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Chromatin Remodeling

Volume 833 of the series Methods in Molecular Biology pp 15-27

Date:

Measuring Dynamic Changes in Histone Modifications and Nucleosome Density during Activated Transcription in Budding Yeast

  • Chhabi K. GovindAffiliated withDepartment of Biological Sciences, Oakland University Email author 
  • , Daniel GinsburgAffiliated withDepartment of Biomedical Sciences, Long Island University
  • , Alan G. HinnebuschAffiliated withLaboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health

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Abstract

Chromatin immunoprecipitation is widely utilized to determine the in vivo binding of factors that regulate transcription. This procedure entails formaldehyde-mediated cross-linking of proteins and isolation of soluble chromatin followed by shearing. The fragmented chromatin is subjected to immunoprecipitation using antibodies against the protein of interest and the associated DNA is identified using quantitative PCR. Since histones are posttranslationally modified during transcription, this technique can be effectively used to determine the changes in histone modifications that occur during transcription. In this paper, we describe a detailed methodology to determine changes in histone modifications in budding yeast that takes into account reductions in nucleosome.

Key words

Activated transcription RNA polymerase II Histone modifications Acetylation Gcn4 Gal4 Saccharomyces cerevisiae Histone acetyltransferase Histone deacetylase complexes