MARCC (Matrix‐Assisted Reader Chromatin Capture): An Antibody‐Free Method to Enrich and Analyze Combinatorial Nucleosome Modifications

Zhangli Su1, John M. Denu1

1 Wisconsin Institutes for Discovery, University of Wisconsin, Madison, Wisconsin
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.32
DOI:  10.1002/0471142727.mb2132s111
Online Posting Date:  July, 2015
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Abstract

Combinatorial patterns of histone modifications are key indicators of different chromatin states. Most of the current approaches rely on the usage of antibodies to analyze combinatorial histone modifications. Here we detail an antibody‐free method named MARCC (Matrix‐Assisted Reader Chromatin Capture) to enrich combinatorial histone modifications. The combinatorial patterns are enriched on native nucleosomes extracted from cultured mammalian cells and prepared by micrococcal nuclease digestion. Such enrichment is achieved by recombinant chromatin‐interacting protein modules, or so‐called reader domains, which can bind in a combinatorial modification‐dependent manner. The enriched chromatin can be quantified by immunoblotting or mass spectrometry for the co‐existence of histone modifications, while the associated DNA content can be analyzed by qPCR or next‐generation sequencing. Altogether, MARCC provides a reproducible, efficient and customizable solution to enrich and analyze combinatorial histone modifications. © 2015 by John Wiley & Sons, Inc.

Keywords: affinity enrichment; antibody‐free; chromatin; histone; PTM; reader domain; MARCC

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Matrix‐Assisted Reader Chromatin Capture (MARCC)
  • Support Protocol 1: Native Nucleosome Preparation from Cell Culture
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Matrix‐Assisted Reader Chromatin Capture (MARCC)

