Steroid hormones are involved in a wide array of physiological processes such as cellular differentiation during development and maintenance of homeostasis in the adult. Steroid hormones act by binding to intracellular receptors that subsequently interact with DNA regulatory elements to activate or repress the transcription of specific genes. cDNAs encoding a large family of thyroid and steroid hormone receptors have been cloned, providing new approaches for understanding steroid receptor action. The glycoprotein hormones (TSH, LH, FSH,hCG) are regulated by thyroid and steroid hormones in classic feedback fashion, thereby providing an ideal model system for examining mechanisms by which these hormones modulate gene expression. This proposal will specifically address interaction of thyroid and glucocorticoid receptors with hormone response elements in the human glycoprotein hormone alpha-gene. Preliminary studies indicate that the alpha-gene contains a negative thyroid response element that resides adjacent to the TATA box. The alpha-gene also contains positive and negative glucocorticoid response elements that overlap previously characterize enhancer elements. These findings indicate that the thyroid and glucocorticoid receptors converge with other cellular transcription factors at common or overlapping DNA target sites, providing a potentially powerful mechanism for modulating alpha-gene transcription. The functional properties of glucocorticoid and thyroid receptors (including thyroid receptor variants) will be explored using gene transfer studies in receptor deficient cell lines. Extensive mutagenesis of the alpha-promoter will be used to define the DNA sequence determinants for the thyroid and glucocorticoid response elements. The convergence of steroid receptor and other cellular signalling pathways at common promoter regions will be dissected using mutational analyses and heterologous constructions. Receptor interaction with cognate DNA response elements will be examined using in vitro binding studies to determine the specificity and affinity of receptor interactions. The DNA sequence determinants for receptor binding will be correlated with mutations that alter hormone responsiveness. In vitro transcription assays will be used to further examine interactions of receptors with partially purified or cloned transcription factors. As a result of these studies, the alpha-gene will provide a valuable model system for understanding multihormonal regulation of gene expression. The mechanims derived from these studies should be applicable to other genes that are regulated by thyroid and steroid hormones.
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