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In Vitro Histone Demethylase Assays

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Histone Methyltransferases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2529))

Abstract

Dynamic histone methylation regulates gene activation and repression. It is involved in proliferation, differentiation, lineage specification, and development. Histone demethylase assays are invaluable in studying histone demethylation substrate recognition, kinetics, regulation, and inhibition by small molecules, many of which are potential therapeutics. Here we describe general procedures to purify recombinant enzymes from different expression hosts, and to prepare a broad range of substrates, as well as to set up a variety of in vitro histone demethylase assays. These assays provide useful tools for discoveries from enzymes to drugs.

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References

  1. Luger K, Mäder AW, Richmond RK, Sargent DF, Richmond TJ (1997) Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389:251–260. https://doi.org/10.1038/38444

    Article  CAS  PubMed  Google Scholar 

  2. Suganuma T, Workman JL (2011) Signals and combinatorial functions of histone modifications. Annu Rev Biochem 80:473–499. https://doi.org/10.1146/annurev-biochem-061809-175347

    Article  CAS  PubMed  Google Scholar 

  3. Brownell JE, Zhou J, Ranalli T, Kobayashi R, Edmondson DG, Roth SY, Allis CD (1996) Tetrahymena histone acetyltransferase a: a homolog to yeast Gcn5p linking histone acetylation to gene activation. Cell 84:843–851. https://doi.org/10.1016/S0092-8674(00)81063-6

    Article  CAS  PubMed  Google Scholar 

  4. Taunton J, Hassig CA, Schreiber SL (1996) A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p. Science 272:408–411. https://doi.org/10.1126/science.272.5260.408

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Li B, Carey M, Workman JL (2007) The role of chromatin during transcription. Cell 128:707–719

    Article  CAS  Google Scholar 

  7. Margueron R, Trojer P, Reinberg D (2005) The key to development: interpreting the histone code? Curr Opin Genet Dev 15:163–176

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  9. Zhang Y, Reinberg D (2001) Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev 15:2343–2360

    Article  CAS  Google Scholar 

  10. Cheng X, Blumenthal RM (2010) Coordinated chromatin control: structural and functional linkage of DNA and histone methylation. Biochemistry 49:2999–3008

    Article  CAS  Google Scholar 

  11. Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA, Casero RA, Shi Y (2004) Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119:941–953. https://doi.org/10.1016/j.cell.2004.12.012

    Article  CAS  PubMed  Google Scholar 

  12. Tsukada YI, Fang J, Erdjument-Bromage H, Warren ME, Borchers CH, Tempst P, Zhang Y (2006) Histone demethylation by a family of JmjC domain-containing proteins. Nature 439:811–816. https://doi.org/10.1038/nature04433

    Article  CAS  PubMed  Google Scholar 

  13. Tu S, Bulloch EMM, Yang L, Ren C, Huang WC, Hsu PH, Chen CH, Liao CL, Yu HM, Lo WS, Freitas MA, Tsai MD (2007) Identification of histone demethylases in Saccharomyces cerevisiae. J Biol Chem 282:14262–14271. https://doi.org/10.1074/jbc.M609900200

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Mosammaparast N, Shi Y (2010) Reversal of histone methylation: biochemical and molecular mechanisms of histone demethylases. Annu Rev Biochem 79:155–179

    Article  CAS  Google Scholar 

  16. Højfeldt JW, Agger K, Helin K (2013) Histone lysine demethylases as targets for anticancer therapy. Nat Rev Drug Discov 12:917–930

    Article  Google Scholar 

  17. Kaniskan HÜ, Martini ML, Jin J (2018) Inhibitors of protein methyltransferases and demethylases. Chem Rev 118:989–1068

    Article  CAS  Google Scholar 

  18. Dimitrova E, Turberfield AH, Klose RJ (2015) Histone demethylases in chromatin biology and beyond. EMBO Rep 16:1620–1639. https://doi.org/10.15252/embr.201541113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Simon MD, Chu F, Racki LR, de la Cruz CC, Burlingame AL, Panning B, Narlikar GJ, Shokat KM (2007) The site-specific installation of methyl-lysine analogs into recombinant histones. Cell 128:1003–1012. https://doi.org/10.1016/j.cell.2006.12.041

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Simon MD, Shokat KM (2012) A method to site-specifically incorporate methyl-lysine analogues into recombinant proteins. In: Methods in enzymology. Academic Press Inc., Cambridge, pp 57–69

    Google Scholar 

  21. Holt M, Muir T (2015) Application of the protein semisynthesis strategy to the generation of modified chromatin. Annu Rev Biochem 84:265–290

    Article  CAS  Google Scholar 

  22. Forneris F, Binda C, Vanoni MA, Mattevi A, Battaglioli E (2005) Histone demethylation catalysed by LSD1 is a flavin-dependent oxidative process. FEBS Lett 579:2203–2207. https://doi.org/10.1016/j.febslet.2005.03.015

