Measuring Dynamic Changes in Histone Modifications and Nucleosome Density during Activated Transcription in Budding Yeast
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 wordsActivated transcription RNA polymerase II Histone modifications Acetylation Gcn4 Gal4 Saccharomyces cerevisiae Histone acetyltransferase Histone deacetylase complexes