This proposal describes the characterization of HSF1, a transcription factor that regulates the heat shock response in humans. HSF1 binds to a specific sequence upstream of heat shock gene promoters and activates transcription. This proposal describes experiments designed to characterize the domains of HSF1 that are responsible for activating transcription, the mechanisms by which these domains function, and the mechanisms that cause these domains to be heat responsive. These experiments are based on the use of GAL4-HSF1 fusion proteins, which have been shown to activate transcription in human cells. The activation domains will be mutated and analyzed for transcriptional activity in vivo (transient and stable transfection) as well as for stimulation of transcriptional elongation in vitro. Functional contacts between the HSF1 activation domains and specific components of the cellular transcriptional machinery will be characterized. Interactions between the activation domains and the regulatory domain of HSF1 will be studied. Post-translational modifications of HSF1, such as phosphorylation, will be investigated. Other cellular factors that interact with the regulatory domain will be sought. These studies may generate new information concerning how HSF1 is activated by stress in humans and how it then activates a set of human genes.
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