Project description:We performed ChIP-seq analyses of Rad2, Mediator (Med17 and Med5) and RNA Polymerase II in Kin28-ts16 mutant, Med17-Q444P and Med17-Q444P/M442L mutants and in an Rpb9 deleted strain.
Project description:How is chromatin architecture established and what role does it play in activation of transcription? We show that a regulatory locus in yeast (the UASg) bears, in addition to binding sites for the activator Gal4, sites bound by the protein RSC. RSC tightly positions a nucleosome, evidently partially unwound, in a structure that facilitates Gal4 binding to its sites. The complex comprises a barrier that suffices to impose characteristic features of chromatin architecture. Removal of RSC allows ordinary nucleosomes to form more broadly over the UASg, and these nucleosomes compete with (but do not exclude) Gal4 binding to its sites. Taken with our previous work, the results show that both prior to and following induction specific DNA binding proteins are the predominant determinants of chromatin architecture at the GAL1/10 genes. RSC/nucleosome complexes are found scattered throughout the yeast genome. We surmise, also, that Gal4 works in higher eukaryotes despite whatever obstacle broadly positioned nucleosomes present. Chromatin was digested under conditions that yielded primarily mononucleosomes, and RSC-bearing fragments were recovered on IgG-beads. Fragments (of size ca. 50-200 bp) were analyzed by paired-end high throughput DNA sequencing (Illumina). This technique determines the sequences found at both ends of each fragment, thus revealing the sizes and genomic origin of these fragments.
Project description:High-resolution genome-wide mapping of the yeast transcription machinery. ChIP-chip was performed to identify the genomic binding locations for each factor. <br><br>Processed data files are also available on the FTP server for this experiment.
Project description:After transcription initiation, RNA polymerase (Pol) II escapes from the promoter and recruits elongation factors. Here we used chromatin immunoprecipitation (ChIP) to elucidate the general initiation-elongation transition of Pol II in yeast. We extended our ChIP-chip occupancy profiling to capping enzymes and the cap-binding complex (CBC). We found that the first two capping enzymes, Cet1 and Ceg1, are recruited near the TSS, whereas the third enzyme, the methyltransferase Abd1, is recruited about 110 nt downstream of the TSS, and CBC is recruited further downstream. ChIP was performed using TAP-tagged S. cerevisiae strains (see protocols).
Project description:A canonical organization of the transcription pre-initiation complex and its regulators. High-resolution genome-wide mapping of the yeast transcription machinery was performed for each factor using ChIP-chip.
Project description:We mapped the genome-wide binding of the flagellar regulators FlhD, FlhC, and FliA in FLAG-tagged derivatives of E. coli K-12 MG1655 using ChIP coupled with deep sequencing (ChIP-seq). We identify new binding sites for each factor.
Project description:CENP-A, a variant of histone H3, is incorporated into centromeric chromatin and plays a role during kinetochore establishment. In fission yeast, the localization of CENP-A is limited to a region spanning 10 to 20 kb of the core domain of the centromere. Here, we report a mutant (rpt3-1) in which this region is expanded to 40 to 70 kb. Likely due to abnormal distribution of CENP-A, this mutant exhibits chromosome instability and enhanced gene silencing. Interestingly, the rpt3+ gene encodes a subunit of the 19S proteasome, which localizes to the nuclear membrane. While Rpt3 associates with centromeric chromatin, the mutant protein has lost this localization. A loss of the cut8+ gene encoding an anchor of the proteasome to the nuclear membrane causes similar phenotypes as observed in the rpt3-1 mutant. Thus, we propose that the proteasome (or its subcomplex) associates with centromeric chromatin and regulates distribution of CENP-A. Chromosomal distributions of differentially expressed centromere protein A in wild-type and a proteasome mutant.