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The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity

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Abstract

Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2. Biochemical and co-crystallization analyses demonstrate that A-196 is a substrate-competitive inhibitor of both SUV4-20 enzymes. In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a first-in-class chemical probe of SUV4-20 to investigate the role of histone methyltransferases in genomic integrity.

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Figure 1: A-196 is potent peptide-site competitive inhibitor of SUV420H1 and SUV420H2.
Figure 2: A-196 directly binds to SUV420H1.
Figure 3: Crystal structure of A-196 bound to SUV420H1.
Figure 4: A-196 inhibits H4K20 di- and trimethylation in cells.
Figure 5: A-196 inhibits 53BP1 foci formation and NHEJ proficiency.

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  • 31 January 2017

    In the version of this article initially published online, the second paragraph of the introduction contained two unnecessary commas, and "Suv4-20h1" was incorrectly listed in place of "Suv4-20h2" in one instance. The first two sentences of the description of mouse Suv4-20h expression should read: "Mouse Suv4-20h1 is ubiquitously expressed throughout embryogenesis and adult homeostasis, and mice that are homozygous null for this gene are perinatal lethal and have incomplete penetrance10. Suv4-20h2 expression, by comparison, is much less abundant during murine development. It is highly restricted in the adult, and Suv4-20h2 homozygous null mice display no apparent defects." These errors have been corrected in the print, PDF and HTML versions of this article.

References

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

    Article  CAS  Google Scholar 

  2. Martin, C. & Zhang, Y. The diverse functions of histone lysine methylation. Nat. Rev. Mol. Cell Biol. 6, 838–849 (2005).

    Article  CAS  Google Scholar 

  3. Lukas, J., Lukas, C. & Bartek, J. More than just a focus: The chromatin response to DNA damage and its role in genome integrity maintenance. Nat. Cell Biol. 13, 1161–1169 (2011).

    Article  CAS  Google Scholar 

  4. Helin, K. & Dhanak, D. Chromatin proteins and modifications as drug targets. Nature 502, 480–488 (2013).

    Article  CAS  Google Scholar 

  5. Herz, H.M., Garruss, A. & Shilatifard, A. SET for life: biochemical activities and biological functions of SET domain-containing proteins. Trends Biochem. Sci. 38, 621–639 (2013).

    Article  CAS  Google Scholar 

  6. Jenuwein, T. & Allis, C.D. Translating the histone code. Science 293, 1074–1080 (2001).

    Article  CAS  Google Scholar 

  7. Plass, C. et al. Mutations in regulators of the epigenome and their connections to global chromatin patterns in cancer. Nat. Rev. Genet. 14, 765–780 (2013).

    Article  CAS  Google Scholar 

  8. Jørgensen, S., Schotta, G. & Sørensen, C.S. Histone H4 lysine 20 methylation: key player in epigenetic regulation of genomic integrity. Nucleic Acids Res. 41, 2797–2806 (2013).

    Article  Google Scholar 

  9. Nishioka, K. et al. 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 (2002).

    Article  CAS  Google Scholar 

  10. Schotta, G. et al. A chromatin-wide transition to H4K20 monomethylation impairs genome integrity and programmed DNA rearrangements in the mouse. Genes Dev. 22, 2048–2061 (2008).

    Article  CAS  Google Scholar 

  11. Yang, H. et al. Preferential dimethylation of histone H4 lysine 20 by Suv4-20. J. Biol. Chem. 283, 12085–12092 (2008).

    Article  CAS  Google Scholar 

  12. Beck, D.B., Oda, H., Shen, S.S. & Reinberg, D. PR-Set7 and H4K20me1: at the crossroads of genome integrity, cell cycle, chromosome condensation, and transcription. Genes Dev. 26, 325–337 (2012).

    Article  CAS  Google Scholar 

  13. Sanders, S.L. et al. Methylation of histone H4 lysine 20 controls recruitment of Crb2 to sites of DNA damage. Cell 119, 603–614 (2004).

    Article  CAS  Google Scholar 

  14. Schotta, G. et al. A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin. Genes Dev. 18, 1251–1262 (2004).

    Article  CAS  Google Scholar 

  15. Benetti, R. et al. Suv4-20h deficiency results in telomere elongation and derepression of telomere recombination. J. Cell Biol. 178, 925–936 (2007).

    Article  CAS  Google Scholar 

  16. Marión, R.M., Schotta, G., Ortega, S. & Blasco, M.A. Suv4-20h abrogation enhances telomere elongation during reprogramming and confers a higher tumorigenic potential to iPS cells. PLoS One 6, e25680 (2011).

