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In Vitro Assays to Measure Histone Methyltransferase Activity Using Different Chromatin Substrates

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Plant Chromatin Dynamics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1675))

Abstract

In vitro histone modification (HM) assays are used to characterize the activity of chromatin-modifying enzymes. These assays provide information regarding the modification sites on histones, the product specificity, and the impact of other histone or nucleotide modifications on enzyme activity. In particular, histone methyltransferase (HMT) assays have been instrumental in elucidating the activity and site specificity of many plant HMT enzymes. In this chapter, we describe a general protocol that can be used to perform HMT assays using different chromatin substrates, detection methods, and enzymes directly purified from plant material or heterologous sources.

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References

  1. Rea S, Eisenhaber F, O'Carroll D, Strahl BD, Sun ZW, Schmid M, Opravil S, Mechtler K, Ponting CP, Allis CD, Jenuwein T (2000) Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406(6796):593–599. doi:10.1038/35020506

    Article  CAS  PubMed  Google Scholar 

  2. Alvarez-Venegas R, Pien S, Sadder M, Witmer X, Grossniklaus U, Avramova Z (2003) ATX-1, an Arabidopsis homolog of trithorax, activates flower homeotic genes. Curr Biol 13(8):627–637

    Article  CAS  PubMed  Google Scholar 

  3. Jackson JP, Lindroth AM, Cao X, Jacobsen SE (2002) Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416(6880):556–560. doi:10.1038/nature731

    Article  CAS  PubMed  Google Scholar 

  4. Jacob Y, Bergamin E, Donoghue MT, Mongeon V, LeBlanc C, Voigt P, Underwood CJ, Brunzelle JS, Michaels SD, Reinberg D, Couture JF, Martienssen RA (2014) Selective methylation of histone H3 variant H3.1 regulates heterochromatin replication. Science 343(6176):1249–1253. doi:10.1126/science.1248357

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Jacob Y, Feng S, LeBlanc CA, Bernatavichute YV, Stroud H, Cokus S, Johnson LM, Pellegrini M, Jacobsen SE, Michaels SD (2009) ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing. Nat Struct Mol Biol 16(7):763–768. doi:10.1038/nsmb.1611

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Chen Z, Grzybowski AT, Ruthenburg AJ (2014) Traceless semisynthesis of a set of histone 3 species bearing specific lysine methylation marks. Chembiochem 15(14):2071–2075. doi:10.1002/cbic.201402313

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Dawson PE, Muir TW, Clark-Lewis I, Kent SB (1994) Synthesis of proteins by native chemical ligation. Science 266(5186):776–779

    Article  CAS  PubMed  Google Scholar 

  8. Couture JF, Dirk LM, Brunzelle JS, Houtz RL, Trievel RC (2008) Structural origins for the product specificity of SET domain protein methyltransferases. Proc Natl Acad Sci U S A 105(52):20659–20664. doi:10.1073/pnas.0806712105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Stutzer A, Liokatis S, Kiesel A, Schwarzer D, Sprangers R, Soding J, Selenko P, Fischle W (2016) Modulations of DNA contacts by linker histones and post-translational modifications determine the mobility and modifiability of nucleosomal H3 tails. Mol Cell 61(2):247–259. doi:10.1016/j.molcel.2015.12.015

    Article  PubMed  Google Scholar 

  10. Kuzmichev A, Nishioka K, Erdjument-Bromage H, Tempst P, Reinberg D (2002) Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein. Genes Dev 16(22):2893–2905. doi:10.1101/gad.1035902

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst P, Jones RS, Zhang Y (2002) Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298(5595):1039–1043. doi:10.1126/science.1076997

    Article  CAS  PubMed  Google Scholar 

  12. Trievel RC, Beach BM, Dirk LM, Houtz RL, Hurley JH (2002) Structure and catalytic mechanism of a SET domain protein methyltransferase. Cell 111 (1):91–103

