Methylation‐Sensitive Single‐Molecule Analysis of Chromatin Structure

Tina B. Miranda1, Theresa K. Kelly1, Karim Bouazoune2, Peter A. Jones1

1 Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, California, 2 Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.17
DOI:  10.1002/0471142727.mb2117s89
Online Posting Date:  January, 2010
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Abstract

Methylation‐sensitive single‐molecule analysis of chromatin structure is a high‐resolution method for studying nucleosome positioning. As described in this unit, this method allows for the analysis of the chromatin structure of unmethylated CpG islands or in vitro–remodeled nucleosomes by treatment with the CpG‐specific DNA methyltransferase SssI (M.SssI), followed by bisulfite sequencing of individual progeny DNA molecules. Unlike nuclease‐based approaches, this method allows each molecule to be viewed as an individual entity instead of an average population. Curr. Protoc. Mol. Biol. 89:21.17.1‐21.17.16. © 2010 by John Wiley & Sons, Inc.

Keywords: chromatin remodeling; nucleosome positioning; methylation footprint

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Treatment of Nuclei with M.SssI
  • Basic Protocol 2: Single Molecule Methylation‐Based Analysis of Nucleosomal DNA Accessibility Alterations Catalyzed by Chromatin‐Remodeling Proteins In Vitro
  • Basic Protocol 3: Bisulfite Conversion of Unmethylated Cytosine Residues to Thymidine
  • Alternate Protocol 1: Rapid Bisulfite Conversion
  • Basic Protocol 4: PCR and Cloning to Obtain Single‐Molecule Resolution of Promoter Architecture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Treatment of Nuclei with M.SssI

  Materials
  • 106 to 107 mammalian cells
  • PBS (phosphate‐buffered saline; appendix 22)
  • RSB Buffer (receive signaling buffer; see recipe)
  • Nonidet P‐40 (NP‐40)
  • 1× M.SssI buffer (see recipe)
  • S‐Adenosylmethionine (SAM; New England Biolabs)
  • 1 M sucrose
  • CpG methyltransferase (M.SssI; New England Biolabs)
  • Stop solution (2× lysis buffer; see recipe)
  • 200 µg/ml proteinase K
  • Dounce homogenizer
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • 37° and 55°C incubators
  • Additional reagents and equipment for trypsinizing cells ( appendix 3F) and purifying DNA by phenol/chloroform extraction and ethanol precipitation (unit 2.1)

Basic Protocol 2: Single Molecule Methylation‐Based Analysis of Nucleosomal DNA Accessibility Alterations Catalyzed by Chromatin‐Remodeling Proteins In Vitro

  Materials
  • Reconstituted nucleosomes (dialyzed against NRB; unit 21.6)
  • NRB (nucleosome remodeling buffer; see recipe)
  • BC 100 buffer (see recipe for BC buffer)
  • 20 mM MgCl 2 (in NRB buffer)
  • 200 mM ADP (in NRB buffer)
  • 32 mM S‐adenosylmethionine (SAM; New England Biolabs)
  • M.SssI (New England Biolabs)
  • Nucleosome remodeling factor (s)/chromatin‐interacting protein(s) (e.g., see Wu and Allis, ) in BC 100 buffer
  • 20 mM ATP/30 mM MgCl 2 (in NRB)
  • Competitor plasmid DNA
  • 4.5% PAA 0.5× TBE gel (optional step)
  • Orange G dye
  • 0.5 µg/ml ethidium bromide
  • TE buffer (see recipe)
  • Low‐retention tubes (ISC Bioexpress)
  • 37° and 30°C incubators
  • UV table (long wavelength; optional step)
  • Plastic wrap
  • Scalpel
  • Additional reagents and equipment for DNA extraction by ethanol precipitation (unit 2.1) and busulfite conversion ( protocol 3)

Basic Protocol 3: Bisulfite Conversion of Unmethylated Cytosine Residues to Thymidine

