Thyrotropin (TSH) is composed of two subunits: an alpha subunit, common to pituitary TSH and pituitary and placental gonadotropins, and a unique bet subunit (TSHbeta). Thyroid hormone and its nuclear receptor (c-erbA) bind to and regulate the transcription of a variety of genes including both TSH subunit genes (TSHalpha, TSHbeta) and the rat growth hormone gene (rGH). However, the molecular mechanisms that determine whether a thyroid hormone DNA response element (TRE) negatively (e.g., TSHbeta) or positively (e.g., rGH) regulates gene expression are completely unknown. The proposed studies will determine whether the location, orientation, or a specific DNA sequence defines a TRE as inhibitory or stimulatory. DNA transfection studies in a variety of cell lines will be performed using chimeric constructs containing inhibitory and stimulatory TREs in a variety of locations and orientations. Moreover, since preliminary evidence suggests that an additional nuclear protein(s), other than the thyroid hormone receptor, is involved in the molecular mechanism of thyroid hormone inhibition of human TSHbeta expression (hTSHbeta) an additional goal of these studies will be to define further the cis-acting elements to which this protein(s) binds, and whether it is cell-specific. DNA transfection studies using deletions and mutations of the human TSHbeta TRE in parallel with DNase I footprinting studies of this TRE with nuclear extracts from TSH-secreting and non-secreting cell lines and tissues will be utilized to accomplish this goal. These studies will provide new insight into thyroid hormone action and allow a more detailed model of thyroid hormone action at any TRE to be developed. Since additional nuclear proteins in the thyrotroph may be involved in the molecular mechanism of thyroid hormone inhibition of hTSHbeta expression, the second major goal of these studies will be to define cis-acting elements responsible for thyroid hormone regulation and thyrotroph-specific expression of the hTSHbeta gene. To accomplish this goal, in vitro DNA transfection studies (utilizing chimeric hTSHbeta constructs) and DNA binding assays (DNase I footprinting) will be correlated with in vivo expression of chimeric hTSHbeta genes in transgenic mice. The long range goal of these studies is to determine the interrelationship between cell- specific expression and thyroid hormone regulation of the hTSHbeta gene in the anterior pituitary. Ultimately, a greater understanding of thyroid hormone action in man may be gained.
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