Project description:A strain harboring two copies of RAP1 is used for a competition-ChIP experiment. One copy of RAP1 is expressed from the endogenous RAP1 promoter and a c-terminal 3X FLAG epitiope tag and the other is expressed from a weakened Galactose inducible promoter and a c-terminal 9X MYC tag. Following induction by 2% galactose Rap1-Myc and Rap1-Flag levels are determined genome wide using ChIP-chip. Time Course ChIP-ChIP experiment, Rap1-Flag IP and Rap1-Myc IP. 10 Time Points (0,10,20,30,40,50,60,90,120,150 Minutes) 2 Biological Replicates. Total Rap1 Occupancy at times 0 and 60 minutes in the time course; 2 Biological Replicates. mRNA expression levels at times 0 and 60 minutes in the time course; 2 biological Replicates.
Project description:A strain harboring two copies of RAP1 is used for a competition-ChIP experiment. One copy of RAP1 is expressed from the endogenous RAP1 promoter and a c-terminal 3X FLAG epitiope tag and the other is expressed from a weakened Galactose inducible promoter and a c-terminal 9X MYC tag. Following induction by 2% galactose Rap1-Myc and Rap1-Flag levels are determined genome wide using ChIP-chip. Time Course ChIP-ChIP experiment, Rap1-Flag IP and Rap1-Myc IP. 13 Time Points (0, 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210, 240 Minutes) 2 Biological Replicates. Total Rap1 Occupancy at times 0 and 60 minutes in the time course; 2 Biological Replicates. mRNA expression levels at times 0 and 60 minutes in the time course; 2 biological Replicates. ChIPs comparing the occupancy of Rap1 in a strain containing two copies of Rap1, one with a Flag tag and one with a Myc tag, and expressed from an identical promoter.
Project description:The relationship between chromatin structure and transposable element (TE) integration is a fundamental question as nucleosomes can either mediate or inhibit TE insertion. The Ty1 element in Saccharomyces cerevisiae preferentially inserts upstream of RNA Polymerase III-transcribed genes, in a manner coincident with nucleosome positioning. Ty1 insertion positions have largely been inferred from nucleosome occupancy maps, but this approach is imperfect. Nucleosome occupancy maps are typically generated from wild-type Saccharomyces cerevisiae cells grown in standard conditions. Many Ty1 studies rely on a galactose-inducible expression system and it is not yet known if Ty1 overexpression from the GAL1 promoter itself alters nucleosome organization under inducing conditions. Here, we used a galactose-inducible Ty1 expression system and genome-wide MNase-seq to determine whether Ty1 donor plasmid transformation and overexpression alter nucleosome occupancy. We find that changes in carbon source do not substantially alter genome-wide nucleosome organization. In contrast, galactose induction of Ty1 expression revealed time-dependent changes in nucleosome occupancy. Nucleosome organization is unchanged across pre-induction, 6-hour galactose exposure, and post-induction repressing conditions, but nucleosome occupancy shifted at 12 and 24 hours, with changes evident at both Pol II and tRNA genes. We also found that Ty1 integration events are rare at early time points, emerge by 12 hours, and accumulate substantially by 24 hours. Ty1 preferentially targets nucleosomal DNA, but prolonged induction shifts insertion positions relative to nucleosomes. We also find that Ty1 preferentially targets nucleosomal DNA that lies in close proximity to histones, with this preference showing time-dependent shifts during induction.
Project description:To assess the relative contributions of the PKA and MAPK pathways in Ras stimulation of filamentation, we examined the change in transcriptional profile following activation of Ras in strains in which one of these two pathways has been severed. These arrays contain the experiments involving WT strain with glucose addition, Ras2G19V strain with galactose induction, tpk2-w strain as a control, and RasG19V activation in the tpk2-w background. Activation of Ras in the Σ1278b background – by galactose addition to a gal1 PGAL10-RAS2G19V strain - causes a massive restructuring of the transcription pattern of the cell, which closely resembles the changes induced by addition of glucose. To assess the extent to which these Ras-induced changes depended on signaling through PKA, we compared the expression profile following induction of activated Ras in a wild type background to that following induction of activated Ras in the bcy1 tpk1 tpk3 tpk2V218G. Keywords: time course
Project description:A strain harboring two copies of RAP1 is used for a competition-ChIP experiment. One copy of RAP1 is expressed from the endogenous RAP1 promoter and a c-terminal 3X FLAG epitiope tag and the other is expressed from a weakened Galactose inducible promoter and a c-terminal 9X MYC tag. Following induction by 2% galactose Rap1-Myc and Rap1-Flag levels are determined genome wide using ChIP-chip.
Project description:A strain harboring two copies of RAP1 is used for a competition-ChIP experiment. One copy of RAP1 is expressed from the endogenous RAP1 promoter and a c-terminal 3X FLAG epitiope tag and the other is expressed from a weakened Galactose inducible promoter and a c-terminal 9X MYC tag. Following induction by 2% galactose Rap1-Myc and Rap1-Flag levels are determined genome wide using ChIP-chip.