Genome‐Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High‐Resolution Histone ChIP, and NCAM

Abstract

Because histones bind DNA very tightly, the location and occupancy of nucleosomes can profoundly affect accessibility of non-histone proteins to chromatin, affecting virtually all DNA-dependent processes, such as transcription, DNA repair, DNA replication and recombination. Therefore, it is often necessary to determine positions and occupancy of nucleosomes to understand how DNA-dependent processes are regulated. Recent technological advancement made such analyses feasible in a genome-wide scale at a high resolution. In addition, we have recently developed a method to measure nuclease accessibility of nucleosomes in a global scale. Here we describe methods to map nucleosome positions, to determine nucleosome density, and to determine nuclease accessibility of nucleosomes using deep sequencing.

Nucleosome occupancy as a novel chromatin parameter for replication origin functions

“…Nucleosome occupancy is often measured by the height of nucleosome signals after micrococcal nuclease (MNase) digestion of chromatin followed by deep sequencing (MNase-seq). However, the levels of nucleosome signals in MNase-seq are strongly affected by the MNase sensitivity of the nucleosome (Weiner et al 2010;Rodriguez and Tsukiyama 2013;Rodriguez et al 2014). This bias is particularly prominent for nucleosomes around nucleosome depleted regions (NDRs), which tend to be sensitive to MNase digestion.…”

“…Cells were harvested and chromatin was prepared as previously described (Rodriguez et al 2014). Thirty microliters of Protein G beads (Invitrogen) was conjugated to 5 µL of H3 C-term rabbit polyclonal antibody (Active Motif).…”

“…Thirty microliters of Protein G beads (Invitrogen) was conjugated to 5 µL of H3 C-term rabbit polyclonal antibody (Active Motif). Bead conjugation and the ChIP were performed as previously described (Rodriguez et al 2014).…”

Chromatin Remodeling Factors Isw2 and Ino80 Regulate Checkpoint Activity and Chromatin Structure in S Phase

“…Sample harvest, preparation, and analysis were performed as described previously (Rodriguez and Tsukiyama 2013;Rodriguez et al 2014). Z-score normalization was performed as described previously (Rodriguez and Tsukiyama 2013) but based on digestion patterns at the +2 to +6 nucleosomes within ORFs genome-wide rather All strains are congenic to W1588-4c with a correction for the weak rad5 allele.…”

“…We have recently developed a method to measure chromatin accessibility to micrococcal nuclease (MNase) in a way normalized for nucleosome occupancy called normalized chromatin accessibility to MNase (NCAM) (Rodriguez and Tsukiyama 2013;Rodriguez et al 2014). To measure NCAM, we digest chromatin by MNase and purify monoucleosomal DNA, followed by hybridization of nucleosomal DNA to high-density tiled microarrays.…”