Molecular determinants of bHLH-PAS transcription factor function

对特定刺激的基因表达是由一大群被称为转录因子的蛋白质来完成的。为了以适当的空间和时间方式指导转录，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。我的研究项目的目的是了解在转录过程中，蛋白质是如何在basic-Helix-Loop-Helix-PAS （bHLH-PAS）转录因子家族中穿梭于基因表达机制之间的。我的目标是了解这些变化如何改变蛋白质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。我选择研究两个受到严格监管的模型系统；芳烃受体（AHR）调控CYP1A1活性；缺氧诱导因子1a （HIF-1a）调节VEGF和EPO的表达。我已经确定，雌激素，通过雌激素受体（ER），通过直接的蛋白质-蛋白质相互作用抑制AHR功能。此外，内质网通过促进AHR靶基因调控元件上的组蛋白去乙酰化酶、HDAC4交换共抑制蛋白RIP140来实现这一功能。在本研究的第一部分中，我建议阐明er介导的ahr调控转录抑制的分子决定因素。我设计了一系列实验，以了解抑制物RIP140的雌激素反应性募集如何调节AHR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。本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调节转录中的作用。我已经证明了共激活剂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依赖性转录活性受到Rb的负调控。总之，这些研究将增加我们对正常AHR生理、血管发生和转录因子功能的理解。

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.