Genome-Wide Approaches to Studying Yeast Chromatin Modifications

Abstract

The genomes of eukaryotic organisms are packaged into nuclei by wrapping DNA around proteins in a structure known as chromatin. The most basic unit of chromatin, the nucleosome, consists of approximately 146 bp of DNA wrapped around an octamer of histone proteins. The placement of nucleosomes relative to a gene can influence the regulation of the transcription of this gene. Furthermore, the N-terminal tails of histone proteins are subjected to numerous post-translational modifications that are also known to influence gene regulation. In recent years, a number of genome-scale approaches to identify modifications to chromatin have been developed. Techniques combining chromatin immunoprecipitation (ChIP) with microarrays (ChIP-chip) and second-generation sequencing (ChIP-Seq) have led to great advances in our understanding of how chromatin modifications contribute to gene regulation. Many excellent protocols related to ChIP-chip have been published recently (Lieb, J. D. (2003) Genome-wide mapping of protein-DNA interactions by chromatin immunoprecipitation and DNA microarray hybridization. Methods Mol. Biol. 224, 99–109.). For this reason, we will focus our attention here on the application of second-generation sequencing platforms to the study of chromatin modifications in yeast. As these genome-scale experiments require both wet-lab and bioinformatic components to reach their full potential, we will detail both the wet-lab protocols and bioinformatic steps necessary to fully conduct genome-scale studies of chromatin modifications.