Ah receptor transcriptional regulation through a novel DNA binding site

Ah 受体通过新的 DNA 结合位点进行转录调控

• 批准号: 7407880
• 负责人: Shelly Renee Wilson
• 金额: $ 2.73万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-11-15 至 2012-11-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7407880
• 关键词: ARNT protein; Affinity Chromatography; Agonist; Aryl Hydrocarbon Receptor; Binding; Binding Sites; Biological Assay; Complex; Consensus; DNA Binding; DNA-Protein Interaction; Dioxins; EMSA; Exposure to; Gene Expression; Gene Targeting; Health; Homeostasis; Human; Link; Liver; Liver Regeneration; Mediating; Mus; Nature; Nuclear; Nuclear Extract; Nuclear Translocation; Physiological; Plasminogen Activator Inhibitor 1; Play; Process; Proteins; Response Elements; Risk Assessment; Role; Series; Site; Toxic Environmental Substances; Transcriptional Regulation; Xenobiotics; design; in vitro Assay; novel; receptor; transcription factor

DESCRIPTION (provided by applicant): Recent studies demonstrate that the aryl hydrocarbon receptor (AhR), a transcription factor, plays an active role in liver homeostasis, and that this process is dysregulated by the environmental toxicant 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). TCDD is a potent AhR agonist responsible for activation and nuclear translocation of AhR from the cytosolic compartment. Previous studies determined that AhR-mediated transcriptional activity requires partnering with the nuclear ARNT protein and DNA binding of this heterodimer to a consensus xenobiotic response element (XRE) linked to target genes. Preliminary evidence examining TCDD induction of plasminogen activator inhibitor-1 (PAI-1), a key protein in liver regeneration, exposed a novel AhR-dependent regulatory mechanism involving DNA binding to a nonconsensus XRE (NC-XRE) independent of the ARNT protein. We hypothesize that the AhR regulates PAI- 1 gene expression by a formerly unrecognized mechanism of action. This study will identify the components of the complex bound to the NC-XRE and characterize the AhR protein-protein and protein- DNA interactions at this site. Specific Aim 1 will employ DNA-affinity chromatography using the NC-XRE binding site characterized by EMSA and functional studies to fractionate the complex from mouse liver nuclear extracts following induction with TCDD. MALDI-TOF/TOF will be used to identify the proteins in the NC-XRE-bound complex. Specific Aim 2 will verify immunologically that the proteins identified in aim 1 are indeed components of the NC-XRE binding complex using EMSA supershift assays and coimmunoprecipitation studies. Studies in Specific Aim 3 will focus on characterizing the nature of AhR interaction in this complex through a series of in vitro assays designed to examine protein-protein and protein-DNA interactions. Collectively, these studies will explore a new mechanism in the growing repertoire of distinct AhR activities that must be resolved if biologists are to fully understand the physiological role of the receptor or obtain reliable human health risk assessments following exposure to TCDD and related environmental toxicants.

描述(申请人提供)：最近的研究表明，芳烃受体(AhR)是一种转录因子，在肝脏稳态中起着积极的作用，并且这一过程受到环境毒物2，3，7，8-四氯二苯并-对二恶英(TCDD)的失调调节。TCDD是一种有效的AhR激动剂，负责激活和核转位胞浆中的AhR。以往的研究确定，AhR介导的转录活性需要与核Arnt蛋白配对，并将这种异源二聚体与与靶基因相连的共识异源反应元件(XRE)的DNA结合。TCDD诱导肝再生的关键蛋白纤溶酶原激活物抑制物-1(PAI-1)的初步证据揭示了一种新的依赖AhR的调控机制，涉及DNA与非共识XRE(NC-XRE)结合，而不是Arnt蛋白。我们假设AhR通过以前未知的作用机制来调节PAI-1基因的表达。本研究将鉴定与NC-XRE结合的复合体的成分，并表征该位点的AhR蛋白质-蛋白质和蛋白质-DNA相互作用。具体目标1将使用以EMSA为特征的NC-XRE结合部位的DNA亲和层析，并进行功能研究，以从TCDD诱导后的小鼠肝核提取物中分离出该复合体。MALDI-TOF/TOF将用于鉴定NC-XRE结合复合体中的蛋白质。特异性AIM 2将通过EMSA超位移分析和免疫共沉淀研究从免疫学上证实AIM 1中鉴定的蛋白质确实是NC-XRE结合复合体的组成部分。具体目标3的研究将侧重于通过一系列旨在检查蛋白质-蛋白质和蛋白质-DNA相互作用的体外试验来表征该复合体中AhR相互作用的性质。总的来说，这些研究将探索不断增长的不同AhR活性的新机制，如果生物学家要充分了解受体的生理作用或在接触TCDD和相关环境毒物后获得可靠的人类健康风险评估，就必须解决这个问题。