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DNA Methyltransferase Probing of Chromatin Structure Within Populations and on Single Molecules

  • Carolina Pardo
  • Scott A. Hoose
  • Santhi Pondugula
  • Michael P. Kladde
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 523)

Abstract

Non-invasive methods for mapping chromatin structure are necessary for creating an accurate view of genome function and dynamics in vivo. Ectopic induction of cytosine-5 DNA methyltransferases (C5 MTases) in Saccharomyces cerevisiae is a powerful technique for probing chromatin structure with minimal disruption to yeast physiology. Accessibility of MTases to their cognate sites is impaired based on the strength and span of the protein–DNA interaction to be probed. Methylated cytosines that resist chemical deamination are detected positively by the PCR-based technique of bisulfite genomic sequencing. PCR amplicons can be sequenced directly yielding an average m5C frequency or accessibility of each target site within the population, a technique termed methyltransferase accessibility protocol (MAP). More recently, the sequencing of cloned molecules in MAP for individual templates (MAPit) enables assignment of the methylation status of each target site along a continuous DNA strand from a single cell. The unique capability to score methylation at multiple sites in single molecules permits detection of inherent structural variability in chromatin. Here, MAPit analysis of the repressed and induced PHO5 promoter of budding yeast, using a C5 MTase with dinucleotide recognition specificity, reveals considerable cell-to-cell heterogeneity in chromatin structure. Substantial variation is observed in the extent to which the MTase gains entry to each of the nucleosomes positioned at PHO5, suggesting differences in their intrinsic thermodynamic stability in vivo. MAPit should be readily adaptable to the analysis of chromatin structure and non-histone protein–DNA interactions in a variety of model systems.

Key words

Chromatin DNA methyltransferases footprinting nucleosomes single-molecule probing transcription yeast 

Notes

Acknowledgments

We are grateful to Randy Morse for the plasmid containing the estrogen-inducible activator, Steve Hanes for the plasmid containing the minimal GAL1 promoter with lexO sites, and the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida for high-throughput sequencing. This work was supported by Public Health Service grant CA095525 from the National Cancer Institute to M.P.K. and in part by a Texas Higher Education Coordinating Board ARP award to M.P.K.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Carolina Pardo
    • 1
  • Scott A. Hoose
    • 1
  • Santhi Pondugula
    • 1
  • Michael P. Kladde
    • 1
  1. 1.Department of Biochemistry and Molecular Biology and UF ShandsCancer Center Program in Cancer Genetics, Epigenetics and Tumor Virology, University of Florida College of MedicineGainesvilleUSA

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