Differential Mechanisms of Nuclear Receptor Regulation by the Coactivator RAC3: A Dissertation

The steroid/thyroid hormone receptor superfamily is a large class of liganddependent transcription factors that plays a critical role in regulating the expression of genes involved in a broad range of physiological functions, including development homeostasis , and reproduction. In the absence of cognate hormone, several receptors are able to repress transcription below the basa11evel via the recruitment of the nuclear receptor corepressors SMRT and NCoR. Upon hormone binding by the receptor, the corepressor complex is dissociated and a coactivator complex is subsequently recruited. This thesis details the mechanisms by which receptor-associated coactivator 3 (RAC3) interacts with nuclear receptors , particularly the vitamin D, estrogen, and retinoid receptors , and modulates their transcriptional activity. It was discovered that these receptors interact with different a-helical LXXLL motifs ofRAC3 in vitro. Mutation of specific motifs differentially impairs the ability ofRAC3 to enhance transcription by the receptors in vivo. In addition , the intrinsic transcriptional activation function ofRAC3 was also characterized. Here , a single LXXLL motif, NR box v , was found to be essential to activation by serving as a binding surface for the general transcriptional integrator CBP/p300. Finally, the cofactor binding pocket of retinoid receptors was characterized. It was demonstrated that, to a large extent , the coactivator pocket of RAa overlaps with the corepressor pocket, with the exception of helix 12 , which is required for coactivator, but not corepressor binding. Recruitment ofRAC3 or SMRT also correlates directly with the ability ofRAa to activate or repress transcription respectively. Intriguingly, it was discovered that the AF-2 domain ofRXa inhibited cofactor binding to RXRa heterodimers, for deletion of this domain dramatically enhanced RAC3 and SMRT binding. In addition, it was demonstrated that the RXa cofactor binding pocket contributed minimally to recruitment of cofactors. Conversely, the AF-2 domain of the partnering monomer and its cofactor pocket were required for these interactions. These findings suggest that the partner of RXa is the primary docking point for co factors at RXa heterodimeric complexes. Taken together , this work contributes significantly to the field of nuclear receptor function in detailing the mechanisms by which the coactivator RAC3 is recruited to nuclear receptors and regulates their transcriptional activity.