THYROID HORMONE RECEPTOR AND GENE EXPRESSION

甲状腺激素受体和基因表达

• 批准号: 2141147
• 负责人: HOWARD C TOWLE
• 金额: $ 11.77万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2141147
• 关键词: DNA binding protein; chemical binding; conformation; dimer; fungal genetics; gel mobility shift assay; gene mutation; genetic regulatory element; genetic transcription; hormone analog; hormone receptor; hormone regulation /control mechanism; molecular cloning; mutant; protein sequence; protein structure function; reporter genes; retinoid binding proteins; thyroid hormones; tissue /cell culture; transcription factor; triiodothyronine

The long range goal of this research is to understand the molecular basis by which the nuclear thyroid hormone receptor (TR) mediates changes in specific gene transcription. TR binds to specific DNA sequences (thyroid hormone response elements or TREs) in both the absence and presence of hormone. Upon binding of hormone, TR undergoes a conformational change that converts it to a transcriptionally active state. The region of TR between the DNA-binding domain and the C-terminal end (residues 170 to 456 of the beta form) is responsible for ligand binding, dimerization and transcriptional activation. Little is currently known regarding these important processes. The focus of this proposal is to identify the critical amino acid residues responsible for these functions and to understand the mechanism of ligand-mediated transcriptional activation by TR. To accomplish these goals, we have established a yeast system in which expression of the reporter gene, beta-galactosidase, is under control of TR. Like many other mammalian transcription factors, TR can function in yeast to activate transcription provided an appropriate DNA recognition site is present. Using the yeast expression system, we have selected for mutations of the C-terminal region of TR that are defective in promoting transcriptional activation. These mutations fall into two general categories: those that fail to bind hormone and those that bind hormone normally, but cannot trans-activate. To evaluate the nature of these functional domains, we will further characterize the mutations leading to defects in hormone-binding and trans-activation. To identify amino acid residues that are in close contact with hormone, we will screen for mutations that alter the ligand-binding specificity of TR (Specific Aim #1). Mutations that bind hormone, but are defective in trans-activation, will be studied to determine whether the defects result from the failure of TR to undergo the ligand-mediated conformational change or lie directly within the trans-activation domain itself (Specific Aim #2). Recently, several laboratories have shown that TR binding and transcriptional activation on certain TREs can be enhanced by heterodimerization with the retinoid X receptor (RXR). We will use the mutations that we have isolated to study the roles of each of the receptor partners in the heterodimer in the activation process (Specific Aim #3). Finally, we will use phenotypic screening in yeast to identify regions of TR that are critical to the process of heterodimerization and to determine whether distinct dimerization domains are involved in interacting with different types of TREs (Specific Aim #4). These studies will allow a better understanding of the process of ligand-mediated transcriptional activation by TR and provide valuable tools for future work to further explore this process.

这项研究的远距离目标是了解分子基础 核甲状腺激素受体（TR）介导了介导的变化 特定的基因转录。 TR与特定的DNA序列结合（甲状腺 激素反应元素或Tres）在不存在和存在的情况下 激素。激素结合后，TR发生构象变化 这将其转换为转录活性状态。 TR区域 在DNA结合域和C末端之间（残基170至456 Beta表格的）负责配体结合，二聚化和 转录激活。目前对这些知之甚少 重要过程。该提议的重点是确定 负责这些功能的关键氨基酸残基和 了解配体介导的转录激活的机制 tr。为了实现这些目标，我们建立了一个酵母系统 报告基因的表达，β-半乳糖苷酶，正在控制 tr。像许多其他哺乳动物的转录因子一样，TR可以在 激活转录的酵母提供了适当的DNA识别 现场存在。使用酵母表达系统，我们选择了 促进有缺陷的TR的C末端区域的突变 转录激活。这些突变分为两个一般 类别：那些无法结合激素和结合激素的人 通常，但不能反式激活。评估这些的性质 功能域，我们将进一步表征导致的突变 激素结合和反式激活中的缺陷。鉴定氨基酸 与激素密切接触的残留物，我们将筛选 改变TR的配体结合特异性的突变（特定目的 ＃1）。结合激素但在反式激活中有缺陷的突变， 将研究以确定缺陷是否导致故障导致 TR进行配体介导的构象变化或直接躺在 在反式激活域本身中（特定的目标＃2）。最近， 几个实验室表明TR结合和转录 可以通过异二聚化来增强某些TRE的激活 视网膜类动物X受体（RXR）。我们将使用孤立的突变 研究每个受体伴侣在异二聚体中的作用 激活过程（特定目标＃3）。最后，我们将使用表型 在酵母中进行筛查以识别TR的区域 异二聚体的过程，并确定是否明显 二聚域与不同类型的相互作用涉及 TRE（特定目标＃4）。这些研究将使人们更好地理解 配体介导的转录激活的过程和 为将来的工作提供有价值的工具，以进一步探索这一过程。