  Materials
  • Plasmid containing cDNA sequence of reader domain (PlasmID, DF/HCC DNA Resource Core; or custom DNA oligos from IDT DNA)
  • ING2 PHD: gcatcccctgtcgagtttgccatcgatcccaatgagcccacctactgcttgtgtaaccaagtgtcctacggggagatgat
  • aggctgtgacaatgaacagtgtcccattgaatggtttcacttttcatgtgtttcactcacctataaacccaaggggaaat
  • ggtattgcccaaagtgtaggggagacaatgagaaaaccatggacaaaagtaccgaaaagacaaaaaaggagagaagagcg
  • agg (ASPVEFAIDPNEPTYCLCNQVSYGEMIGCDNEQCPIEWFHFSCVSLTYKPKGKWYCPKCRGDNEKTMDKSTEKTKKERRAR)
  • AIRE PHD: cagaagaatgaggacgagtgtgccgtgtgtcgggacggcggggagctcatctgctgtgacggctgccctcgggccttcca
  • cctggcctgcctgtcccctccgctccgggagatccccagtgggacctggaggtgctccagctgcctgcaggcaacagtcc
  • aggaggtgcagccccgggcagaggag (QKNEDECAVCRDGGELICCDGCPRAFHLACLSPPLREIPSGTWRCSSCLQATVQEVQPRAEE)
  • ATRX ADD: GGGCTTCATGGGATTGTGAGCTGCACTGCTTGTGGACAACAGGTCAATCATTTTCAAAAAGATTCCATTTATAGACACCCTTCATTGCAAGTTCTTATTTGTAAGAATTGCTTTAAGTATTACATGAGTGATGATATTAGCCGTGACTCAGATGGAATGGATGAACAATGTAGGTGGTGTGCGGAAGGTGGAAACTTGATTTGTTGTGACTTTTGCCATAATGCTTTCTGCAAGAAATGCATTCTACGCAACCTTGGTCGAAAGGAGTTGTCCACAATAATGGATGAAAACAACCAATGGTATTGCTACATTTGTCACCCAGAGCCTTTGTTGGACTTGGTCACTGCATGTAACAGCGTATTTGAGAATTTAGAACAGTTGTTGCAGCAA (GLHGIVSCTACGQQVNHFQKDSIYRHPSLQVLICKNCFKYYMSDDISRDSDGMDEQCRWCAEGGNLICCDFCHNAFCKKCILRNLGRKELSTIMDENNQWYCYICHPEPLLDLVTACNSVFENLEQLLQQ)
  • HaloTag E. coli expression vector (pFN29K N‐term His 6HaloTag T7 Flexi Vector; Promega, cat. no. G8331 or pFC30K C‐term His 6HaloTag T7 Flexi Vector; Promega, cat. no. G8381)
  • HaloTag expression control vector (pH6HTN His 6HaloTag T7 vector; Promega, cat. no. G7971)
  • DNA polymerase (Q5 Hot Start High‐Fidelity 2× master mix; New England Biolabs, cat. no. M0494)
  • Nuclease‐free water (UltraPure DNase/RNase‐free distilled water; Life Technologies, cat. no. 10977‐015)
  • 1% agarose DNA gel
  • 50× TAE buffer
  • DNA gel stain (SYBR Safe DNA Gel Stain, Life Technologies, cat. no. S33102)
  • Gel purification kit (GeneJet Gel Extraction Kit; Thermo Scientific, cat. no. K0692)
  • Restriction enzymes (10× Flexi Enzyme Blend; Promega, cat. no. R1851) containing:
    • 5× Flexi digestion buffer
    • Flexi enzyme blend (SgfI and PmeI)
    • Carboxy Flexi enzyme blend (PmeI and EcoICRI)
  • Ligation‐independent cloning kit (In‐Fusion HD Cloning System; Clontech, cat. no. 649646) containing:
    • 5× infusion enzyme mix
  • Stella competent cells (Clontech)
  • Ice
  • 2× YT liquid medium containing 50 μg/ml kanamycin
  • LB agar plates containing 50 μg/ml kanamycin
  • GeneJet Plasmid Miniprep Kit (Thermo Scientific, cat. no. K0503)
  • BL21 (DE3) chemically competent E. coli cells (Life Technologies, cat. no. C6000‐03)
  • Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG)
  • 2.0 M Zinc sulfate solution in water (Sigma‐Aldrich, cat. no. 83265), optional
  • Nickel resin (Ni Sepharose 6 Fast Flow; GE Life Sciences, cat. no. 17‐5318)
  • Resuspension buffer (see recipe)
  • Lysozyme from chicken egg white (Sigma‐Aldrich, cat. no. L6876)
  • HBS (see recipe)
  • Wash buffer (see recipe)
  • Elution buffer (see recipe)
  • Dialysis buffer 1 (see recipe)
  • Dialysis buffer 2 (see recipe)
  • Dialysis buffer 3 (see recipe)
  • Liquid nitrogen
  • HaloLink resin (Promega, cat. no. G1914)
  • Nucleosome binding buffer (see recipe)
  • Native nucleosomes extracted from cell culture (see protocol 2Support Protocol)
  • MARCC elution buffer 1 (see recipe)
  • TEV protease (HaloTEV; Promega, cat. no. G6602)
  • MARCC elution buffer 2 (see recipe), optional
  • GeneJet PCR Purification kit (Thermo Scientific, cat. no. K0702)
  • Isopropanol
  • 200‐μl PCR tubes
  • Pipets
  • Real‐time thermal cycler (Bio‐Rad, cat. no. CFX96)
  • NanoVue Plus Spectrophotometer (GE Life Sciences)
  • 42°C water bath
  • 37°C floor shaker/incubator
  • DNA sequencing facility
  • Spectrophotometer (Shimadzu, cat. no. UV1800)
  • 4‐liter flasks
  • 50‐ml flasks
  • Floor centrifuge (Sorvall Evolution RC with rotor F8‐6 × 1000 y and F21‐8 × 50 y, Thermo Scientific)
  • Benchtop centrifuge (Eppendorf, cat. no. 5810R)
  • F806 × 1000 y and F21‐8 × 50 y rotors
  • 50‐ml Falcon tubes
  • Sonicator (Sonic Dismembrator Model 505 with ¼‐in. probe; Fisher Scientific)
  • Dialysis tubing (SnakeSkin Dialysis Tubing, 10 K MWCO; Thermo Scientific, cat. no. PI‐68100)
  • Centrifugal filters for protein concentration (Amicon 10 K Ultra‐15; Millipore, cat. no. UFC901024)
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge with cooler (Sorvall Legend Micro 21R; Thermo Scientific)
  • Tube rotators
  • Nalgene Rapid‐Flow sterile disposable bottle top filters with PES membrane (0.2 micron, 500 ml; Thermo Scientific, cat. no. 595‐3320)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5; Voytas ), determining protein concentration (unit 10.1; Simonian and Smith, ), analyzing protein purity by SDS‐PAGE (unit 10.2; Gallagher, ), and Coomassie staining (unit 10.6; Sasse and Gallagher, )

Support Protocol 1: Native Nucleosome Preparation from Cell Culture

  Materials
  • Cell pellets of 2 × 107 MCF‐7 cells
  • Buffer A (see recipe)
  • 2× lysis buffer (see recipe)
  • Ice
  • Sucrose cushion buffer (see recipe)
  • Nuclei storage buffer (see recipe)
  • 1 M calcium chloride (CaCl 2)
  • Micrococcal nuclease (MNase; New England Biolabs, cat. no. M0247S)
  • 0.5 M EDTA
  • Chromatin recover buffer (see recipe)
  • Dialysis cassette (Slide‐A‐Lyzer Dialysis cassette 10 K MWCO; Thermo Scientific, cat. nos. 66385 or 66382)
  • Dialysis buffer 3 (see recipe)
  • 15‐ml Falcon tubes
  • Tabletop centrifuge (Eppendorf, cat. no. 5810R)
  • Vortex mixers
  • 50‐ml tubes
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge with cooler (Sorvall Legend Micro 21R; Thermo Scientific)
  • Spectrophotometer (Shimadzu, cat. no. UV1800)
  • 37°C water bath
  • Tube rotators
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Figures

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Literature Cited

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Internet Resources
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