Transcription Factor Interactions in Inhibin Regulation

这项研究提案的总体目标是在分子水平上了解转录因子和它们的共同调节蛋白之间的相互作用如何在生殖组织中介导激素依赖基因的表达。这些研究将使用抑制素α亚基编码的基因进行，抑制素是一种性腺蛋白，调节垂体促卵泡激素(FSH)的合成和分泌。抑制素基因的表达受FSH调控，途径涉及cAMP依赖的蛋白激酶(PKA)的激活和转录因子CREB(cAMP反应元件(CRE)结合蛋白)的磷酸化，CREB与抑制素α亚单位启动子中非典型的CRE结合。FSH的作用依赖于孤儿核受体类固醇生成因子-1(SF-1)，并被其增强，SF-1与抑制素α亚单位基因中Cre附近的非典型靶点结合。目前尚不清楚这种相互依赖的性质是什么，也不知道这些功能相互作用是如何作为正常生殖周期中发生的荷尔蒙和细胞变化的结果而调节的。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这两种蛋白质在抑制素α亚单位启动子上的直接相互作用来解决这些问题。目标2中的实验将探讨这样的假设，即转录共激活因子CBP和SRC-1可能通过促进包括这些共调节蛋白在内的高阶蛋白质复合体的形成，部分介导CREB和SF-1之间的功能相互依赖。最后，目标3中的实验将使用基于核磁共振的方法来研究重要的蛋白质-DNA和蛋白质-蛋白质相互作用的结构基础。这些研究将提供机械性的见解，将增加我们对生殖周期中卵巢基因调控的了解，并将适用于理解对人类健康具有重要意义的生殖疾病中可能发生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.