A Novel Paradigm for Aryl Hydrocarbon Receptor Signaling

芳基烃受体信号传导的新范例

• 批准号: 8896257
• 负责人: Cornelis Johan Elferink
• 金额: $ 23.25万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8896257
• 关键词: ARNT protein; Abbreviations; Amino Acids; Aryl Hydrocarbon Receptor; Binding; Biological; Biological Assay; Biology; Bromodeoxyuridine; CDK2 gene; Cell Cycle Arrest; Cell Cycle Progression; ChIP-seq; Co-Immunoprecipitations; Complex; Coupled; Cyclin-Dependent Kinases; DNA; DNA Binding; DNA Binding Domain; DNA-Protein Interaction; Data; Dimethyl Sulfoxide; Dioxins; EMSA; Environmental Pollutants; Environmental Pollution; Evaluation; Event; Exposure to; Functional disorder; Future; G1 Phase; Gene Expression; Gene Expression Profile; Gene Targeting; General Population; Genetic Transcription; Genomics; Green Fluorescent Proteins; Health; Helix-Turn-Helix Motifs; Hepatic; Heterodimerization; Human; Immune System Diseases; Injury; Knock-out; Ligands; Liver Regeneration; Malignant Neoplasms; Mediating; Mediator of activation protein; Molecular; Outcome; Partial Hepatectomy; Pathway interactions; Plasminogen Activator Inhibitor 1; Process; Proteins; Receptor Signaling; Recombinant Proteins; Recruitment Activity; Research; Research Design; Response Elements; Risk; Shotgun Sequencing; Signal Transduction; Site; Tertiary Protein Structure; Testing; Tetrachlorodibenzodioxin; Time; Toxic effect; Transactivation; Tumor Suppressor Proteins; Work; Xenobiotics; activating transcription factor; chromatin immunoprecipitation; deep sequencing; design; fetal bovine serum; human ARNT protein; inhibitor/antagonist; innovation; mutant; novel; pollutant; prevent; public health relevance; receptor; receptor binding; receptor function; retinoblastoma tumor suppressor; transcriptome sequencing

 DESCRIPTION (provided by applicant): The Aryl hydrocarbon Receptor (AhR) is a mediator of xenobiotic toxicity, best recognized for conveying the deleterious human health effects following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related environmental contaminants. Mechanistically, the AhR is known to function as a ligand-activated transcription factor that binds to a canonical xenobiotic response element (XRE) in association with its heterodimerization partner, the AhR nuclear translocator (Arnt) protein. However, within the repertoire of AhR target genes identified in recent years, many lack a clearly defined XRE, highlighting the growing realization that AhR-mediated gene expression appears to involve additional mechanisms distinct from the well-characterized process involving the XRE. We recently identified a non-consensus XRE (NC-XRE) that recruits the AhR in conjunction with a novel DNA binding partner, the Krüppel-like factor 6 (KLF6) tumor suppressor. DNA binding and functional studies confirmed that NC-XRE binding by the AhR-KLF6 complex is independent of the Arnt protein. The toxicological ramification of TCDD-induced AhR-KLF6 activity is revealed in liver regeneration studies. Our previous work established that TCDD inhibits normal liver regeneration following injury, by suppressing G1 phase cyclin-dependent kinase (CDK) activity, specifically CDK2. Increased CDK2 activity is necessary for G1 phase cell cycle progression, and inhibition of CDK2 activity leads to cell cycle arrest. New data demonstrate that the TCDD-induced inhibition of liver regeneration is directly tied to AhR-dependent expression of the CDK2 inhibitor, p21Cip1. The results confirm that p21Cip1 is an AhR target gene, and that its induction relies on NC-XRE-mediated AhR-KLF6 activity. A preliminary analysis of the AhR-KLF6 protein-DNA interaction revealed that deletion of the AhR's DNA binding domain required for XRE binding did not prevent receptor binding to the NC-XRE. Hence, we hypothesize that AhR-NC-XRE binding and function is fundamentally different from signaling through the XRE. In keeping with our long-term objective to understand the pathophysiology of AhR activity, this proposal will examine two specific aims designed to characterize this novel NC-XRE regulatory complex as a platform for future studies designed to explore AhR-KLF6 signaling. Specific aim 1 describes a detailed characterization and functional analysis of the NC-XRE protein-DNA complex. Specific Aim 2 will use ChIP-seq and RNA-seq to provide a global genomic assessment of the functional NC-XRE sites regulated by the AhR/KLF6 transcriptional complex. Identification of this novel AhR complex represents a paradigm shift in our understanding of AhR biology and TCDD toxicity.

 描述（由申请人提供）：芳基碳氢化合物受体 (AhR) 是一种异生物质毒性介质，最被认可的是在暴露于 2,3,7,8-四氯二苯并-对二恶英（TCDD，二恶英）和相关环境污染物后对人类健康产生有害影响。从机制上讲，AhR 被认为是一种配体激活转录因子，与典型的异生素反应元件 (XRE) 结合，并与其异二聚化伙伴，AhR 核易位蛋白 (Arnt) 蛋白结合。然而，在近年来确定的 AhR 靶基因库中，许多缺乏明确定义的 XRE，这突显出人们日益认识到 AhR 介导的基因表达似乎涉及与涉及 XRE 的充分表征过程不同的其他机制。我们最近发现了一种非共识 XRE (NC-XRE)，它可以将 AhR 与一种新型 DNA 结合伴侣——Krüppel 样因子 6 (KLF6) 肿瘤抑制因子结合起来。 DNA 结合和功能研究证实 AhR-KLF6 复合物的 NC-XRE 结合独立于 Arnt 蛋白。肝脏再生研究揭示了 TCDD 诱导的 AhR-KLF6 活性的毒理学后果。我们之前的工作表明，TCDD 通过抑制 G1 期细胞周期蛋白依赖性激酶 (CDK) 活性，特别是 CDK2 来抑制损伤后的正常肝再生。 CDK2 活性的增加是 G1 期细胞周期进展所必需的，抑制 CDK2 活性会导致细胞周期停滞。新数据表明，TCDD 诱导的肝再生抑制与 CDK2 抑制剂 p21Cip1 的 AhR 依赖性表达直接相关。结果证实 p21Cip1 是 AhR 靶基因，其诱导依赖于 NC-XRE 介导的 AhR-KLF6 活性。对 AhR-KLF6 蛋白-DNA 相互作用的初步分析表明，XRE 结合所需的 AhR DNA 结合域的删除并不能阻止受体与 NC-XRE 的结合。因此，我们假设 AhR-NC-XRE 结合和功能与通过 XRE 发出的信号有根本不同。为了与我们了解 AhR 活动的病理生理学的长期目标保持一致，该提案将研究两个具体目标，旨在表征这种新型 NC-XRE 调控复合物，作为未来研究的平台，旨在探索 AhR-KLF6 信号传导。具体目标 1 描述了 NC-XRE 蛋白质-DNA 复合物的详细表征和功能分析。具体目标 2 将使用 ChIP-seq 和 RNA-seq 对 AhR/KLF6 转录复合物调控的功能性 NC-XRE 位点进行全局基因组评估。这种新型 AhR 复合物的鉴定代表了我们对 AhR 生物学和 TCDD 毒性理解的范式转变。