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: 7531785
• 负责人: Shelly Renee Wilson
• 金额: $ 2.75万
• 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/7531785
• 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（由申请人提供）：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，并且该过程被环境毒物2，3，7，8-四氯二苯并-对-二恶英（TCDD）失调。TCDD是一种有效的AhR激动剂，负责AhR从胞质隔室的激活和核转位。以前的研究确定，AhR介导的转录活性需要与核ARNT蛋白和DNA结合，这种异源二聚体的共识异生素反应元件（XRE）连接到靶基因。初步证据检查TCDD诱导纤溶酶原激活物抑制剂-1（派-1），肝再生的关键蛋白，暴露了一种新的AhR依赖的调节机制，涉及DNA结合到一个非共识XRE（NC-XRE）独立的ARNT蛋白。我们推测，AhR调节派- 1基因表达的一个以前未被认识到的行动机制。本研究将鉴定与NC-XRE结合的复合物的组分，并表征该位点的AhR蛋白-蛋白和蛋白- DNA相互作用。具体目标1将采用DNA亲和色谱法，使用NC-XRE结合位点，通过EMSA和功能研究表征，以在用TCDD诱导后从小鼠肝细胞核提取物中分离复合物。MALDI-TOF/TOF将用于鉴定NC-XRE结合复合物中的蛋白质。特异性目标2将使用EMSA超位移测定和免疫共沉淀研究从免疫学上验证目标1中鉴定的蛋白质确实是NC-XRE结合复合物的组分。在具体目标3的研究将集中在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这种复合物中AhR相互作用的性质。总的来说，这些研究将探索一个新的机制，在不断增长的剧目不同的AhR活动，必须解决，如果生物学家要充分了解受体的生理作用或获得可靠的人类健康风险评估后，暴露于TCDD和相关的环境毒物。