MECHANISM OF PROGESTERONE-INDUCED GENE ACTIVATION

本研究计划的长远目标是探讨 孕酮的分子作用机制， 在女性生殖中的作用 核武器通过其核 受体调节特定细胞基因的表达， 生殖组织 人类孕酮受体（hPR） 有两种不同的分子形式hPR-A和hPR-b。 最近的研究 利用PR-B和甲状腺之间的转录干扰 激素受体（TR），表明一个额外的辅因子，称为 共激活因子，与激活的PR和基础 转录器是胆固醇依赖基因所必需的 activation.该提案的具体目标是：1。识别 有效的甾体酮依赖性 通过hPR-B的反式激活。 一些最近被孤立的候选人 辅活化剂如SRC-1、TIF-1、RIP-140、RIP 160、TIF-2和SUG-1， 将测试他们的能力，以减轻转录 瞬时共表达时PR-B和TR之间的干扰 转染实验。候选蛋白的功能将是 进一步分析，通过消耗细胞的无细胞转录提取物， 假定的辅激活因子，然后重建孕酮- 通过加回纯化的辅激活因子介导的反式激活 蛋白 2. 定义重组所需的组件 在纯化的重组质粒中靶基因的甾体酮依赖性反式激活 无细胞系统 一种明确的孕酮调节的无细胞 反式激活系统将从高度纯化的 的职能作用， 共活化剂。 参与功能相互作用的PR-B结构域 将通过诱变作图。 3. 分析 PR-B、共激活剂和蛋白质之间的功能性相互作用 基础转录器的组成部分，导致 转录起始的增强。 先前的研究表明 PR促进了前起始复合物（PIC）的组装， 靶向启动子。 组装之间的功能相互作用 复合物和配体结合的PR在逐步产生 PIC和共激活剂对这些的调节作用 相互作用将通过蛋白质-蛋白质 相互作用和无细胞转录实验。 拟议 研究将更好地了解 PR正常细胞生理学和分子生物学的基因调控 基础异常的Pr信号通路，导致女性 生殖障碍和不育症。

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.