  Materials
  • DNA (2 to 4 µg in nuclease‐free water; see protocol 1 or 2)
  • Restriction enzymes (e.g., HindIII, BamHI, and EcoRI)
  • 3 M NaOH, prepare fresh
  • Hydroquinone (Sigma)
  • Sodium bisulfite
  • Wizard miniprep kit (Promega)
  • 5 M sodium acetate (NaOAc)
  • Ethanol
  • Glycogen
  • Heating block−80°C or −20°C freezer
  • Microcentrifuge

Alternate Protocol 1: Rapid Bisulfite Conversion

  Materials
  • Genomic DNA (e.g., from protocol 2)
  • Restriction enzymes (e.g., HindIII, BamHI and EcoRI)
  • 3 M NaOH
  • NaHSO 3 (Wako)
  • (NH 4) 2SO 3H 2O (Wako)
  • 50% (NH 4)HSO 3 (Wako)
  • Wizard miniprep kit (Promega)
  • 5 M sodium acetate (NaOAc)
  • Ethanol
  • 20 mg/ml glycogen
  • Heating block
  • Microcentrifuge

Basic Protocol 4: PCR and Cloning to Obtain Single‐Molecule Resolution of Promoter Architecture

  Materials
  • Bisulfate‐converted DNA (see protocol 3 or the protocol 4)
  • TOPO TA cloning kit (Invitrogen)
  • Miniprep kits and/or TempliPhi DNA amplification kit (GE Healthcare)
  • Additional reagents and equipment for PCR (unit 15.1)
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Figures

