The goal of this application is to specifically relate TR biochemistry and molecular biology to the cellular and molecular physiological effects of T3. It has become clear that regulation by TR involves repression by unliganded TR as well as ligand dependent activation, and this repression is thought be dependent on the recruitment of corepressor proteins by the unliganded TR. The first specific aim is to determine mechanisms by which different regions of TR regulate both interactions with corepressors N-CoR and SMRT, and ligand-independent and -dependent heterodimerization with retinoid X receptor (RXR). It is hypothesized that RXR differentially recognizes the liganded and unliganded conformations of TR, and this will be tested using mutations based on the known structure of the TR ligand binding domain.
The second aim i s to determine the ability of RXR to regulate functional interactions with N-CoR and SMRT and with candidate coactivators. It is hypothesized that the differential recognition of liganded and unliganded forms of TR by RXR results in regulation of repression as well as transactivation. This will be tested by determining the requirement for RXR in corepressor and coactivator binding and function.
The third aim i s determine the principles underlying AF-2 dependent transactivation by TR, and it is hypothesized that DNA binding and RXR heterodimerization increase the specificity of coactivator binding. The contributions of the DNA binding site as well as RXR heterodimerization in regulating coactivator function will be determined. The fourth specific aim is to identify mechanisms by which N-CoR and SMRT inhibit basal transcription. It is hypothesized that each of multiple repression domains in N-CoR recruits proteins that stabilize the repression complex and/or function to repress transcription. Such proteins will be cloned and characterized using dihybrid screens in yeast. These studies will provide insight into hormone action in general, and enhance understanding of many pathophysiological states.
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