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:The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which requires a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in S. cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1, CAF-1, Rtt106, Spt6, and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA.
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. Equal amount of mono-nucleosomal DNA and sized matched naked DNA prepared from formaldehyde fixed cells (Yuan et al. 2005), was labeled with Cy5 and Cy3 respectively and hybridized to the microarray. Each Hybridization was done in triplicate from three independent biological samples in each condition. Enrichment of mono-nucleosomal DNA over naked genomic DNA is a measure of nucleosome density calculated as Log2(nucDNA/nakedDNA).