Transcription Factor Interactions in Inhibin Regulation

该研究计划的总体目标是在分子水平上了解转录因子及其协同调节蛋白之间的相互作用如何介导生殖组织中激素依赖性基因表达。 这些研究将使用编码抑制素α亚基的基因进行，抑制素是一种调节垂体促卵泡激素（FSH）合成和分泌的性腺蛋白。抑制素基因表达由 FSH 通过涉及 cAMP 依赖性蛋白激酶 (PKA) 激活和转录因子 CREB（cAMP 响应元件 (CRE) 结合蛋白）磷酸化的途径调节，CREB ​​与抑制素 α 亚基启动子中的非典型 CRE 结合。 FSH 作用与孤儿核受体类固醇生成因子 1 (SF-1) 共同依赖并增强，SF-1 与抑制素 α 亚基基因中与 CRE 相邻的非典型靶位点结合。 目前尚不清楚这种相互依赖性的本质是什么，或者这些功能相互作用是如何由于正常生殖周期中发生的激素和细胞变化而受到调节的。 目标 1 中的实验将通过研究激素刺激后 CREB ​​和 SF-1 占据启动子的时间过程以及测试抑制素 α 亚基启动子上两种蛋白质之间的直接相互作用来解决这些问题。 目标 2 中的实验将研究以下假设：转录共激活因子 CBP 和 SRC-1 可能通过促进包括这些共调节蛋白在内的高级蛋白复合物的形成，部分介导 CREB ​​和 SF-1 之间观察到的功能相互依赖性。 最后，目标 3 中的实验将使用基于 NMR 的方法研究功能重要的蛋白质-DNA 和蛋白质-蛋白质相互作用的结构基础。 这些研究将提供机制见解，增加我们对生殖周期期间卵巢基因调控的了解，并将适用于了解对人类健康重要的生殖疾病中可能出现的基因表达和调控缺陷。

The overall objective of this research proposal is to understand at a molecular level how interactions between transcription factors and their co-regulatory proteins mediate hormone-dependent gene expression in reproductive tissues. These studies will be performed using the gene encoding the alpha subunit of inhibin, a gonadal protein that modulates pituitary follicle-stimulating hormone (FSH) synthesis and secretion. Inhibin gene expression is regulated by FSH through a pathway involving the activation of the cAMP-dependent protein kinase (PKA) and the phosphorylation of the transcription factor CREB (cAMP-response element (CRE) binding protein), which binds to an atypical CRE in the inhibin alpha subunit promoter. FSH action is co-dependent on and augmented by the orphan nuclear receptor steroidogenic factor-1 (SF-1), which binds to an atypical target site adjacent to the CRE in the inhibin alpha subunit gene. It remains unknown what the nature of this interdependence is or how these functional interactions are regulated as a consequence of the hormonal and cellular changes that occur during a normal reproductive cycle. Experiments in aim 1 will address these issues by investigating the time course of promoter occupancy by CREB and SF-1 following hormone stimulation and by testing for direct interactions between the two proteins on the inhibin alpha subunit promoter. Experiments in aim 2 will investigate the hypothesis that the transcriptional coactivators CBP and SRC-1 may mediate in part the functional interdependence observed between CREB and SF-1 by facilitating formation of a higher-order protein complex including these co-regulatory proteins. Finally, experiments in aim 3 will investigate the structural basis for functionally important protein-DNA and protein-protein interactions using NMR-based approaches. These studies will provide mechanistic insights that will increase our knowledge of ovarian gene regulation during the reproductive cycle and will be applicable to understanding defects in gene expression and regulation that may occur in reproductive disease of importance to human health.