Project description:The association of the RSC remodeler complex with the chromatin depends on the prefoldin-like Bud27 and determines the nucleosome positioning and polyadenylation sites usage in Saccharomyces cerevisiae
Project description:The goal of this study is to analyse genome-wide the binding profile of Bud27 prefoldin-like to the genes or other genomic regions in order to know which genes could be transcriptionally regulated by this protein and to elucidate the contribution of Bud27 to the transcription. Average normalised ChIP-seq signal for Bud27 prefoldin-like binding in a S.cerevisiae wild-type strain.
Project description:Most yeast genes have a nucleosome-depleted region (NDR) at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. We have examined the interplay between RSC (a conserved essential SWI/SNF-type complex that determines NDR size) and the ISW1, CHD1 and ISW2 nucleosome spacing enzymes in chromatin organization and transcription, using isogenic strains lacking all combinations of these enzymes. The contributions of these remodelers to chromatin organization are largely combinatorial, distinct and non-redundant, supporting a model in which the +1 nucleosome is positioned by RSC and then used as a reference nucleosome by the spacing enzymes. Defective chromatin organization correlates with altered RNA polymerase II (Pol II) distribution. RSC-depleted cells exhibit low levels of elongating Pol II and high levels of terminating Pol II, consistent with defects in both termination and initiation, suggesting that RSC facilitates both. Cells lacking both ISW1 and CHD1 show the opposite Pol II distribution, suggesting elongation and termination defects. These cells have extremely disrupted chromatin, with high levels of close-packed di-nucleosomes near the 5’-ends of genes. We propose that ISW1 and CHD1 facilitate Pol II elongation by separating close-packed nucleosomes and by eliminating long linkers to prevent cryptic initiation.
Project description:Nucleosome arrays begin at nucleosome-free promoter regions (NFRs) and regulate gene expression. Reconstituting such organization throughout a genome with purified proteins is a critical challenge in establishing biochemical mechanisms for chromosome assembly. Here we establish a four-step hierarchical building plan for yeast genomic nucleosome organization using only purified components: genomic DNA, histones, site-specific organizing factors Abf1 and Reb1, and the chromatin remodelers RSC, ISW2, INO80, and ISW1a. First, RSC makes NFRs by translating promoter poly(dA:dT) tracts into directional nucleosome removal. Second, +1 nucleosomes are positioned by INO80 at most genes potentially involving DNA shape, or by ISW2 using gene-specific Abf1 and Reb1. Third, INO80 or ISW2 create arrays with wide spacing. Fourth, ISW1a tightens the spacing and creates properly positioned arrays. We conclude that entire genomes use a simple set of rules and proteins, without transcription, to build a common chromatin architecture. In this study, nucleosomes were assembled using Salt Gradient Dialysis (SGD) on yeast genomic DNA library. Assembled nucleosomes were either left untreated (labelled as "SGD", control), treated with whole cell extract (WCE), mutant extracts (rsc3ts WCE, isw1 isw2 chd1 WCE), purified remodelers; singly or in combinations (RSC, ISW1a, ISW1b, ISW2, INO80, CHD1, SWI/SNF), combinations of mutant extracts and chromatin remodelers or combination of General Regulatory Factors (Abf1, Reb1) and chromatin remodelers. The resulting nucleosome positions were mapped genome-wide using MNase-(anti-H3-ChIP)-Seq.
Project description:The classic view of nucleosome organization at active promoters is that two well-positioned nucleosomes flank a nucleosome-depleted region (NDR). However, this view has been recently challenged by contradictory reports as to whether a distinct set of wider (≳150 bp) NDRs instead contain unusually unstable Micrococcal Nuclease-sensitive “fragile” particles, thought to be nucleosomal because of their size. To determine the composition of fragile particles we introduce CUT&RUN.ChIP, in which targeted nuclease cleavage and release is followed by chromatin immunoprecipitation. We find that fragile particles represent the occupancy and action of the RSC nucleosome remodeler acting on dynamically unwrapped nucleosomal intermediates. We also find that general regulatory factors (GRFs) bind to partially unwrapped nucleosomal intermediates at NDRs. We propose that RSC-engagement and its action cause nucleosomes to unravel, and subsequent binding of GRFs constitute a dynamic cycle of nucleosome deposition and clearance at the subset of wide Pol II promoter NDRs.