Project description:This experiment aims to map nucleosome positions and comparison of the same in WT NORMAL GROWTH vs WT-NUTRIENT STARVATION/isw1∆2∆ MUTANT/rsc4-∆4 MUTANT in Saccharomyces cerevisiae using a custom designed tiling array on Agilent plat form. The corresponding platform is submitted to GEO under Geo-ID GPL15842. 60mer probes with variable tiling density were designed for all the genes transcribed by RNA polymerase III. Each gene is tiled along with its 1kb downstream and upstream region with the exceptions of RPR1, SCR1, RDN5(1-6) and SNR52. Mononucleosomal DNA and size matched naked DNA was competitively hybridized to the array. Data was extracted and normalized log ratios were calculated using Agilent sofware. Normalized log2 ratio data was used in MLM to detection nucleosome positions.
Project description:We have investigated the genome-wide occupancy of Sas3p by ChIP-Chip, using tiled microarrays. Using this technique, it has been described that H3K14 and H3K9 acetylation is enriched at promoter regions and transcriptional start sites of active genes. Considering that Sas3p is a HAT whose main target in vitro is H3K14 we expected to detect Sas3 binding largely to promoter regions of genes. Surprisingly, we found that Sas3p is associated to the coding regions of genes, with a peak enrichment located) within the 5’ half of the ORF, and this enrichment drops substantially toward the 3’ region of the ORF. This result is very similar to that obtained for Yng1 genome-wide occupancy, also a component of the NuA3 complex, suggesting that this complex could be involved in transcriptional elongation, at least, in an initial step of the process.
Project description:A six array study using total gDNA recovered from two separate cultures of each of three different strains of Saccharomyces cerevisiae (YB-210 or CRB, Y389 or MUSH, and Y2209 or LEP) and two separate cultures of Saccharomyces cerevisiae DBY8268. Each array measures the hybridization of probes tiled across the Saccharomyces cerevisiae genome.
Project description:SPO11-promoted DNA double-strand breaks (DSBs) formation is a crucial step for meiotic recombination, and it is indispensable to detect the broken DNA ends accurately for dissecting the molecular mechanisms behind. Here, we report a novel technique, named DEtail-seq (DNA End tailing followed by sequencing), that can directly and quantitatively capture the meiotic DSB 3’ overhang hotspots at single-nucleotide resolution.
Project description:Background: Chromatin remodeling complexes facilitate the access of enzymes that mediate transcription, replication or repair of DNA by modulating nucleosome position and/or composition. Ino80 is the DNA-dependent Snf2-like ATPase subunit of a complex whose nucleosome remodeling activity requires actin-related proteins, Arp4, Arp5 and Arp8, as well as two RuvB-like DNA helicase subunits. Budding yeast mutants deficient for Ino80 function are not only hypersensitive to reagents that induce DNA double strand breaks, but also to those that impair replication fork progression. Results: To understand why ino80 mutants are sensitive to agents that perturb DNA replication, we used chromatin immunoprecipitation to map the binding sites of the Ino80 chromatin remodeling complex on four budding yeast chromosomes. We found that Ino80 and Arp5 binding sites coincide with origins of DNA replication and tRNA genes. In addition, Ino80 was bound at 67% of the promoters of genes that are sensitive to ino80 mutation. When replication forks were arrested near origins in the presence of hydroxyurea (HU), the presence of the Ino80 complex at stalled forks and at unfired origins increased dramatically. Importantly, the resumption of DNA replication after release from a HU block was impaired in the absence of Ino80 activity. Mutant cells accumulated double-strand breaks as they attempted to restart replication. Consistently, ino80-deficient cells, although proficient for checkpoint activation, delay recovery from the checkpoint response. Conclusions: The Ino80 chromatin remodeling complex is enriched at stalled replication forks where it promotes the resumption of replication upon recovery from fork arrest. Keywords: ChIP-chip