    Google Scholar 

  13. Zhang X, Tamaru H, Khan SI, Horton JR, Keefe LJ, Selker EU, Cheng X (2002) Structure of the Neurospora SET domain protein DIM-5, a histone H3 lysine methyltransferase. Cell 111 (1):117–127

    Google Scholar 

  14. Dyer PN, Edayathumangalam RS, White CL, Bao Y, Chakravarthy S, Muthurajan UM, Luger K (2004) Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol 375:23–44

    Article  CAS  PubMed  Google Scholar 

  15. Luger K, Rechsteiner TJ, Flaus AJ, Waye MM, Richmond TJ (1997) Characterization of nucleosome core particles containing histone proteins made in bacteria. J Mol Biol 272(3):301–311. doi:10.1006/jmbi.1997.1235

    Article  CAS  PubMed  Google Scholar 

  16. Voigt P, Reinberg D (2011) Histone tails: ideal motifs for probing epigenetics through chemical biology approaches. Chembiochem 12(2):236–252. doi:10.1002/cbic.201000493

    Article  CAS  PubMed  Google Scholar 

  17. Simon RH, Felsenfeld G (1979) A new procedure for purifying histone pairs H2A + H2B and H3 + H4 from chromatin using hydroxylapatite. Nucleic Acids Res 6(2):689–696

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Eickbush TH, Moudrianakis EN (1978) The histone core complex: an octamer assembled by two sets of protein-protein interactions. Biochemistry 17(23):4955–4964

    Article  CAS  PubMed  Google Scholar 

  19. Thastrom A, Lowary PT, Widlund HR, Cao H, Kubista M, Widom J (1999) Sequence motifs and free energies of selected natural and non-natural nucleosome positioning DNA sequences. J Mol Biol 288(2):213–229. doi:10.1006/jmbi.1999.2686

    Article  CAS  PubMed  Google Scholar 

  20. Yuan W, Wu T, Fu H, Dai C, Wu H, Liu N, Li X, Xu M, Zhang Z, Niu T, Han Z, Chai J, Zhou XJ, Gao S, Zhu B (2012) Dense chromatin activates polycomb repressive complex 2 to regulate H3 lysine 27 methylation. Science 337(6097):971–975. doi:10.1126/science.1225237

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We want to thank Jean-François Couture (University of Ottawa) for his advice on some of the assays described and Chantal LeBlanc for her helpful comments on this chapter. Work in the Voigt lab is supported by the Wellcome Trust and the Royal Society (joint Grant Ref. 104175/Z/14/Z, Sir Henry Dale Fellowship to PV) and has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-STG grant agreement No. 639253). The Wellcome Trust Centre for Cell Biology is supported by core funding from the Wellcome Trust (Grant Ref. 092076).

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Author notes

  1. Yannick Jacob and Philipp Voigt contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, Yale University, 165 Prospect St., New Haven, CT, 06511-2106, USA

    Yannick Jacob

  2. Wellcome Trust Centre for Cell Biology, The University of Edinburgh, Michael Swann Building, Max Born Crescent, Edinburgh, EH9 3BF, UK

    Philipp Voigt

Authors
  1. Yannick Jacob
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  2. Philipp Voigt
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Correspondence to Philipp Voigt .

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Editors and Affiliations

  1. Department of Molecular Biology, Wageningen University & Research, Wageningen, The Netherlands

    Marian Bemer

  2. Department of Plant and Microbial Biology, Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland

    Célia Baroux

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Jacob, Y., Voigt, P. (2018). In Vitro Assays to Measure Histone Methyltransferase Activity Using Different Chromatin Substrates. In: Bemer, M., Baroux, C. (eds) Plant Chromatin Dynamics. Methods in Molecular Biology, vol 1675. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7318-7_20

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  • DOI: https://doi.org/10.1007/978-1-4939-7318-7_20

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7317-0

  • Online ISBN: 978-1-4939-7318-7

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