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Related Experiment Videos

Mapping chromatin structure in vivo using DNA methyltransferases.

Walter J Jessen1, Archana Dhasarathy, Scott A Hoose

  • 1Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128, USA.

Methods (San Diego, Calif.)
|March 25, 2004
PubMed
Summary

This study introduces two novel methods using DNA methyltransferases (C5 DMTases) to analyze chromatin organization and DNA-bound factors. These techniques, free enzyme probing and targeted gene methylation (TAGM), offer sensitive detection of chromatin structure and factor interactions.

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Area of Science:

  • Molecular Biology
  • Epigenetics
  • Biochemistry

Background:

  • Cytosine-5 DNA methyltransferases (C5 DMTases) are crucial for analyzing DNA methylation patterns and chromatin structure.
  • Bisulfite sequencing is a standard PCR-based method for assaying cytosine methylation in accessible DNA regions.
  • Understanding chromatin organization and factor interactions in vivo is essential for gene regulation studies.

Purpose of the Study:

  • To present two complementary methods utilizing C5 DMTases for probing chromatin organization and footprinting DNA-bound factors.
  • To detail the application of a free DNA methyltransferase as a diffusible probe for nucleosome structure and factor interactions.
  • To describe the targeted gene methylation (TAGM) approach for sensitive detection of DNA-binding factor activity.

Main Methods:

Related Experiment Videos

  • Utilizing the DNA methyltransferase CviPI (GC specificity) as a diffusible probe to map nucleosome structure.
  • Implementing targeted gene methylation (TAGM) by fusing C5 DMTases to DNA-binding factors for sensitive detection.
  • Developing methods for constructing C5 DMTase-expressing Saccharomyces cerevisiae strains and analyzing chromatin.
  • Providing protocols for genomic DNA isolation, bisulfite treatment, and bisulfite sequencing.

Main Results:

  • Demonstrated the efficacy of free CviPI enzyme probing in mapping chromatin changes and factor interactions at cis-regulatory sequences (GAL1 and PHO5).
  • Showcased the sensitivity of the TAGM method for detecting DNA-binding factor occupancy.
  • Presented experimental data and theoretical considerations supporting both DMTase probing techniques.
  • Successfully applied these methods in Saccharomyces cerevisiae models.

Conclusions:

  • The described C5 DMTase-based strategies provide powerful and sensitive tools for in vivo chromatin analysis.
  • These methods enable detailed mapping of nucleosome organization and DNA-binding factor interactions.
  • The protocols facilitate the application of C5 DMTases, including M.SssI, for diverse epigenetic studies.