Thyrotropin (TSH) is the pituitary glycoprotein hormone which controls the growth and function of the thyroid gland. It is a dimeric glycoprotein consisting of two nonidentical and noncovalently associated alpha and beta subunits which are encoded by separate genes on different chromosomes. The regulation of TSH is predominantly by stimulatory and inhibitory factors from the hypothalamus (thyrotropin releasing hormone, dopamine, and somatostatin) and negative feedback inhibition by circulating thyroid hormone. Our preliminary studies have shown that many of these regulators have strong effects at the level of TSH subunit gene transcription. The current proposal will extend these results by discovering molecular mechanisms which govern TSH subunit gene expression. To perform these investigations, two interrelated specific aims will be addressed. First, a knowledge of TSH subunit gene organization, sequence, and chromatin structure is necessary to implement focused studies on TSH subunit gene regulation. We have therefore cloned the murine TSH-beta and alpha-subunit genes and we will now characterize their structure concentrating on domains of functional importance. Second, we will utilize these genes and the knowledge gained about their organization and sequence to study TSH subunit gene regulation. These studies will be performed by introducing chimeric genes into cells and monitoring their expression. We will create deletion-mutation constructs to analyze control elements within the 5' flanking regions of both the TSH-beta and alpha-subunit gene. To perform these studies, we have cloned a new permanent cell line from a murine thyrotropic tumor which is species, cell, and hormone specific. With these important tools, we will address molecular and genetic requirements which impart basal TSH-beta and alpha- subunit expression including tissue specificity. Once this information is known, we will determine those genetic elements which confer regulation by thyroid hormone and dopamine. These studies will provide fundamental information on TSH regulation that is directly applicable to other pituitary hormone genes.
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