Nucleosome maps in S. cerevisiae at varying phosphate concentrations
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ABSTRACT: Genome wide maps of nucleosome occupancy in yeast have been produced through deep sequencing of nuclease-protected DNA. These maps have been obtained from crosslinked chromatin in vivo at varying phosphate concentrations (no phoshate and 10mM phosphate concentration). Here, we analyze these maps in combination with existing TF binding data (Harbison et al., Nature, 2004, 431(7004):99-104), and with new gene expression experiments reported here (GSE26770). We also confirm previous conclusions that the intrinsic,sequence dependent binding of nucleosomes helps determine the localization of TF binding sites. High-throughput sequencing of yeast nucleosomal DNA at varying phosphate concentration. Yeast S. cerevisiae (strain: PHO4-MYC::TRP1; CBF1-3HA::LEU2) in log phase was grown in PNB medium with no phosphate for 3 hours and then shifted to various phosphate concentration (0mM and 10 mM) for 80 minutes. After micrococcal nuclease treatment, reverse crosslink and gel purification mononucleosome were isolated. The sequencing by Illumina Solexa following manufacturer protocol has provided mononucleosomal maps in yeast genome (Oct. 2003 SGD/sacSer1 genome release) at two phosphate concentrations (starvation and normal).
Project description:Genome wide maps of nucleosome occupancy in yeast have been produced through deep sequencing of nuclease-protected DNA. These maps have been obtained from crosslinked chromatin in vivo at varying phosphate concentrations (no phoshate and 10mM phosphate concentration). Here, we analyze these maps in combination with existing TF binding data (Harbison et al., Nature, 2004, 431(7004):99-104), and with new gene expression experiments reported here (GSE26770). We also confirm previous conclusions that the intrinsic,sequence dependent binding of nucleosomes helps determine the localization of TF binding sites.
Project description:In this study, we determined the expression profiles of Pho4 and Cbf1 targeted genes in phosphate perturbation. Yeast S. cerevisiae in log phase was grown in PNB medium with no phosphate for 3 hours and then shift to various phosphate concentration (from 0 to 10 mM) for 80 minutes.
Project description:The evolutionarily conserved ATP-dependent chromatin remodeling enzyme Fun30 has recently been shown to play important roles in heterochromatin silencing and DNA repair. However, how Fun30 remodels nucleosomes is not clear. Here we report a nucleosome sliding activity of Fun30 and its role in transcriptional repression. We observed that Fun30 repressed the expression of genes involved in amino acid and carbohydrate metabolism, the stress response, and meiosis. In addition, Fun30 was localized at the 5M-bM-^@M-2 and 3M-bM-^@M-2 ends of genes and within the open reading frames of its targets. Consistent with its role in gene repression, we observed that Fun30-target genes lacked histone modifications often associated with gene activation and showed an increased level of ubiquitinated histone H2B. Furthermore, genome-wide nucleosome mapping analysis revealed that the length of the nucleosome-free region at the 5M-bM-^@M-2 end of a subset of genes was changed in Fun30-depleted cells. In addition, the positions of the -1, +2 and +3 nucleosomes at the 5M-bM-^@M-2 end of target genes were significantly shifted, while position of the +1 nucleosome remained largely unchanged in the fun30M-NM-^T mutant. Finally, we demonstrated that affinity purified single-component Fun30 exhibited nucleosome sliding activity in an ATP-dependent manner. These results define a role for Fun30 in regulation of transcription and indicate that Fun30 remodels chromatin at the 5M-bM-^@M-2 end of genes by sliding promoter proximal nucleosomes. Micrococcal nuclease digested mononucleosome DNA from wild type and fun30D cells were sequenced by illumina Genome Analyzer. Genome-wide nucleosome positioning and occupancy were analyzed.
