Project description:H3 ChIP and input DNA were hybridized to Affymetrix GeneChip S. cerevisiae Tiling 1.0R Array Genome-wide mapping of nucleosomes generated by micrococcal nuclease (MNase) suggests that yeast promoter and terminator regions are very depleted of nucleosomes, predominantly because their DNA sequences intrinsically disfavor nucleosome formation. However, MNase has strong DNA sequence specificity that favors cleavage at promoters and terminators and accounts for some of the correlation between occupancy patterns of nucleosomes assembled in vivo and in vitro. Using an improved method for measuring nucleosome occupancy in vivo that does not involve MNase, we confirm that promoter regions are strongly depleted of nucleosomes, but find that terminator regions are much less depleted than expected. Unlike at promoter regions, nucleosome occupancy at terminators is strongly correlated with the orientation of and distance to adjacent genes. In addition, nucleosome occupancy at terminators is strongly affected by growth conditions, indicating that it is not primarily determined by intrinsic histone-DNA interactions. Rapid removal of RNA polymerase II (Pol II) causes increased nucleosome occupancy at terminators, strongly suggesting a transcription-based mechanism of nucleosome depletion. However, the distinct behavior of terminator regions and their corresponding coding regions suggests that nucleosome depletion at terminators is not simply associated with passage of Pol II, but rather involves a distinct mechanism linked to 3’ end formation.
Project description:We have used a high-resolution tiled microarray, with single-nucleosome resolution, to investigate the in vivo occurrence of combinations of 12 histone modifications on thousands of nucleosomes in actively growing S. cerevisiae. We found that histone modifications do not occur independently; there are roughly two groups of co-occurring modifications. We find no evidence for a deterministic code of many discrete states, but instead see blended, continuous patterns that distinguish nucleosomes at one location (promoter nucleosomes, for example) from those at another location (over the 3’ ends of coding regions, for example). These results are consistent with the idea of a simple, redundant histone code, in which multiple modifications share the same role. Keywords: ChIP-chip
Project description:We report the genome-wide localization of Sgo1p in mitosis of Saccharomyces cerevisiae using ChIP-seq. The high resolution mapping clearly shows a tripartite domain of Sgo1p in each mitotic chromosome. This domain requires the wildtype tension sensing motif (TSM) of histone H3.
Project description:We report change in the nucleosome occupancy and accessibility upon deletion of ATP-dependent chromatin remodellers (ISW1, ISW2 & CHD1) in Saccharomyces cerevisiae.
Project description:Mapping of nucleosomes, the basic DNA packaging unit in eukaryotes, is fundamental for understanding genome regulation as nucleosomes modulate DNA access by their positioning along the genome. A cell population nucleosome map requires two observables: nucleosome positions along the DNA (“Where?”) and nucleosome occupancies across the population (“In how many cells?”). All available genome-wide nucleosome mapping techniques are yield methods as they score either nucleosomal (e.g., MNase-seq, chemical cleavage-seq) or non-nucleosomal (e.g., ATAC-seq) DNA but lose track of the total DNA population for each genomic region. Therefore, they only provide nucleosome positions and maybe compare relative occupancies between positions but cannot measure absolute nucleosome occupancy, which is the fraction of all DNA molecules occupied at a given position and time by a nucleosome. Here, we established two orthogonal and thereby crossvalidating approaches to measure absolute nucleosome occupancy across the Saccharomyces cerevisiae genome via restriction enzymes and DNA methyltransferases. The resulting high-resolution (9 bp) map shows uniform absolute occupancies. Most nucleosome positions are occupied in most cells: 97% of all nucleosomes called by chemical cleavage-seq have a mean absolute occupancy of 90 ± 6% (± SD). Depending on nucleosome position calling procedures, there are 57-60,000 nucleosomes per yeast cell. The few low absolute occupancy nucleosomes do not correlate with highly transcribed gene bodies, but with increased presence of the nucleosome-evicting RSC chromatin remodeling complex there and are enriched upstream of highly transcribed or regulated genes. Our work provides a quantitative method and reference frame in absolute terms for future chromatin studies.
Project description:Mapping of nucleosomes, the basic DNA packaging unit in eukaryotes, is fundamental for understanding genome regulation as nucleosomes modulate DNA access by their positioning along the genome. A cell population nucleosome map requires two observables: nucleosome positions along the DNA (“Where?”) and nucleosome occupancies across the population (“In how many cells?”). All available genome-wide nucleosome mapping techniques are yield methods as they score either nucleosomal (e.g., MNase-seq, chemical cleavage-seq) or non-nucleosomal (e.g., ATAC-seq) DNA but lose track of the total DNA population for each genomic region. Therefore, they only provide nucleosome positions and maybe compare relative occupancies between positions but cannot measure absolute nucleosome occupancy, which is the fraction of all DNA molecules occupied at a given position and time by a nucleosome. Here, we established two orthogonal and thereby crossvalidating approaches to measure absolute nucleosome occupancy across the Saccharomyces cerevisiae genome via restriction enzymes and DNA methyltransferases. The resulting high-resolution (9 bp) map shows uniform absolute occupancies. Most nucleosome positions are occupied in most cells: 97% of all nucleosomes called by chemical cleavage-seq have a mean absolute occupancy of 90 ± 6% (± SD). Depending on nucleosome position calling procedures, there are 57-60,000 nucleosomes per yeast cell. The few low absolute occupancy nucleosomes do not correlate with highly transcribed gene bodies, but with increased presence of the nucleosome-evicting RSC chromatin remodeling complex there and are enriched upstream of highly transcribed or regulated genes. Our work provides a quantitative method and reference frame in absolute terms for future chromatin studies.