Videos

Literature Cited

   Appanah, R., Dickerson, D.R., Goyal, P., Groudine, M., and Lorincz, M.C. 2007. An unmethylated 3′ promoter‐proximal region is required for efficient transcription initiation. PLoS Genet. 3:e27.
   Bock, C., Reither, S., Mikeska, T., Paulsen, M., Walter, J., and Lengauer, T. 2005. BiQ Analyzer: Visualization and quality control for DNA methylation data from bisulfite sequencing. Bioinformatics 21:4067‐4068.
   Boeger, H., Griesenbeck, J., Strattan, J.S., and Kornberg, R.D. 2003. Nucleosomes unfold completely at a transcriptionally active promoter. Mol. Cell 11:1587‐1598.
   Boeger, H., Griesenbeck, J., Strattan, J.S., and Kornberg, R.D. 2004. Removal of promoter nucleosomes by disassembly rather than sliding in vivo. Mol. Cell 14:667‐673.
   Bouazoune, K., Miranda, T.B., Jones, P.A., and Kingston, R.E. 2009. Analysis of individual remodeled nucleosomes reveals decreased histone‐DNA contacts created by hSWI/SNF. Nucleic Acids Res. (In press).
   Fatemi, M., Pao, M.M., Jeong, S., Gal‐Yam, E.N., Egger, G., Weisenberger, D.J., and Jones, P.A. 2005. Footprinting of mammalian promoters: Use of a CpG DNA methyltransferase revealing nucleosome positions at a single molecule level. Nucleic Acids Res. 33:e176.
   Gal‐Yam, E.N., Jeong, S., Tanay, A., Egger, G., Lee, A.S., and Jones, P.A. 2006. Constitutive nucleosome depletion and ordered factor assembly at the GRP78 promoter revealed by single molecule footprinting. PLoS Genet. 2:e160.
   Gonzalgo, M.L. and Jones, P.A. 1997. Rapid quantitation of methylation differences at specific sites using methylation‐sensitive single nucleotide primer extension (Ms‐SNuPE). Nucleic Acids Res. 25:2529‐2531.
   Gonzalgo, M.L. and Liang, G. 2007. Methylation‐sensitive single‐nucleotide primer extension (Ms‐SNuPE) for quantitative measurement of DNA methylation. Nat. Protoc. 2:1931‐1936.
   Hinshelwood, R.A., Melki, J.R., Huschtscha, L.I., Paul, C., Song, J.Z., Stirzaker, C., Reddel, R.R., and Clark, S.J. 2009. Aberrant de novo methylation of the p16INK4A CpG island is initiated post gene silencing in association with chromatin remodeling and mimics nucleosome positioning. Hum. Mol. Genet. (In press).
   Kladde, M.P. and Simpson, R.T. 1994. Positioned nucleosomes inhibit Dam methylation in vivo. Proc. Natl. Acad. Sci. U.S.A. 91:1361‐1365.
   Kladde, M.P. and Simpson, R.T. 1996. Chromatin structure mapping in vivo using methyltransferases. Methods Enzymol. 274:214‐233.
   Kladde, M.P., Xu, M., and Simpson, R.T. 1996. Direct study of DNA‐protein interactions in repressed and active chromatin in living cells. Embo J. 15:6290‐6300.
   Lin, J.C., Jeong, S., Liang, G., Takai, D., Fatemi, M., Tsai, Y.C., Egger, G., Gal‐Yam, E.N., and Jones, P.A. 2007. Role of nucleosomal occupancy in the epigenetic silencing of the MLH1 CpG island. Cancer Cell 12:432‐444.
   Lomvardas, S. and Thanos, D. 2001. Nucleosome sliding via TBP DNA binding in vivo. Cell 106:685‐696.
   Lorch, Y., LaPointe, J.W. and Kornberg, R.D. 1987. Nucleosomes inhibit the initiation of transcription but allow chain elongation with the displacement of histones. Cell 49:203‐210.
   Lusser, A. and Kadonaga, J.T. 2003. Chromatin remodeling by ATP‐dependent molecular machines. Bioessays 25:1192‐1200.
   Rando, O.J. and Chang, H.Y. 2009. Genome‐wide views of chromatin structure. Annu. Rev. Biochem. 78:245‐271.
   Renbaum, P., Abrahamove, D., Fainsod, A., Wilson, G.G., Rottem, S., and Razin, A. 1990. Cloning, characterization, and expression in Escherichia coli of the gene coding for the CpG DNA methylase from Spiroplasma sp. strain MQ1(M.SssI). Nucleic Acids Res. 18:1145‐1152.
   Shiraishi, M. and Hayatsu, H. 2004. High‐speed conversion of cytosine to uracil in bisulfite genomic sequencing analysis of DNA methylation. DNA Res. 11:409‐415.
   Studitsky, V.M., Clark, D.J., and Felsenfeld, G. 1995. Overcoming a nucleosomal barrier to transcription. Cell 83:19‐27.
   Studitsky, V.M., Walter, W., Kireeva, M., Kashlev, M., and Felsenfeld, G. 2004. Chromatin remodeling by RNA polymerases. Trends Biochem. Sci. 29:127‐135.
   Tost, J., Dunker, J., and Gut, I.G. 2003. Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing. Biotechniques 35:152‐156.
   Tsukiyama, T., Becker, P.B. and Wu, C. 1994. ATP‐dependent nucleosome disruption at a heat‐shock promoter mediated by binding of GAGA transcription factor. Nature 367:525‐532.
   Wang, R.Y., Gehrke, C.W., and Ehrlich, M. 1980. Comparison of bisulfite modification of 5‐methyldeoxycytidine and deoxycytidine residues. Nucleic Acids Res. 8:4777‐4790.
   Workman, J.L. and Kingston, R.E. 1998. Alteration of nucleosome structure as a mechanism of transcriptional regulation. Annu. Rev. Biochem. 67:545‐579.
   Wu, C. and Allis, C.D. 2004. Chromatin and Chromatin Remodeling Enzymes, Part C (Methods in Enzymology). Academic Press, San Diego.
   Xu, Y.H., Manoharan, H.T. and Pitot, H.C. 2007. CpG PatternFinder: A Windows‐based utility program for easy and rapid identification of the CpG methylation status of DNA. Biotechniques 43:334‐342.
Internet Resources
  http://biq‐analyzer.bioinf.mpi‐sb.mpg.de/
  This program aligns bisulfite‐converted sequences and removes poorly converted and duplicate sequences.
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