The regulation of promoter activity by nuclear receptors requires the assembly of large multi-subunit complexes that either directly impact the basal transcriptional machinery or modify core histones to affect chromatin structure and remodeling. The mechanisms responsible for maintaining the highly ordered dynamics of protein binding to hormonally responsive promoters have not been definitively established. We have recently developed a novel in situ fluorescence recovery after photobleaching (FRAP) assay that led to the identification of molecular chaperones and their associated co-chaperones as nuclear mobility factors for the glucocorticoid receptor (GR). This assay will be exploited to provide additional mechanistic insights into the role of chaperones in nuclear dynamics of GR. The major hypothesis to be tested in this proposal is that molecular chaperones play a major role in steroid receptor dynamics at target sites within the nucleus via their regulation of receptor nuclear mobility, chromatin exchange and hormone exchange.
Specific Aim 1 seeks to identify the role of individual chaperones and the pathway of chaperone complex assembly that mediates their activity as steroid receptor nuclear mobility factors. Chaperone protein assembly will be manipulated in the context of a novel in situ nuclear mobility assay and effects on steroid receptor/chaperone complex formation assessed by co-immunoprecipitation assays.
Specific Aim 2 will determine whether molecular chaperones participate in the dynamic exchange of GR and other factors at receptor target sites within the nucleus. The in situ FRAP assay will be used to determine whether specific chaperone complexes are required for the dynamic exchange of GR and various receptor cofactors from a functional target gene. Chromatin immunoprecipitation assays will also be used with permeabilized cells to examine chaperone effects on GR and cofactor recruitment and cycling on the chromatin of target genes. Finally, Specific Aim 3 will determine whether molecular chaperones are required for the exchange of hormone from chromatin- bound GR. The effects of molecular chaperones on hormone exchange on nuclear GRs will be assessed in permeabilized cells using assays that distinguish hormone release from hormone exchange. Chaperone effects on hormone exchange at a specific target site will be revealed using a fluorescent GR ligand. Public Health Relevance: Hsp90 inhibitors such as geldanamycin (GA) are being evaluated for breast cancer therapy due primarily to the their selective action in cancer cells on hsp90 client proteins such as estrogen receptor, p53 and various kinases. However, the development of therapeutic anti-cancer drugs directed against hspQO or other chaperones has not taken into account the newly discovered roles for these proteins in chromatin dynamics or histone modification, which is the subject of this proposal. ? ? ?
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