Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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A genome-wide housekeeping role for TFIID and a highly stress-related role for SAGA in Saccharomyces cerevisiae

ABSTRACT: TFIID and SAGA are the only two known yeast complexes that modify chromatin and deliver TBP to promoters. Previous genome wide expression studies indicated that TFIID and SAGA positively regulate most but not all yeast genes. Using a relatively low noise microarray approach, we have re-examined the genome-wide dependence on TFIID and SAGA. We find that TFIID and SAGA contribute to the expression of virtually the entire genome, with TFIID being preferred at ~90% of the genes, and SAGA being preferred at ~10%. SAGA-dominated genes were found to overlap substantially with a previously described set of highly active genes that are attenuated in part by the TBP regulator NC2, and an auto-inhibitory function of TFIID. These SAGA-dominated genes also encompass most of the previously reported âTAF-independentâ genes. These results build upon and refine the generally held view that activators recruit either TFIID or SAGA to promoters which then bind and acetylate nucleosomes locally, thereby enhancing TBP delivery to the TATA box. Promoter-specific differences in the ability to alleviate auto-inhibitory activities associated with TFIID and SAGA might contribute to the preferential use one complex versus the other. Keywords = Chromatin Immunoprecipitation Keywords = genome-wide binding

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Frank Pugh

PROVIDER: E-GEOD-1061 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Despite being one of the first eukaryotic transcriptional regulatory elements identified, the sequence of a native TATA box and its significance remain elusive. Applying criteria associated with TATA boxes we queried several Saccharomyces genomes and arrived at the consensus TATA(A/T)A(A/T)(A/G). Approximately 20% of yeast genes contain a TATA box. Strikingly, TATA box-containing genes are associated with responses to stress, are highly regulated, and preferentially utilize SAGA rather than TFIID when compared to TATA-less promoters. Transcriptional regulation in yeast appears to be mechanistically bipolar, possibly reflecting a need to balance inducible stress-related responses with constitutive housekeeping functions. A strain containing amino terminal HA-tagged TBP and its parental untagged counterpart BY4741 (Resgen) were grown at 23?C in CSM to OD600 = 0.8-1.0. Cultures were shifted to 37?C for 5 minutes (to mimic heat treatments used elsewhere (Huisinga and Pugh, 2004)), then crosslinked at 23?C with 1% formaldehyde. ChIP was performed as described previously (Strahl-Bolsinger et al., 1997) with the some modification. Following cell disruption with glass beads, the chromatin was partially purified by centrifugation (Kurdistani and Grunstein, 2003). HA-TBP was immunopurified with 12CA5 antibody. After elution and reversal of the crosslinks, samples were subjected to non-specific amplification, labeling, and array hybridization as described (Bohlander et al., 1992; Chitikila et al., 2002). The amplification procedure was modified as follows. Following 15 round B amplification cycles, DNA was purified via QIAquick PCR Purification kit. Ten cycles were used in round C. Intergenic microarrays were generated by PCR amplification of entire intergenic regions of all yeast genes, then spotted onto glass slides. Spot intensities were filtered to remove any signal that was less then one standard deviation above local background. Three replicates were performed and their log2 ratios (enriched/input) were averaged. Keywords = Chromatin Immunoprecipitation Keywords = genome-wide binding

Project description:TATA-binding protein (TBP) is central to the regulation of transcription initiation. Recruitment of TBP to target genes can be positively regulated by one of two basal transcription factor complexes: SAGA or TFIID. Negative regulation of TBP promoter association can be performed by Mot1 or the NC2 complex. Recent evidence suggest that Mot1, NC2, and TBP form a DNA-dependent protein complex. Here, we compare the functions of Mot1 and NC2beta during basal and activated transcription using the anchor-away technique for conditional nuclear depletion. Genome-wide expression analysis indicates that both proteins regulate a highly similar set of genes (r2=0.8). Upregulated genes were enriched for SAGA occupancy, while downregulated genes preferred TFIID binding. Mot1p and NC2beta depletion during heat shock resulted in a failure to downregulate gene expression after initial activation, which was accompanied by increased TBP and RNA pol II promoter occupancies. Depletion of Mot1p or NC2beta displayed preferential synthetic lethality with the TBP-interaction module of SAGA. Our results suggest that Mot1 and NC2beta cooperate in vivo to regulate TBP function, and that they are involved in maintaining basal expression levels as well as in resetting gene expression after induction by stress. In this study 3 S. cerevisiae strains were grown with and without addition of rapamycin. For each experimental factor two independent colonies were inoculated in Complete Synthetic Medium (CSM) with 2% glucose. Overnight cultures were diluted to 0.3 OD600 in 50 ml medium and grown to 1 OD600. Cultures were then grown for 60 minutes in the absence or presence of rapamycin at 1ug/ml (LC laboratories). Cells were harvested by centrifugation at 4000 rpm for 3 minutes and were frozen in liquid nitrogen. Amplified cRNA samples were labeled with either cy3 or cy5 and put on microarray together with an oppositely labeled common reference sample consisting of cRNA of untreated wildtype strain.

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A genome-wide housekeeping role for TFIID and a highly stress-related role for SAGA in Saccharomyces cerevisiae

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