PHYSIOLOGY OF THYROID HORMONE DEPENDENT GENE EXPRESSION

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Thyroid hormone is essential for normal human growth, normal brain development and function and for constituitive thermogenesis. Most of the active thyroid hormone, 3,5, 3' triiodothyronine (T3), in intact individuals and all of this in the 2-3% of the U.S. population with hypothyroidism, is generated from thyroxine (T4) by 5' monodeiodination. This reaction is catalyzed by the selenoenzymes, Types 1 and 2 deiodinase (D1 and D2) products of the dio1 and 2 genes respectively. Inactivation of T4 and T3 especially in the brain and feto-placental unit, occurs by 5 deiodination catalyzed by another selenoenzyme, Type 3 deiodinase (D3) as well as by the bifunctional D1. Both D1 and D3 have been cloned and both are regulated positively by T3 at the mRNA level through direct mechanisms which do not require new protein synthesis. These critical enzymes are essential parts of an internal feedback loop acting to maintain T3 homeostasis. They can function independently of the hypothalamic- pituitary axis. This project will analyze the regulation of dio1 and 3 as models of genes directly and positively regulated by T3. Specific Aim I will explore the mechanism for regulation of the action of TRE2 in the human dio1 gene and determine the mechanism for negative regulation of dio1 expression by T3 in specific cell types. These studies will employ transient expression assays of homologous promoter-reporter constructs with and without co-expressed thyroid hormone receptor (TR) or other transcription factors. Specific Aim II will delineate the nature of the TR-TRE interaction and how it is affected by T3 using in vivo genomic footprinting of synthetic and endogenous genes by ligation-mediated PCR. Specific Aim III will focus on the mechanism for regulation of D3 expression by thyroid hormone including cloning of the 5' flanking region, studies of changes in D3 expression in various brain regions with altered thyroid status and iodine deficiency using in situ hybridization and immunohistochemistry, studies in cell culture systems, and in vivo genomic footprinting. By these techniques we will gain new knowledge of how T3 directly regulates the transcription of the genes encoding the enzymes which catalyze essential steps in thyroid hormone activation and inactivation.