Project description:ChIP-seq of Abf1, TATA-binding protein, and RNA Polymerase II in wild type and abf1 temperature sensitive mutant yeast (Saccharomyces cerevisiae)
Project description:Abf1 and Rap1 are General Regulatory Factors that contribute to transcriptional activation of a large number of genes, as well as to replication, silencing, and telomere structure in yeast. In spite of their widespread roles in transcription, the scope of their functional targets genome-wide has not been previously determined. We have used microarrays to examine the contribution of these essential GRFs to transcription genome-wide, by using ts mutants that dissociate from their binding sites at 37 C. We combined this data with published ChIP-chip studies and motif analysis to identify probable direct targets for Abf1 and Rap1. We also identified a substantial number of genes likely to bind Rap1 or Abf1, but not affected by loss of GRF binding. Interestingly, the results strongly suggest that Rap1 can contribute to gene activation from farther upstream than can Abf1. Also, consistent with previous work, more genes that bind Abf1 are unaffected by loss of binding than those that bind Rap1. Finally, we showed for several such genes that the Abf1 C-terminal region, which contains the putative activation domain, is not needed to confer this peculiar "memory effect" that allows continued transcription after loss of Abf1 binding. Keywords: Genetic modification (ts mutant versus wild type)
Project description:We grew wild type and corresponding mutant abf1 and rap1 temperature sensitive yeast at 25 C, then for one hour after shifting to 37 C. We then prepared nucleosomal (mostly mononucleosomal) DNA and hybridized to tiling arrays. Three abf1 ts mutant and corresponding wild type, and three rap1 ts mutant and corresponding wild type (note not the same wild type; BY4741 for Rap1, but TMY86 for Abf1)
Project description:We grew wild type and corresponding mutant abf1 and rap1 temperature sensitive yeast at 25 C, then for one hour after shifting to 37 C. We then prepared nucleosomal (mostly mononucleosomal) DNA and hybridized to tiling arrays.
Project description:Abf1 has been defined as a general regulatory factor involved in determining chromatin structure in the yeast Saccharomyces cerevisiae. As such, it plays a major role in a range of functions including DNA replication, transcriptional activation and gene silencing, as well as DNA repair. Previous studies have identified an Abf1 DNA consensus binding sequence and Abf1 binding at up to several hundred locations throughout the genome. Depletion of Abf1 in the cell, however, results in altered nucleosome structure at many thousands of sites throughout the yeast genome, suggesting a far greater role for Abf1 in chromatin structure and dynamics. Here, we examine genome-wide Abf1 binding using ChIP-Chip to measure its chromatin occupancy. Using a novel software package, Sandcastle, we detect 3,821 genomic positions at which Abf1 binds. We conducted a detailed bioinformatic analysis of these novel Abf1 binding sites, defining variations in the current consensus sequence and identifying many more genomic locations at which Abf1 can be found. These observations define the sites at which Abf1 occupancy determines chromatin structure, providing a framework for understanding how processes such as replication, transcription and DNA repair are organised within the genome.
Project description:Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic reaction which includes an excess production of extracellular matrix and interacts with integrin adhesion receptors. Integrin adhesion complexes formed by HPDE cells (H6c7), with and without expression of mutant KRas G12V, were isolated and analysed by LC-MSMS
Project description:Abf1 and Rap1 are General Regulatory Factors that contribute to transcriptional activation of a large number of genes, as well as to replication, silencing, and telomere structure in yeast. In spite of their widespread roles in transcription, the scope of their functional targets genome-wide has not been previously determined. We have used microarrays to examine the contribution of these essential GRFs to transcription genome-wide, by using ts mutants that dissociate from their binding sites at 37 C. We combined this data with published ChIP-chip studies and motif analysis to identify probable direct targets for Abf1 and Rap1. We also identified a substantial number of genes likely to bind Rap1 or Abf1, but not affected by loss of GRF binding. Interestingly, the results strongly suggest that Rap1 can contribute to gene activation from farther upstream than can Abf1. Also, consistent with previous work, more genes that bind Abf1 are unaffected by loss of binding than those that bind Rap1. Finally, we showed for several such genes that the Abf1 C-terminal region, which contains the putative activation domain, is not needed to confer this peculiar "memory effect" that allows continued transcription after loss of Abf1 binding. Experiment Overall Design: Three replicates each of rap1 ts and abf1 ts yeast were grown at 25 C and shifted to 37 C, along with matched wild type controls, prior to RNA isolation. The ts mutants lose DNA binding at 37 C.
Project description:The abf1-1 mutant and wild type cells were grown at 30 degrees C in YPAD until OD600 reached 1.0-1.2. The same volume of 42 degrees C preheated YPAD was added for each flask. After 45 min incubation at 36 degrees C, cells were harvested and RNA was isolated. Four repeats of microarray results are in this series. Keywords: repeat sample