Thyroid-stimulating hormone (TSH) is one of a family of pituitary glycoprotein hormones that include luteinizing hormone (LH), follicle-stimulating hormone (FSH), as well as placental chorionic gonadotropin (CG). These hormones regulate the metabolic processes required for growth, reproduction and development. The hormones consist of two linked, dissimilar subunits, alpha and beta. In a given species, the structures of the alpha subunits are identical. Beta subunits differ greatly in structure and confer biologic specificities to the hormone. The goals are to understand gene structure, organization and regulation, and the cellular mechanisms used in the expression of the alpha and beta subunit genes; particularly how the expression of the genes are coordinately regulated and :pressed in a tissue-specific manner. Cyclic AMP markedly stimulates the transcription the CG subunit genes in a placental (JEG) cell line. Mutational deletion/expression and CAT expression competition assays have defined several cis-elements in the alpha gene, one of which is a potent cAMP-response element as well as a cell-preferential enhancer. The sequence consists of two direct 18 bp tandem repeated sequences, and each repeat sequence contains a dyad symmetrical octamer. These two cAMp-response elements act synergistically, both with each other and a downstream promoter element, in mediating both basal and cAMP-stimulated transcription. The hypothesis to be tested is that the cAMP- regulated and cell-specific expression of the hCG-alpha gene involves complex interactions of several different DNA binding proteins, both with each other and with at least three DNA sequence elements, all within 180 bps upstream from the transcriptional CAP-site. The studies proposed will employ mutational analyses to identify the precise regulatory sequences including both tissue-specific enhancer and cAMP-response elements well as promoter and phorbol ester-mediated response elements. The trans-acting DNA binding proteins that cooperatively activate expression of these genes will be identified and characterized. Efforts will be made to isolate the 5' flanking region of the CG#- 5 gene containing the cAMP-response elements, enhancer and other regulatory regions and to begin analyses of the cis elements and interactive DNA binding proteins. A major long-term goal of these studies is to utilize this understanding of the molecular and cellular biology of glycoprotein hormone gene expression to provide an understanding of human disease.
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