Thyrotropin (TSH) is the pituitary glycoprotein hormone which controls the growth and function of the thyroid gland. It is produced solely by thyrotrope cells, one of five terminally differentiated pituitary cell types. The TSH molecule is composed of two nonidentical glycosylated subunits, alpha and TSHbeta, which are noncovalently associated. These two subunits arise independently from separate genes located on different chromosomes. The alpha-subunit gene is expressed not only in TSH cells but also pituitary gonadotropes and placental chorionic gonadotropes. In contrast, TSH-beta gene expression is restricted only to thyrotropes. The studies outlined in this grant proposal are firmly based on previous studies from this laboratory in which the gene for the murine TSH-beta subunit has been isolated and sequenced, its promoter-region characterized, the important functional cis-acting DNA elements identified, and the putative areas of protein-DNA interactions on the promoter determined. Our proposed investigations will be to first define the DNA sequences contained in the functionally most important footprint of the TSH-beta promoter which are responsible for thyrotrope-specific expression of this gene. We will then analyze the role of our newly discovered thyrotrope-specific variant isoform of the Pit-1 protein. The studies are designed to understand precisely the role of this novel factor on TSH-beta promoter activation. We will then attempt to reconstitute TSH- beta promoter activation in stably transfected cells with the unique Pit-1 isoforms. For these studies, we will utilize another unique reagent developed in this laboratory, an isogenic alpha-TSH cell line which has lost the ability to express the TSH-beta subunit gene. Finally, we will utilize our precise understanding of the cis-acting determinants of the TSH-beta promoter to identify and clone other novel DNA-binding factors in thyrotrope cells which activate the TSH-beta promoter. These projects will utilize the most modern techniques of cell biology and molecular endocrinology. Recombinant DNA technology will be coupled with novel methods analyzing gene expression in unique cell lines. These studies will provide fundamental information on pituitary cell differentiation and cell-specific gene expression which is directly applicable to human thyroid-pituitary disease.
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