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）：芳烃受体（AhR）是异生物质毒性的介体，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传递有害的人类健康影响。从机制上讲，AhR已知作为配体激活的转录因子发挥作用，其结合与其异源二聚化伴侣AhR核转运蛋白（Arnt）蛋白相关的典型异源反应元件（XRE）。然而，在近年来鉴定的AhR靶基因库中，许多缺乏明确定义的XRE，突出了越来越多的认识，AhR介导的基因表达似乎涉及与涉及XRE的良好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），它招募AhR与一种新的DNA结合伴侣，Krüppel样因子6（KLF 6）肿瘤抑制因子。DNA结合和功能研究证实，AhR-KLF 6复合物与NC-XRE的结合不依赖于Arnt蛋白。TCDD诱导的AhR-KLF 6活性的毒理学衍生物在肝再生研究中被揭示。我们以前的工作证实，TCDD通过抑制G1期细胞周期蛋白依赖性激酶（CDK）活性，特别是CDK 2，抑制损伤后的正常肝再生。增加的CDK 2活性对于G1期细胞周期进展是必需的，并且抑制CDK 2活性导致细胞周期停滞。新的数据表明，TCDD诱导的肝再生抑制与CDK 2抑制剂p21 Cip 1的AhR依赖性表达直接相关。结果证实p21 Cip 1是AhR靶基因，并且其诱导依赖于NC-XRE介导的AhR-KLF 6活性。对AhR-KLF 6蛋白-DNA相互作用的初步分析显示，XRE结合所需的AhR DNA结合结构域的缺失不会阻止受体与NC-XRE的结合。因此，我们假设AhR-NC-XRE结合和功能与通过XRE的信号传导根本不同。为了与我们了解AhR活性的病理生理学的长期目标保持一致，该提案将研究两个特定目标，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-KLF 6信号传导。具体目标1描述了NC-XRE蛋白-DNA复合物的详细表征和功能分析。Specific Aim 2将使用ChIP-seq和RNA-seq对AhR/KLF 6转录复合物调控的功能性NC-XRE位点进行全球基因组评估。这种新的AhR复合物的鉴定代表了我们对AhR生物学和TCDD毒性的理解的范式转变。