ABSTRACT: CRAC of yeast RNA polymerase II in various thermosensitive strains at permissive and non-permissive temperature and anchor-away strains with the addition of rapamycin.
Project description:We obtained RNA polymerase II occupancy profile across the genome of S.cerevisiae in several strains, Wild type at 25 and 37 degrees C, RNA15-1 at 25 and 37 degrees C, anchor-away parental strain plus rapamycin, Reb1 anchor-away plus rapamycin, Rap1 anchor-away plus rapamycin, Nrd1-AID plus auxin, and nrd1-AID without auxin. This allowed us to compare polymerase profiles when these proteins are depleted from the nucleus.
Project description:We obtained RNA polymerase II occupancy profiles across the genome of S.cerevisiae strains: Abf1 anchor-away cells after addition of rapamycin for different time points (including no addition of rapamycin) and Rap1-AID auxin-degron cells after addition of auxin for different time points (including no addition of auxin) . This allowed us to compare polymerase occupancy profiles when these proteins are depleted from the nucleus or degraded.
Project description:Mediator subunits Med17 (Srb4) and Med15 (Gal11) were subjected to ChIP in wild type yeast, med18 (srb5), med20 (srb2), and med2 med3 med15 (triple mutant) yeast, all in kin28-anchor away yeast after 1 hr rapamycin treatment to evict Kin28 from the nucleus. This results in inactivation of Kin28, which reduces Mediator turnover at promoters and allows stronger Mediator ChIP signal at promoters. <br></br>Both Med17 and Med15 were tagged with myc and precipitated using the monoclonal antibody against c-myc. Please note that a specific control for rapamycin treatment was not performed in this experiment, as that has been well documented in the literature for this experiment, using what is called the anchor away method. The untagged sample is a control for non-specific immunoprecipitation.
Project description:Transcriptome analyses of thermosensitive exosome mutants from permissive temperature to various time exposure to restrictive temperature.
Project description:To investigate whether the action of Dbp2 on Xrn1-sensitive lncRNAs depends on a nuclear or cytoplasmic localization, we used the anchor-away approach to deplete a Dbp2-FRB-GFP fusion from the nucleus upon rapamycin treatment.
Project description:The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyper-activation of the mTOR pathway in human cancers, novel strategies to enhance TOR pathway inhibition are highly desirable. We used a yeast-based high-throughput chemical genetic screen to identify small-molecule enhancers of rapamycin (SMERs) and used whole genome expression analysis to identify their mechanisms of action. We incubated various temperature-sensitive yeast strains at either the permissive (room temperature) or non-permissive (35°C) temperature for 1 hour prior to RNA extraction and hybridization on Affymetrix microarrays. Comparison of expression profiles enabled identification of gene-signatures specific to inhibition of each gene product.
Project description:As part of a study on the function of Rap1 at HML and MAT in Saccharomyces cerevisiae, we performed an Anchor Away experiment on Rap1, inducibly depleting it from the nucleus upon addition of Rapamycin, followed by a ChIP-seq time-course. This was done in a strain that had SNPs introduced to HMLalpha to make it distinguishable from MATalpha. Samples were spiked with a constant amount (5% by OD) of a S.paradoxus strain that also expressed 2xV5-Rap1. Reads from the S.paradoxus sample served as the normalization factor. Peaks were then fit to a non-linear regression model to extract the k(off) and apparent residence time in vivo.
Project description:Comparative Dynamic Transcriptome Analysis (cDTA) enables global analysis of newly synthesized RNA as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111) and reveals defects in transcription with much higher sensitivity than conventional steady-state methods. cDTA was carried out as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111), using the S. cerevisiae heterozygous Med17/med17delta strain (Euroscarf) transfected with plasmids pRS315-SRB4 or pRS315-srb4-ts as described in Larivi̬re et al. Nature. 2012 (DOI:10.1038/nature11670), and Y40343-wildtype (Euroscarf) or Med18-FRB-KanMX6 (Euroscarf) strains. Heatshock of SRB4 and srb4-ts strains was applied for 18 or 60 minutes at 37C prior to RNA labeling as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111). To deplete the Med18 subunit from the nucleus, anchor-away experiments were performed by rapamycin treatment (1 ug/ml in 200 mL YPD) for 18 or 60 minutes at 30C prior to RNA labeling as described in Sun et al. Mol. Cell. 2013 (DOI:10.1016/j.molcel.2013.09.010). Data analysis was as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111).
