Installation of Site‐Specific Methylation into Histones Using Methyl Lysine Analogs

Matthew D. Simon1

1 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.18
DOI:  10.1002/0471142727.mb2118s90
Online Posting Date:  April, 2010
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Chromatin structure is influenced by post‐translational modifications on histones, the principal basic protein component of chromatin. In order to study one of these modifications, lysine methylation, in the context of reconstituted chromatin, this unit describes the installation of analogs of methyl lysine residues into recombinant histones. The modification site is specified by mutating the lysine of interest to cysteine. The mutant histones are expressed and purified, and the cysteine residue alkylated to produce N‐methyl aminoethylcysteine, an isosteric analog of methyl lysine. Using different alkylating reagents, it is possible to install analogs of mono‐, di‐, or trimethyl lysine. While these analogs are not identical to methyl lysine residues, they show similar biochemical properties to their natural counterparts. The ease of synthesis of methyl lysine analog (MLA) histones, especially on a large scale, makes them particularly useful reagents for studying the effects of histone lysine methylation on chromatin structure, biophysics and biochemistry. Curr. Protoc. Mol. Biol. 90:21.18.1‐21.18.10. © 2010 by John Wiley & Sons, Inc.

Keywords: methyl lysine analog; histone; methylation; methyl lysine; chromatin

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Histones for Alkylation
  • Basic Protocol 2: Alkylation Reactions to Produce Methyl Lysine Analog Histones
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of Histones for Alkylation

  • Inclusion bodies from cells expressing cysteine mutant histones (unit 21.6)
  • DMSO
  • Unfolding buffer (see recipe)
  • Sephacryl S‐200 (26/60) column (GE Healthcare)
  • SAU1000 buffer (see recipe)
  • 3 µM 2‐mercaptoethanol (2‐ME) in H 2O
  • Liquid N 2
  • 35‐ml Oak Ridge tubes
  • Spatula
  • Stir bar and magnetic stirrer
  • 50‐ml conical tubes (e.g., BD Falcon)
  • FPLC that can be run at room temperature (e.g., ÄKTA from GE Healthcare)
  • Dialysis membrane (MWCO 3500 and MWCO 6000 to 8000 have been successfully used; also see appendix 3C)
  • 250‐ml centrifuge bottles
  • 15‐ml conical tubes (e.g., BD Falcon; if needed)
  • Lyophilizer
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2), dialysis ( appendix 3C), and spectrophotometric determination of protein concentration (unit 10.1)

Basic Protocol 2: Alkylation Reactions to Produce Methyl Lysine Analog Histones

  • 10 mg histone pellet (from protocol 1)
  • Alkylation buffer (see recipe)
  • 1 M dithiothreitol (DTT; prepare immediately before use from fresh, solid DTT that has been stored at –20°C protected from air)
  • 2‐mercaptoethanol (2‐ME)
  • (2‐bromoethyl)‐trimethylammonium bromide (Aldrich, cat. no. 117196)
  • (2‐chloroethyl)‐dimethylammonium chloride (Aldrich, cat. no. D141208)
  • (2‐chloroethyl)‐methylammonium chloride (Karl Industries, cat. no. M200;
  • (2‐bromoethyl)‐ammonium bromide (Fluka, cat. no. 06670)
  • Centrifuge
  • 50°C heat block
  • PD‐10 Columns (GE Healthcare, cat. no. 17‐0851‐01)
  • Additional reagents and equipment for spectrophotometric determination of protein concentration (unit 10.1) and mass spectrometry (unit 10.21)
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Literature Cited

Literature Cited
   He, S., Bauman, D., Davis, J.S., Loyola, A., Nishioka, K., Gronlund, J.L., Reinberg, D., Meng, F., Kelleher, N., and McCafferty, D.G. 2003. Facile synthesis of site‐specifically acetylated and methylated histone proteins: Reagents for evaluation of the histone code hypothesis. Proc. Natl. Acad. Sci. U.S.A. 100:12033‐12038.
   Kang, T.J., Yuzawa, S., and Suga, H. 2008. Expression of histone H3 tails with combinatorial lysine modifications under the reprogrammed genetic code for the investigation on epigenetic markers. Chem. Biol. 15:1740‐1750.
   Kenyon, G.L. and Bruice, T.W. 1977. Novel sulfhydryl reagents. Methods Enzymol. 47:407‐430.
   Luger, K., Rechsteiner, T.J., Flaus, A.J., Waye, M.M., and Richmond, T.J. 1997. Characterization of nucleosome core particles containing histone protein made in bacteria. J. Mol. Biol. 272:301‐311.
   Luger, K., Rechsteiner, T.J., and Richmond, T.J. 1999. Preparation of nucleosome core particle from recombinant histones. Methods Enzymol. 304:3‐19.
   Martin, C. and Zhang, Y. 2005. The diverse functions of histone lysine methylation. Nat. Rev. Mol. Cell Biol. 6:838‐849.
   Shogren‐Knaak, M.A., Fry, C.J., and Peterson, C.L. 2003. A native peptide ligation strategy for deciphering nucleosomal histone modifications. J. Biol. Chem. 278:15744‐15748.
   Simon, M., Chu, F., Racki, L.R., de la Cruz, C.C., Burlingame, A.L., Panning, B., Narlikar, G., and Shokat, K. 2007. The site‐specific installation of methyl‐lysine analogs into recombinant histones. Cell 128:1003‐1012.
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