MECHANISM OF ACTIVATION BY NUCLEAR RECEPTOR COACTIVATORS

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The steroid-thyroid hormone receptor (or nuclear receptor) superfamily includes the steroid, thyroid, vitamin A, and vitamin D receptors. Deficiencies in these receptors and their associated signaling pathways are associated with many genetic diseases, and steroid receptors have important roles in the etiology and treatment of breast, prostate, and lymphoid cancers. These hormone controlled transcriptional activators bind to specific enhancer elements assoicated with the genes they regulate and thereby promote the assembly or activation of a functional transcription pre-initiation complex. Transcriptional activation by nuclear receptors (NR) is mediated by a new family of three proteins, the Nuclear Receptor Coactivators (NRCoA); the three 150-160-kDa proteins are SRC-1; GRIP1 or TIF2; and p/CIP (which also has several other names). The functional domains of these proteins are still under investigation but include a centrally located NR interaction domain; one transcriptional activation domain that contains a binding site for the coactivator CBP or p300; a second transcriptional activation domain near the C-terminus that is CBP-independent; and a histone acetyltransferase (HAT) activity in the C-terminal domain. Little is known about the mechanism by which these NRCoAs mediate transcriptional activation of NRs. Our working hypothesis is that the C-terminal region of the NRCoAs, which contains transactivation and HAT activities, and the N- terminal region, which is the most highly conserved region among the three NRCoAs, play important roles in coactivator activity by making direct or indirect contact with proteins in the transcription machinery and/or chromatin. We have recently developed a series of transient transfection assays that demonstrate the coactivator function of NRCoAs in mammalian cells; in these assays deletion of the N-terminal or the C-terminal domain dramatically alters the function of the NRCoAs. Here we propose to map in detail the subdomains associated with these activities and to identify and characterize specific cellular proteins that associate with these domains and mediate their activities. The NRCoAs represent a new step in the NR signaling pathways that activate transcription of specific genes in response to specific hormones. The proposed investigations will identify the next step(s) in the pathway. The long range goal is to completely define the protein components that transmit the activating signal from the activated, DNA-bound NRs to the transcription machinery.