Unit

UNIT 21.22 Simultaneous Single-Molecule Mapping of Protein-DNA Interactions and DNA Methylation by MAPit

  1. Carolina E. Pardo,
  2. Russell P. Darst,
  3. Nancy H. Nabilsi,
  4. Amber L. Delmas,
  5. Michael P. Kladde

Published Online: 1 JUL 2011

DOI: 10.1002/0471142727.mb2122s95

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Pardo, C. E., Darst, R. P., Nabilsi, N. H., Delmas, A. L. and Kladde, M. P. 2011. Simultaneous Single-Molecule Mapping of Protein-DNA Interactions and DNA Methylation by MAPit. Current Protocols in Molecular Biology. 95:21.22:21.22.1–21.22.18.

Author Information

  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 History

  1. Published Online: 1 JUL 2011
  2. Published Print: JUL 2011

Abstract

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