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Mot1 regulation of promoter binding by TBP varies with gene expression levels and stress independently of coactivator-dependence [ChIP-Seq]

ABSTRACT: Mot1 is an essential Snf2-family ATPase in budding yeast that regulates transcription by dissociating the general initiation factor TBP (TATA-binding protein) from DNA. Previous studies showed that in optimum growth conditions Mot1 preferentially removes TBP from stress-responsive promoters that utilize the coactivator SAGA to enhance TBP binding at TFIID-dependent promoters of “housekeeping” genes. In stress conditions of amino acid starvation, by contrast, we found that Mot1 promotes high-level TBP occupancy at genes activated by transcription factor Gcn4, enriched for SAGA-dependent promoters, and at highly-transcribed subsets of constitutively expressed SAGA- and TFIID-dependent genes, while suppressing TBP occupancies at lowly transcribed genes regardless of SAGA/TFIID dependence. Importantly, the response to Mot1 depletion for genes induced by starvation or oxidative stress switched from decreased to increased TBP occupancies when transcribed at low basal levels in non-stressed cells. Notably, reduced TBP binding on Mot1 depletion impairs transcription of highly expressed TFIID genes but not highly expressed SAGA/stress-activated genes, implying that promoter activation by SAGA produces a surfeit of incomplete preinitiation complexes dependent on Mot1 for their formation.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE299870 | GEO | 2025/09/25

REPOSITORIES: GEO

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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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Mot1 regulation of promoter binding by TBP varies with gene expression levels and stress independently of coactivator-dependence [ChIP-Seq]

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