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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

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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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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Materials

Basic Protocol 1: Preparation of Histones for Alkylation

Materials

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 H₂ O
Liquid N₂

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

Materials

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; http://www.karlindustries.com/)
(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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Figures

Figure 21.18.1 Methyl lysine analogs are made by alkylating unique cysteine residues in full‐length histones. By using different alkylating reagents, analogs can be made of (A ) trimethyl lysine, (B ) dimethyl lysine, (C ) monomethyl lysine, or (D ) unmethylated lysine.

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Literature Cited

Literature Cited
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