Simultaneous Single‐Molecule Mapping of Protein‐DNA Interactions and DNA Methylation by MAPit

Carolina E. Pardo1, Russell P. Darst1, Nancy H. Nabilsi1, Amber L. Delmas1, Michael P. Kladde1

1 Department of Biochemistry and Molecular Biology and UF Shands Cancer Center Program in Cancer Genetics, Epigenetics and Tumor Virology, University of Florida College of Medicine, Gainesville, Florida
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.22
DOI:  10.1002/0471142727.mb2122s95
Online Posting Date:  July, 2011
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Sites of protein binding to DNA are inferred from footprints or spans of protection against a probing reagent. In most protocols, sites of accessibility to a probe are detected by mapping breaks in DNA strands. As discussed in this unit, such methods obscure molecular heterogeneity by averaging cuts at a given site over all DNA strands in a sample population. The DNA methyltransferase accessibility protocol for individual templates (MAPit), an alternative method described in this unit, localizes protein‐DNA interactions by probing with cytosine‐modifying DNA methyltransferases followed by bisulfite sequencing. Sequencing individual DNA products after amplification of bisulfite‐converted sequences permits assignment of the methylation status of every enzyme target site along a single DNA strand. Use of the GC‐methylating enzyme M.CviPI allows simultaneous mapping of chromatin accessibility and endogenous CpG methylation. MAPit is therefore the only footprinting method that can detect subpopulations of molecules with distinct patterns of protein binding or chromatin architecture and correlate them directly with the occurrence of endogenous methylation. Additional advantages of MAPit methylation footprinting as well as considerations for experimental design and potential sources of error are discussed. Curr. Protoc. Mol. Biol. 95:21.22.1‐21.22.18. © 2011 by John Wiley & Sons, Inc.

Keywords: chromatin; nucleosomes; DNA methylation; DNA methyltransferases; footprinting; single‐molecule analysis

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Table of Contents

  • Introduction
  • Basic Protocol 1: Probing Mammalian Nuclear Chromatin with DNMTs
  • Support Protocol 1: Verification of Methylation of DNA by M.CviPI
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Probing Mammalian Nuclear Chromatin with DNMTs

  • Trypsin‐EDTA solution (see recipe), 37°C
  • Mammalian cell lines cultured under appropriate experimental conditions in tissue culture plates or flasks
  • Cell growth medium (store up to 4 months at 4°C), 37°C
  • Phosphate buffered saline (PBS; appendix 22), ice cold
  • 0.4% (w/v) trypan blue solution (store indefinitely at room temperature)
  • 80 U/µl M.CviPI fused to maltose binding protein (MBP, New England Biolabs) or fused to glutathione‐S‐transferase (GST, Zymo Research); store in 20‐µl aliquots up to 1 year at −20°C in non‐frost‐free freezer; see recipe for dilutions)
  • DNMT dilution buffer (see recipe), ice cold
  • DNMT storage buffer (see recipe), ice cold
  • Cell resuspension buffer (see recipe), ice cold
  • Cell lysis buffer (see recipe), ice cold
  • Methylation buffer (see recipe)
  • Methylation stop buffer (see recipe), room temperature
  • 20 mg/ml proteinase K (store up to 4 months at −20°C in non‐frost‐free freezer)
  • Phenol/chloroform solution (see recipe)
  • 10.0 M ammonium acetate, pH 8.0 ( appendix 22)
  • Absolute and 70% (v/v) ethanol (see recipe; store indefinitely at room temperature)
  • 0.1× TE buffer (see recipe)
  • Refrigerated microcentrifuge
  • Hemacytometer or automated cell‐counting device
  • Light microscope
  • 1.7‐ml microcentrifuge tubes
  • 37° and 50°C water baths
  • Additional reagents and equipment for bisulfite sequencing (unit 7.9)
NOTE: Reagents should be prepared in sterile disposable labware. Use only distilled water in all steps and solutions. Nuclei isolation and methylation buffers should be freshly prepared on the day of the experiment. DTT, PMSF, and SAM should be added to solutions immediately before use to avoid oxidation or hydrolysis. M.CviPI activity is strongly dependent on fresh addition of DTT.
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Key References
  Kladde et al., 1996. See above.
  First demonstration of the utility of M.SssI for detection of nucleosome position and transcription factor binding.
  Fatemi et al., 2005. See above.
  First demonstrations of the use of C‐5 DNMTs in single‐molecule footprinting.
  Jessen et al., 2006. See above.
  First documented use of MAPit with M.CviPI, yielding simultaneous detection of chromatin accessibility and endogenous m5CG at the single‐molecule level.
  Kilgore et al., 2007. See above.
  Development of MethylViewer program for rapid analysis of MAPit datasets.
  Pardo et al., 2010. See above.
Internet Resources
  Site for download of MethylViewer program and detailed usage instructions.
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