Glucocorticoid receptors, upon association with their cognate steroid ligands, bind to specific DNA sites, thereby activating receptor-dependent transcriptional enhancer elements that in turn stimulate initiation from nearby promoters. The long-term objectives of this study are to determine the molecular mechanisms by which steroid receptors regulate gene expression, to define how these mechanisms might be related to other modes of gene control, and to infer pathways by which coordinated networks of regulated genes might arise and evolve. In the present application, recombinant DNA methods will be used to construct an extensive series of mutations in discrete regions of DNA that are bound specifically by pure glucocorticoid receptor in vitro and are active in vivo as receptor-dependent enhancers. Together with intact, cleaved, and mutant receptor proteins, we will define at the nucleotide level the characteristics of specific receptor: DNA interactions, the consequences of those interactions on DNA and chromatin conformation in vivo and in vitro, and the precise relationships of the binding and template structure alterations to the mechanism of transcriptional enhancement. In addition, nonreceptor factors essential for hormonal regulation will be sought and characterized by biochemical, cell biological, and somatic cell genetic procedures. Understanding the mechanisms that modulate gene expression is central to the problems of human disease and developmental defects; steroid hormones, in particular, regulate a broad spectrum of developmental and physiological phenomena, and are widely used pharmacological agents for immunosuppression and cancer chemotherapy. (D)
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