Transcription Factor Interactions in Inhibin Regulation

本研究计划的总体目标是在分子水平上了解转录因子及其共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的表达。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。抑制素基因的表达受FSH调控，其途径涉及camp依赖性蛋白激酶（PKA）的激活和转录因子CREB （cAMP-response element (CRE) binding protein）的磷酸化，CREB与抑制素α亚基启动子中的非典型CRE结合。FSH的作用是由孤儿核受体甾体生成因子-1 （SF-1）共同依赖和增强的，SF-1结合到抑制素α亚基基因中靠近CRE的非典型靶点。目前尚不清楚这种相互依赖的本质是什么，也不知道在正常的生殖周期中，激素和细胞的变化是如何调节这些功能相互作用的。目的1的实验将通过研究激素刺激后CREB和SF-1占用启动子的时间过程，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。目的2的实验将研究转录共激活因子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的假设。最后，目标3中的实验将使用基于核磁共振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中卵巢基因调控的认识，并将适用于理解对人类健康重要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.