Heat Shock and Steroid Receptor Signaling

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DESCRIPTION: (provided by applicant) The glucocorticoid receptor (GR) and heat shock factor 1 (HSF1) are transcription factors that control the response of cells to corticosteroids and cell stress, respectively. Because the inactive forms of GR and HSF1 are maintained by similar, if not identical, Hsp90-based protein-folding complexes, our laboratory has investigated the nature of cross-talk between these otherwise independent signal pathways. We have documented a dramatic and non-reciprocal regulation between GR and HSF1 in cells exposed to glucocorticoid hormone and stress. Using cells stably- transfected with GR- and HSF1 -responsive promoters, we find that heat shock and other forms of stress greatly enhance hormone-induced GR-mediated gene expression, while glucocorticoid hormones suppress the activity of HSF-1. We now have evidence that stress potentiation of GR requires intrinsic HSF1 activity, and that glucocorticoid hormone inhibition of HSF1 requires genomic action on the part of GR. Most intriguing is our latest finding that a protein-protein interaction between GR and HSF1 can occur in cells exposed to hormone and stress. As this interaction appears to occur in the nuclear compartment when the activated forms of GR and HSF1 are present, we now propose a model for mutual regulation in which a direct (or indirect) binding of GR and HSF1 at their respective promoters can cause either the heat shock potentiation of GR, or the GR repression of HSF1, or both. In Specific Aim 1 of this proposal, we will test this model in a variety of ways, most notably, through use of the chromatin immunoprecipitation assay - an assay used to detect proteins and protein complexes localized to promoters in vivo. In Specific Aim 2, we will explore additional mechanisms to explain stress potentiation of GR transactivity, including stress-induced alterations to the GR heterocomplex and to co-regulator recruitment at a GR-regulated promoter. In addition, we will further characterize the role of HSF1 activity in stress potentiation of the receptor through use of constitutively-active and dominant-negative forms of HSF1. In Specific Aim 3, we investigate GR repression of HSF1 through use of GR mutants expressing variable transactivation and transrepression properties, by mapping the presence of putative GR-binding sites on an HSF1-responsive promoter, and by assessing alterations to various properties of HSF1 activity at its promoter.