MECHANISM OF PROGESTERONE-INDUCED GENE ACTIVATION

这项研究建议的长期目标是探索 中枢孕酮作用的分子机制 在女性生殖中的作用。黄体酮通过其细胞核起作用 调节特定细胞基因表达的受体 生殖组织。人孕酮受体(HPR)存在 有两种不同的分子形式，hPR-a和hpr-b。最新研究 在PR-B和甲状腺之间进行转录干扰 激素受体(TR)，表明另一个辅因子(S)，称为 辅助激活剂，与被激活的PR和基础 孕酮依赖基因的转录装置是必需的 激活。这项建议的具体目的是：1.确定 有效的孕酮依赖所需的辅活化子(S) HPR-B的转录激活作用一些最近被孤立的候选人 共激活剂，如SRC-1、TIF-1、RIP-140、RIP 160、TIF-2和SUG-1， 将接受测试以确定它们是否有能力解除转录 PR-B和TR在瞬时共表达时的干扰 转基因实验。候选蛋白质的功能将是 通过耗尽无细胞转录提取液进一步分析 可能是辅活化子，然后是孕酮重组- 通过重新添加纯化的辅活化子来介导反式激活 蛋白。2.定义重组所需的组件 黄体酮依赖的目的基因在纯化的小鼠体内反式激活 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 评估推定的功能作用的组件 协促进者(S)。参与功能相互作用的PR-B结构域 与辅活化子的关系将通过突变进行定位。3.分析 PR-B、辅活化子和细胞因子之间的功能相互作用 基础转录装置的组成部分，它导致 增强转录的启动。先前的研究表明 该PR促进了预引发复合体(PIC)在 靶向启动子。装配之间的功能相互作用 络合物和配体结合PR在逐步生成过程中的作用 PIC及共激活剂对这些信号的调节作用 相互作用将通过蛋白质-蛋白质的组合来研究 相互作用和无细胞转录实验。建议数 研究将使我们更好地理解 PR正常细胞生理和分子水平对基因的调控 导致女性的PR信号通路异常的基础 生殖障碍和不孕不育。

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.