This proposal describes a characterization of the factor that regulates the heat shock response in humans. The heat shock response occurs after a variety of stresses, plays a protective role in humans following stress due to chemical (e.g., ethanol) insults, and may play a role in protecting cells after physical trauma has placed them in a stressful environment (e.g., post-ischemia). The heat shock response is remarkably conserved, occurring in all studied organisms, and therefore provides an excellent model for understanding the molecular mechanisms by which mammals transduce environmental signals to activation of specific genes. The mechanisms by which these environmental signals are transduced are likely to have similarities with the mechanisms that transduce growth and developmental signals to the cell. An understanding of the function of heat shock factor therefore has implications that extend beyond heat shock. Human heat shock factor (HSF) binds to a regulatory sequence (heat shock element, HSE) found upstream of heat-induced promoters. The HSE is both necessary and sufficient for heat shock regulation. Human HSF has been purified to homogeneity and shown to be post-translationally modified in its DNA-binding capacities following heat shock. A human cDNA encoding HSF has been isolated and antibodies specific to HSF have been produced. These reagents will be used to dissect the DNA-binding and transcriptional activation functions of HSF and to characterize heat-activation of this factor. Heat shock factor must interact with DNA that has been formed into chromatin in the intact cell. Experiments designed to characterize the interaction of HSF on nucleosome assembled templates are therefore emphasized in this proposal. In vitro systems will be used to determine what other factors are necessary to allow HSF to efficiently bind to DNA that has been formed into nucleosomes. Both nucleosome assembly protocols and other transcription protocols will be used to define the step(s) in the transcription process that are activated by HSF. Once these characteristics have been established, the domains of HSF responsible for DNA binding and transcriptional activation will be mutationally dissected. Thermodynamic studies will characterize potential cooperative binding of HSF to the HSE, and a domain of the protein responsible for any cooperative interaction will be characterized. The domain(s) responsible for heat-induction of DNA-binding will be found, and the requirement for factors other than HSF in that induction will be determined. Finally, the amino acids where HSF is phosphorylated will be identified and the role of phosphorylation in heat-activation of HSF will be determined by a mutational approach.
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