., K.C., B.G., R.P., M.B., A.T., X.C. and O.G. discussed studies; D.L. and O.G. designed studies, analyzed data, and wrote the paper; D.L. and A.J.K. contributed independently to the work; and all authors discussed and commented on the manuscript.

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Correspondence to Or Gozani.

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C.K., R.K.P. and K.L. are employees of GlaxoSmithKline.

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Supplementary Figures 1–25, Tables 1–3 and Data (PDF 7458 kb)

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Expression array data set. (XLS 467 kb)

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Levy, D., Kuo, A., Chang, Y. et al. Lysine methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling. Nat Immunol 12, 29–36 (2011). https://doi.org/10.1038/ni.1968

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