The long-term goal of this research proposal is to explore the molecular mechanism of action of progesterone which plays a central role in female reproduction. Progesterone acts through its nuclear receptors which regulate the expression of specific cellular genes in reproductive tissues. The human progesterone receptor (hPR) exists in two distinct molecular forms, hPR-A and hPR-b. Recent studies employing transcriptional interference between PR-B and thyroid hormone receptor (TR), indicated that an additional cofactor(s), termed coactivator, acting in concert with the activated PR and the basal transcription apparatus is necessary for progesterone-dependent gene activation.
The Specific Aims of this proposal are: 1. To identify the coactivator(s) that is required for efficient progesterone-dependent transactivation by hPR-B. A number of recently-isolated candidate coactivators such as, SRC-1, TIF-1, RIP-140, RIP 160.TIF-2, and SUG-1, will be tested for their ability to relieve the transcriptional interference between PR-B and TR when coexpressed in transient transfection experiments. The function of a candidate protein will be analyzed further by depleting cell-free transcriptional extracts of the putative coactivator followed by reconstitution of progesterone- mediated transactivation by adding back the purified coactivator protein. 2. To define the components required to reconstitute progesterone-dependent transactivation of a target gene in a purified cell-free system. A well-define progesterone-regulated cell-free transactivation system will be reconstituted from highly purified components to assess the functional role of the putative coactivator(s). The PR-B domain involved in functional interaction with the coactivator will be mapped by mutagenesis. 3. To analyze the functional interactions between PR-B, the coactivator and the components of the basal transcription apparatus that leads to the enhancement of initiation of transcription. Previous studies indicated that PR facilitates the assembly of a preinitiation complex (PIC) at the target promoter. The functional interactions between the assembling complex(es) and the ligand-bound PR during stepwise generation of the PIC and the modulatory effects of the coactivator on these interactions will be investigated by a combination of protein-protein interaction and cell-free transcription experiments. The proposed research will develop a better understanding of the mechanisms of gene regulation by PR normal cellular physiology and the molecular basis of abnormalities in the Pr signalling pathway that lead to female reproductive disorders and infertility.
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