Role of yFACT in transcriptional initiation in Saccharomyces cerevisiae

The yeast Nhp6 protein is an architectural transcription factor in the budding yeast Saccharomyces cerevisiae. Genetic experiments suggest that Nhp6 can affect gene expression both positively and negatively, depending on the gene. In this dissertation, I have explored the functions of Nhp6 in relation to the yFACT chromatin reorganizing complex. The results show that the transcriptional regulation by Nhp6 in regulating binding of the TATA-binding protein (TBP) is mediated through the yFACT complex. A genetic screen conducted in yeast identified TBP mutations that are lethal in the absence of Nhp6, but viable in a NHP6+ strain. Further analysis of these TBP mutations showed a functional interaction among Spt3, TBP and Nhp6 in regulating essential functions in vivo, including formation of the complex of TBP and TFIIA with DNA. Using both genetic and biochemical assays, we have shown that Nhp6, histone acetylation by Gcn5, and chromatin remodeling by the Swi/Snf chromatin remodeling complex, have a role in TBP-TFIIA complex formation. We have also explored functional relationships between Nhp6 and Gcn5 with negative regulators of TBP binding such as Mot1 and the Ccr4/Not complex. We show that Mot1 and Ccr4/Not also have a positive role in formation of the TBP-TFIIA complex in vivo. Previous work suggested that posttranslational histone methylation at H3-K36 regulates transcription at the elongation step. Our results show that this histone modification also negatively regulates yFACT mediated TBP and RNA polymerase II binding at the promoter regions of some genes. The ATP-dependent chromatin remodeler Chd1 physically and genetically interacts with components of the yFACT complex. In vivo Chd1 has been implicated in negative regulation of transcription. We provide evidence that shows an opposite role of Chd1 in the yFACT mediated stimulation of TBP and RNA polymerase II binding at promoter regions. The role of components of the yFACT complex in regulating transcription at the promoter region is discussed.