    Article  Google Scholar 

  17. Hsiao, K.Y. & Mizzen, C.A. Histone H4 deacetylation facilitates 53BP1 DNA damage signaling and double-strand break repair. J. Mol. Cell Biol. 5, 157–165 (2013).

    Article  CAS  Google Scholar 

  18. Tuzon, C.T. et al. Concerted activities of distinct H4K20 methyltransferases at DNA double-strand breaks regulate 53BP1 nucleation and NHEJ-directed repair. Cell Rep. 8, 430–438 (2014).

    Article  CAS  Google Scholar 

  19. Ma, A. et al. Discovery of a selective, substrate-competitive inhibitor of the lysine methyltransferase SETD8. J. Med. Chem. 57, 6822–6833 (2014).

    Article  CAS  Google Scholar 

  20. Blum, G. et al. Small-molecule inhibitors of SETD8 with cellular activity. ACS Chem. Biol. 9, 2471–2478 (2014).

    Article  CAS  Google Scholar 

  21. Nguyen, H. et al. LLY-507, a cell-active, potent, and selective inhibitor of protein-lysine methyltransferase SMYD2. J. Biol. Chem. 290, 13641–13653 (2015).

    Article  CAS  Google Scholar 

  22. Schapira, M. Structural chemistry of human SET domain protein Methyltransferases. Curr. Chem. Genomics 5 (Suppl. 1), 85–94 (2011).

    Article  CAS  Google Scholar 

  23. Barsyte-Lovejoy, D. et al. (R)-PFI-2 is a potent and selective inhibitor of SETD7 methyltransferase activity in cells. Proc. Natl. Acad. Sci. USA 111, 12853–12858 (2014).

    Article  CAS  Google Scholar 

  24. Wu, H. et al. Crystal structures of the human histone H4K20 methyltransferases SUV420H1 and SUV420H2. FEBS Lett. 587, 3859–3868 (2013).

    Article  CAS  Google Scholar 

  25. Pesavento, J.J., Yang, H., Kelleher, N.L. & Mizzen, C.A. Certain and progressive methylation of histone H4 at lysine 20 during the cell cycle. Mol. Cell. Biol. 28, 468–486 (2008).

    Article  CAS  Google Scholar 

  26. Gunn, A. & Stark, J.M. I-SceI-based assays to examine distinct repair outcomes of mammalian chromosomal double strand breaks. Methods Mol. Biol. 920, 379–391 (2012).

    Article  CAS  Google Scholar 

  27. Han, L. & Yu, K. Altered kinetics of nonhomologous end joining and class switch recombination in ligase IV-deficient B cells. J. Exp. Med. 205, 2745–2753 (2008).

    Article  CAS  Google Scholar 

  28. Yan, C.T. et al. IgH class switching and translocations use a robust non-classical end-joining pathway. Nature 449, 478–482 (2007).

    Article  CAS  Google Scholar 

  29. Ramachandran, S. et al. The SAGA deubiquitination module promotes DNA repair and class switch recombination through ATM and DNAPK-mediated γH2AX formation. Cell Rep. 15, 1554–1565 (2016).

    Article  CAS  Google Scholar 

  30. Abbas, T. et al. CRL4(Cdt2) regulates cell proliferation and histone gene expression by targeting PR-Set7/Set8 for degradation. Mol. Cell 40, 9–21 (2010).

    Article  CAS  Google Scholar 

  31. Centore, R.C. et al. CRL4(Cdt2)-mediated destruction of the histone methyltransferase Set8 prevents premature chromatin compaction in S phase. Mol. Cell 40, 22–33 (2010).

    Article  CAS  Google Scholar 

  32. Oda, H. et al. Regulation of the histone H4 monomethylase PR-Set7 by CRL4(Cdt2)-mediated PCNA-dependent degradation during DNA damage. Mol. Cell 40, 364–376 (2010).

    Article  CAS  Google Scholar 

  33. Tardat, M. et al. The histone H4 Lys 20 methyltransferase PR-Set7 regulates replication origins in mammalian cells. Nat. Cell Biol. 12, 1086–1093 (2010).

    Article  CAS  Google Scholar 

  34. McKay, D.J. et al. Interrogating the function of metazoan histones using engineered gene clusters. Dev. Cell 32, 373–386 (2015).