    Article  CAS  PubMed  Google Scholar 

  23. Vaillancourt FH, Yin J, Walsh CT (2005) SyrB2 in syringomycin E biosynthesis is a nonheme FeII α-ketoglutarate- and O2-dependent halogenase. Proc Natl Acad Sci U S A 102:10111–10116. https://doi.org/10.1073/pnas.0504412102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Gelperin DM, White MA, Wilkinson ML, Kon Y, Kung LA, Wise KJ, Lopez-Hoyo N, Jiang L, Piccirillo S, Yu H, Gerstein M, Dumont ME, Phizicky EM, Snyder M, Grayhack EJ (2005) Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev 19:2816–2826. https://doi.org/10.1101/gad.1362105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Klose RJ, Yan Q, Tothova Z, Yamane K, Erdjument-Bromage H, Tempst P, Gilliland DG, Zhang Y, Kaelin WG (2007) The retinoblastoma binding protein RBP2 is an H3K4 demethylase. Cell 128:889–900. https://doi.org/10.1016/j.cell.2007.02.013

    Article  CAS  PubMed  Google Scholar 

  26. Cao R, Zhang Y (2004) SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex. Mol Cell 15:57–67. https://doi.org/10.1016/j.molcel.2004.06.020

    Article  CAS  PubMed  Google Scholar 

  27. Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489. https://doi.org/10.1093/nar/11.5.1475

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Fang J, Wang H, Zhang Y (2003) Purification of histone methyltransferases from HeLa cells. Methods Enzymol 377:213–226. https://doi.org/10.1016/S0076-6879(03)77012-8

    Article  Google Scholar 

  29. Luger K, Rechsteiner TJ, Richmond TJ (1999) Preparation of nucleosome core particle from recombinant histones. Methods Enzymol 304:3–19. https://doi.org/10.1016/S0076-6879(99)04003-3

    Article  CAS  PubMed  Google Scholar 

  30. Dyer PN, Edayathumangalam RS, White CL, Bao Y, Chakravarthy S, Muthurajan UM, Luger K (2003) Reconstitution of nucleosome Core particles from recombinant histones and DNA. Methods Enzymol 375:23–44. https://doi.org/10.1016/S0076-6879(03)75002-2

    Article  Google Scholar 

  31. Neely KE, Hassan AH, Wallberg AE, Steger DJ, Cairns BR, Wright APH, Workman JL (1999) Activation domain-mediated targeting of the SWI/SNF complex to promoters stimulates transcription from nucleosome arrays. Mol Cell 4:649–655. https://doi.org/10.1016/S1097-2765(00)80216-6

    Article  CAS  PubMed  Google Scholar 

  32. Nishioka K, Reinberg D (2003) Methods and tips for the purification of human histone methyltransferases. Methods 31:49–58. https://doi.org/10.1016/S1046-2023(03)00087-2

    Article  CAS  PubMed  Google Scholar 

  33. Lizcano JM, Unzeta M, Tipton KF (2000) A spectrophotometric method for determining the oxidative deamination of methylamine by the amine oxidases. Anal Biochem 286:75–79. https://doi.org/10.1006/abio.2000.4782

    Article  CAS  PubMed  Google Scholar 

  34. Kleeberg U, Klinger W (1982) Sensitive formaldehyde determination with NASH’s reagent and a “tryptophan reaction”. J Pharmacol Methods 8:19–31. https://doi.org/10.1016/0160-5402(82)90004-3

    Article  CAS  PubMed  Google Scholar 

  35. Simon MD (2010) Installation of site-specific methylation into histones using methyl lysine analogs. Curr Protoc Mol Biol, Chapter 21:Unit 21.18.1-10

    Google Scholar 

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Acknowledgments

The author is very grateful to Dr. Ming-Daw Tsai and all Tsai lab members, OSU Campus Chemical Instrument Center, and Dr. Danny Reinberg lab.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA

    Shengjiang Tu

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  1. Shengjiang Tu
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Correspondence to Shengjiang Tu .

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Editors and Affiliations

  1. Institute Curie, INSERM U934/CNRS UMR 3215, Paris, France

    Raphaël Margueron

  2. Institute Curie, INSERM U934/CNRS UMR 3215, Paris, France

    Daniel Holoch

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Tu, S. (2022). In Vitro Histone Demethylase Assays. In: Margueron, R., Holoch, D. (eds) Histone Methyltransferases. Methods in Molecular Biology, vol 2529. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2481-4_3

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  • DOI: https://doi.org/10.1007/978-1-0716-2481-4_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2480-7

  • Online ISBN: 978-1-0716-2481-4

  • eBook Packages: Springer Protocols

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