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 is expressed not only in TSH cells but also in pituitary gonadotropes and placental cells. In contrast, TSHbeta gene expression is restricted only to thyrotropes. The studies proposed in this grant are a direct extension of our important previous contributions in which the murine TSHbeta gene was identified and sequenced, its promoter region characterized, the important functional cis-acting DNA elements determined, and important transcription factors binding to these regions identified. Two of these transcription factors are Pit-1 and GATA-2 which interact in a unique complex on the TSHbeta promoter. An additional factor, TRAP220, is a non-DNA binding coactivator/mediator. We have now shown that TRAP220 functionally synergizes with the interaction between Pit-1 and GATA-2 resulting in expression levels approaching that of the endogenous TSHbeta gene. Our proposed studies are designed to precisely define the molecular basis for the observed synergistic interaction of Pit-1 and GATA-2 with TRAP220. We will first map the respective domains on Pit-1 and GATA-2 that are responsible for their ability to functionally synergize, and for their interaction in the presence and absence of the proximal TSHbeta promoter by chromatin immunoprecipitations. We will next precisely define the specific amino acid residues on each protein that are involved in dynamic multiprotein interactions by nuclear magnetic resonance spectroscopy. Finally, we will establish the physiological in vivo, thyrotrope-specific, roles of GATA-2 and TRAP220 by developing unique transgenic models of conditional gene deletions utilizing a ligand-regulated cre-recombinase at the TSHbeta gene locus and determine their effect on endogenous TSHbeta gene expression. These projects will utilize the most advanced techniques of molecular and cell biology incorporated into structural studies and transgenic technology. These studies will provide new fundamental knowledge on the structural interactions between multiple classes of transcription factors and the impact of these interactions on normal physiology.
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