The overall goal is to increase our understanding of how thyroid hormone (T3) regulates gene expression. T3 binds to receptors (TRs), which bind to T3 response elements (TREs) in specific target genes. TREs generally consist of two (or more) binding sites (half sites) arranged as a direct repeat, inverted repeat, or everted repeat. TRs can bind to TREs as homodimers or as heterodimers with retinoid X receptors (RXRs); the relative biological importance of each of these dimer forms is uncertain. TRs regulate transcription via two domains, AF-1 and AF-2. The function of AF-1 is poorly understood. AF-2 functions by interacting with other proteins, generally known as coactivators and corepressors. T3 alters the conformation of the TR, thereby affecting which proteins interact with this receptor. Our data suggest that certain genes are regulated by TR homodimers and others by RXR-TR heterodimers, and that this is determined by the sequence of the TRE. In addition, our data suggest that TR homodimers and RXR-TR heterodimers have different coactivator requirements, and that half site orientation further influences coactivator requirements. These issues will be studied in yeast and in mammalian cells. Yeast are uniquely valuable because they lack the above proteins. Hence, TR, RXR, and various coactivators can be added back in defined ways to determine their effects on gene expression. Additionally, yeast are amenable to genetic manipulations that are essentially impossible in mammalian cells. However, confirmation of the findings in yeast must be made in mammalian cells, to demonstrate biological relevance.
Three specific aims will be addressed: 1) Assess the mechanism of coactivator-independent (AF-1) TR function in yeast; 2) Assess the role of TRE structure and homodimers versus heterodimers in defining coactivator requirements in yeast; 3) Determine whether the key findings in the above aims apply to mammalian cells. The results should further our understanding of how T3 affects a broad range of metabolic processes in health and disease states such as hyperthyroidism and hypothyroidism.
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