Project description:Open chromatin provides access to a wide spectrum of DNA binding proteins for DNA metabolism processes such as transcription, repair, recombination, and replication. In this regard, open chromatin profiling has been widely used to identify the location of regulatory regions, including promoters, enhancers, insulators, silencers, replication origins, and recombination hotspots. Regulatory DNA elements are made accessible by nucleosome-depeleted states. Thus, nucleosome remodelling and modification should be intimately coupled with open chromatin formation and regulation. However, our knowledge of nucleosome regulation is largely limited to promoter regions, which comprise only a subset of all regulatory loci in the genome. In order to examine nucleosome patterns in open chromatin regions, we performed micrococcal nuclease (MNase) sequencing for a laboratory strain of yeast. Nucleosome occupancy profiled by Micrococcal nuclease (MNase) digestion
Project description:Yeast knockout strains were constructed by the Yeast Deletion Project. Three biological replicates were analyzed for each strain. 5 ml cultures were inoculated at OD600 = 0.2 from saturated overnight cultures and grown to mid-log phase (OD600 = 0.6 - 0.8) in YPD media 37 or in phosphate-depleted media 38 at 30C. Cells were harvested by centrifugation and washed with nuclease-free water (Ambion). Total RNA was isolated immediately after harvest using the Ribopure Yeast RNA Isolation Kit (Ambion). 5 mg of total RNA was used to generate labeled probes with standard Affymetrix protocols.
Project description:We describe a Hi-C based method, Micro-C, in which micrococcal nuclease is used instead of restriction enzymes to fragment chromatin, enabling nucleosome resolution chromosome folding maps. Analysis of Micro-C maps for budding yeast reveals abundant self-associating domains similar to those reported in other species, but not previously observed in yeast. These structures, far shorted than topologically-associating domains in mammals, typically encompass one to five genes in yeast. Strong boundaries between self-associating domains occur at promoters of highly transcribed genes, and regions of rapid histone turnover that are typically bound by the RSC chromatin-remodeling complex. Investigation of chromosome folding in mutants confirms roles for RSC, “gene looping” factor Ssu72, Mediator, H3K56 acetyltransferase Rtt109, and N-terminal tail of H4 in folding of the yeast genome. This approach provides detailed structural maps of a eukaryotic genome and our findings provide insights into the machinery underlying chromosome compaction. Chromatin is fragmented by Mnase, subsequenct nucleosomal end repair, and a modified two-step method for purfiying ligation products. Using Illumina paired-end sequencing maps Micro-C library and generates nucleosome resolution contact maps. The readme.txt file contains additional description of how each processed data file was generated.
Project description:We present Micrococcal Nuclease digestion maps of S. cerevisiae through the progression of the Yeast Metabolic Cycle. We demonstrate that nucleosome positions at many promoters are dynamic, and remodeling events at promoters have significant consequences with respect to gene expression. Examination of nucleosome positions and transcriptional output through metabolic oscillations in budding yeast.
Project description:In this study, the proteome-level effects of the deletion of the gene encoding Hmt1p, the predominant yeast arginine methyltransferase, were measured using SILAC (stable isotope labeling by amino acids in cell culture), to clarify if the abundance levels of any proteins were disrupted by systemic loss of arginine methylation. Analysis revealed that numerous proteins were found to be differentially abundant between wild-type yeast and Δhmt1, and functional analysis revealed perturbation of phosphate signalling and repression of the PHO pathway.
Project description:Wild type steady state chemostats cultures, in dilution rate of 0.3[1/hr] or 0.1[1/hr], and phosphate concentration in the feeding vessel of 0.3 or 0.1mM Pi Wild type cells were grown in a chemostat at a dilution rate of 0.3[1/hr] or 0.1[1/hr], and phosphate concentration in the feeding vessel of 0.3 or 0.1mM Pi. Cells were allowed to reach steady state and then harvested. Total RNA was extracted using MasterPure™ Kit (Epicentre). The samples were amplified, labeled, hybridized to yeast dual color expression microarrays and scanned, all using standard Agilent protocols, reagents, and instruments. The scanned images were analyzed using SpotReader software (Niles Scientific).
Project description:In this study we investigated the transcriptional response of the yeast Saccharomyces cerevisiae to potassium starvation. To this end yeast cells were grown for 60 min in media without potassium or in media with a standard potassium concnetration (50 mM KCl). Using Serial Analysis of Gene Expression (SAGE)-tag sequencing the effect of potassium starvation on the transcriptome was determined. 4 samples of cells grown in media without potassium and 4 samples of cells grown in the presence of potassium were analyzed.