Abstract
The paradigm of activation via ordered recruitment has evolved into a complicated picture as the influence of coactivators and chromatin structures on gene regulation becomes understood. We present here a comprehensive study of many elements of activation of ADH2 and FBP1, two glucose-regulated genes. We identify SWI/SNF as the major chromatin-remodeling complex at these genes, whereas SAGA (Spt-Ada-Gcn5-acetyltransferase complex) is required for stable recruitment of other coactivators. Mediator plays a crucial role in expression of both genes but does not affect chromatin remodeling. We found that Adr1 bound unaided by coactivators to ADH2, but Cat8 binding depended on coactivators at FBP1. Taken together, our results suggest that commonly regulated genes share many aspects of activation, but that gene-specific regulators or elements of promoter architecture may account for small differences in the mechanism of activation. Finally, we found that activator overexpression can compensate for the loss of SWI/SNF but not for the loss of SAGA.
Highlights
One of the reasons for this evolving picture of activation is the finding that many coactivator complexes can play multiple roles
Coactivators Are Required for Stable Cat8 Binding—We previously established that SAGA, SWI/SNF, and Mediator occupy the promoters of ADH2 and FBP1 and contribute to their activation [12]
chromatin immunoprecipitation (ChIP) assays for Adr1 and Cat8 in coactivator deletion strains showed that Adr1 binding at ADH2 was unaffected by these mutations, but Cat8 binding at FBP1 was reduced (Fig. 1, A and B)
Summary
Yeast Strains and Growth of Cultures—All strains used in this study were derived from W303 and are described in supplemental Table S1. Strains overexpressing Adr were created by introducing a plasmid (pNKA1-U) based on pKD17, which expresses Adr from the ADH1 promoter, with only a minor modification from its original form [19], in the various coactivator mutant backgrounds. These strains were grown in synthetic media lacking the appropriate amino acid for plasmid selection with either 5% glucose (repressed) or 0.05% glucose (derepressed). MRNA Isolation and QPCR—mRNA was isolated from strains grown in either repressing (YPD (yeast extract/peptone/ dextrose) with 5% glucose) or derepressing (YPD with 0.05% glucose) media for the time indicated and processed as detailed in a previous study [12]. The Southern blots were probed using either a 32P-labeled probe as in Tachibana [16] or using the AP direct labeling and detection system from GE, following the manufacturer’s instructions
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