    Article  CAS  Google Scholar 

  35. Beck, D.B. et al. The role of PR-Set7 in replication licensing depends on Suv4-20h. Genes Dev. 26, 2580–2589 (2012).

    Article  CAS  Google Scholar 

  36. Hartlerode, A.J. et al. Impact of histone H4 lysine 20 methylation on 53BP1 responses to chromosomal double strand breaks. PLoS One 7, e49211 (2012).

    Article  CAS  Google Scholar 

  37. Senisterra, G.A. et al. Screening for ligands using a generic and high-throughput light-scattering-based assay. J. Biomol. Screen. 11, 940–948 (2006).

    Article  CAS  Google Scholar 

  38. Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D Biol. Crystallogr. 67, 293–302 (2011).

    Article  CAS  Google Scholar 

  39. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  40. Wlodek, S., Skillman, A.G. & Nicholls, A. Automated ligand placement and refinement with a combined force field and shape potential. Acta Crystallogr. D Biol. Crystallogr. 62, 741–749 (2006).

    Article  CAS  Google Scholar 

  41. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  42. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  43. Bricogne, G. et al. BUSTER v. 1.11.2. (Global Phasing Ltd., 2011).

  44. Wesemann, D.R. et al. Immature B cells preferentially switch to IgE with increased direct Sμ to Sɛ recombination. J. Exp. Med. 208, 2733–2746 (2011).

    Article  CAS  Google Scholar 

  45. Deenick, E.K., Hasbold, J. & Hodgkin, P.D. Switching to IgG3, IgG2b, and IgA is division linked and independent, revealing a stochastic framework for describing differentiation. J. Immunol. 163, 4707–4714 (1999).

    CAS  PubMed  Google Scholar 

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Acknowledgements

AbbVie acknowledgments: We thank V. Abraham and M. Smith of AbbVie for high-content microscopy expertise and S. Kennedy of the Structural Genomics Consortium (SGC) for technical support. The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA), Janssen, Merck & Co., Novartis Pharma AG, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome Trust. 7TM, kinase, and ion channel off-target selectivity screening was kindly supplied by Eurofins-Cerep. Further Ki determinations and receptor binding profiles were generously provided by the National Institute of Mental Health's Psychoactive Drug Screening Program, Contract # HHSN-271-2013-00017-C (NIMH PDSP). The NIMH PDSP is directed by B.L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer J. Driscoll at NIMH, Bethesda, Maryland, USA. Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. J.C.R. is supported by the American Cancer Society (RSG117619) and an NCI Cancer Center Support Grant (P30CA014089). G.S. is supported by the Deutsche Forschungsgemeinschaft (SFB1064/A11).

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Authors and Affiliations

Authors

Contributions

M.A.J. and K.M.C. developed and conducted the high-throughput screen. Y.D., R.F.S., and M.R.M. designed compounds. M.T. generated 3D protein homology models and small-molecule docking/computational models. A.K.U. produced protein and protein crystals and C.G.J. performed X-ray structure determination and analysis. A.K.U. performed ITC analysis and Thermal Shift analyses. N.B.S., V.M, and M.A.A. performed in vitro biochemical studies. K.D.B. and L.M.L. performed high-content microscopy cellular methyl mark and proliferation assays. G.S. and A.N. performed immunofluorescence analyses in MEFs and ES cells. C.T.T. and J.C.R. performed DNA-damage response, 53BP1, NHEJ and HDR reporter assays. C.L. and A.M. performed the class switch recombination assays. T.R.H.M. and D.B.-L. performed histone marks analysis, toxicity and sensitization experiments. F.L. performed all lead optimization screening and IC50 determinations and mechanism-of-action studies. F.L. and M.S.E. performed selectivity assays. M.V. designed experiments, reviewed data and led in vitro assays. P.J.B., V.S., C.H.A., K.D.B., M.A., C.S., A.R.S., G.G.C., J.C.R., and W.N.P. designed studies and interpreted results. T.M., D.B.-L., K.D.B., and W.N.P. wrote the paper.

Corresponding authors

Correspondence to Dalia Barsyte-Lovejoy or William N Pappano.

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Competing interests

This study was sponsored by AbbVie. AbbVie contributed to the study design, research, and interpretation of data, writing, reviewing, and approving the publication. K.D.B., A.K.U., C.G.K., M.A.J., K.M.C., L.M.L., Y.D., N.B.S., V.M., M.A., R.F.S., M.T., C.S., M.R.M., A.R.S., G.G.C. and W.N.P. were employees of AbbVie at the time of the study.

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Bromberg, K., Mitchell, T., Upadhyay, A. et al. The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity. Nat Chem Biol 13, 317–324 (2017). https://doi.org/10.1038/nchembio.2282

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