Molecular determinants of bHLH-PAS transcription factor function

基因对特定刺激的反应是由一大群被称为转录因子的蛋白质来执行的。为了以适当的时空方式指导转录，这些蛋白质必须指导染色质上大型蛋白质机器的协调组装和拆卸。我的研究计划的目的是了解在转录过程中，基本-螺旋-环-螺旋-PAS(bHLH-PAS)家族转录过程中蛋白质是如何往返于基因表达机制的。我的目标是了解这些变化如何改变蛋白质的功能，以响应不同的信号。这些信息将有助于合理设计利用转录因子功能并直接针对其活动的治疗方法。我选择研究两个严格调控的模型系统：芳香烃受体(AHR)调节的CYP1A1活性；以及缺氧诱导因子-1a(HIF-1a)调节的血管内皮生长因子和促红细胞生成素的表达。我已经证实，雌激素通过雌激素受体-α(ER)，通过直接的蛋白质-蛋白质相互作用来抑制AHR的功能。此外，ER通过促进AHR靶基因调控元件上的组蛋白去乙酰化酶HDAC4和共抑制蛋白RIP140的交换来实现这一点。在这项研究的第一部分，我建议阐明ER介导的AHR调节转录抑制的分子决定因素。我设计了一系列实验，以了解雌激素响应的抑制子RIP140如何调节AHR的活性，并确定HDAC4在维持RIP140处于有利于AHR介导的基因转录的状态中所起的作用。这项提案的第二部分概述了我的计划，以阐明视网膜母细胞瘤蛋白(RB)在HIF-1a调控转录中的作用。我已经证明，共激活因子TRIP230对于HIF-1a的转录激活是必不可少的。此外，我有证据表明，介导的HIF-1a依赖的转录活性受到Rb的负面调控。总之，这些研究将增加我们对正常的AHR生理、血管生成和转录因子功能的了解。

The expression of genes in response to specific stimuli is carried out by a large group of proteins known as transcription factors. In order to direct transcription in an appropriate spatial and temporal fashion, these proteins must direct the coordinated assembly and disassembly of large protein machines on chromatin. The objective of my research program is to understand how proteins are shuttled to and from gene expression machinery during transcription by the basic-Helix-Loop-Helix-PAS (bHLH-PAS) family of transcription factors. It is my goal to understand how these changes alter protein function in response to different signals. This information will aid in the rational design of therapies that exploit transcription factor function and are directly targeted at their activities. I have chosen to study two tightly regulated model systems; aryl hydrocarbon receptor (AHR)-regulated CYP1A1 activity; and, hypoxia-inducible factor-1a (HIF-1a)-regulated VEGF and EPO expression. I have established that estrogens, through the estrogen receptor-alpha (ER), repress AHR function via direct protein-protein interactions. Furthermore, ER does so by facilitating an exchange for the histone deacetylase, HDAC4 for the co-repressor protein, RIP140 at the regulatory elements of AHR target genes. In part 1 of this study, I propose to elucidate the molecular determinants of ER-mediated repression of AHR-regulated transcription. I have designed a series of experiments to understand how estrogen-responsive recruitment of the repressor RIP140 modulates AHR activity and to determine the role HDAC4 plays in maintaining RIP140 in a state conducive to AHR-mediated gene transcription. The second part of this proposal outlines my plans to elucidate the role of the retinoblastoma protein (Rb) on HIF-1a regulated transcription. I have demonstrated that the co-activator TRIP230 is essential for transcriptional activation by HIF-1a. In addition, I have evidence that mediated HIF-1a-dependent transcriptional activity is negatively regulated by Rb. Together, these studies will increase our understanding of normal AHR physiology, vasculogenesis, and transcription factor function in general.