This renewal application by Dr. Gross is designed to address how transcriptional activators stimulate promoter activity in vivo. In the yeast Saccharomyces cerevisiae, heat shock factor (HSF) activates transcription of at least two heat shock genes--HSP82 and HSC82-- by alleviating nucleosomal repression of their upstream regulatory regions. Unknown is how this protein--or any protein-- mediates local disruption of unfolding of promoter chromatin structure, the essential initial step of transcriptional activation. Dr. Gross proposes to identify the activation domains of yeast HSF (yHSF) that direct chromatin remodeling in vivo. First, using a genetic suppression assay, the investigator wishes to select nucleosomes. Sequencing of such dominant HSF1 suppressors should permit identification of the native remodeling domain; a complementary HSF1 domain deletion analysis will be conducted to confirm this assignment. Second, Dr. Gross proposes to characterize structural and functional phenotypes associated with the HSF1 mutants, and ascertain their role in both establishing and maintaining the nucleosome-free phenotype at a target heat shock promoter. Third, to address the mechanism by which the remodeling domain works, the applicant will biochemically reconstitute the HSP82 promoter into a stable dinucleosomal complex using purified components. Reconstitution will be attempted using both HeLa and S. cerevisiae core histone, with the goal being a full homologous system. Dr. Gross will then ask whether recombinant yHSF can bind and disrupt this reconstituted complex, whether this activity is mediate (or enhanced) by the presence of SWI/SNF complex and ATP, whether it is attenuated by the presence of hsp70 or mutant histone, and whether gain-of-function yHSF mutant polypeptides are more effective in in vitro remodeling that wild-type yHSF. Finally, the applicant will the ability of the putative yHSF remodeling domain to interact with SWI/SNF subunits, as well as individual core histone and components of the general transcription complex, by performing GST-pull down experiments. The health relatedness of this project derives from the fact that a large number of human diseases, including many cancers, correlate with missense mutations in promoter-specific transcription factors. Moreover, the SWI/SNF complex is known to associate with proteins that either enhance or suppress the formation of human tumors.
