Transcription in human cells is regulated by numerous mechanisms. The rate at which RNA polymerase forms a productive complex at a promoter, the rate at which polymerase initiates transcription, and the efficiency with which polymerase elongates are all steps that are subject to regulation. These processes can be inhibited by nucleosomes, the basic packaging unit of DNA in the eukaryotic nucleus. Because of these inhibitory effects of nucleosomes, transcription can also be regulated by altering the nucleosomes at the promoter by covalent modification (e.g., acetylation) or by non-covalent remodeling of nucleosome structure. Non-covalent remodeling of nucleosome structure occurs in an ATP-dependent process that can be catalyzed by several different remodeling complexes. The purpose of this research is to characterize the mechanism of regulation of the human HSP70 promoter by its major activator, heat shock factor 1 (HSF1). Transcription from the HSP70 promoter is regulated at several levels, including the level of transcriptional elongation. HSF1 is capable of stimulating transcriptional elongation and is also capable of directing chromatin remodeling. The experiments proposed will investigate the molecular interactions that are required for HSF1 to stimulate transcriptional elongation and chromatin remodeling. These experiments will characterize the domains of HSF1 that are required for both of these steps, they will use in vitro systems to determine which other factors are involved in these processes, and they will determine which cellular factors directly contact HSF1. In addition, in vivo approaches will be used to buttress the findings made in vitro. Both the in vitro and in vivo approaches will address the potential role for ATP-dependent chromatin remodeling complexes in regulation of HSP70. The overall goal is to determine how HSF1 directs chromatin remodeling, how HSF1 stimulates transcriptional elongation, and how these two processes relate to each other.
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