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(申请人提供)：芳烃受体(AhR)是一种外来生物毒性的介体，最著名的是传递暴露于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的有害影响。从机制上讲，AhR是一种配体激活的转录因子，与其异源二聚体蛋白AhR核转运蛋白(ArnT)结合在一起，与典型的异源生物反应元件(XRE)结合。然而，在近年来发现的AhR靶基因库中，许多基因缺乏明确定义的XRE，这突显了人们越来越认识到AhR介导的基因表达似乎涉及不同于涉及XRE的特征良好的过程的额外机制。我们最近发现了一个非共识XRE(NC-XRE)，它与一个新的DNA结合伙伴Krüppel样因子6(KLF6)肿瘤抑制因子一起招募AhR。DNA结合和功能研究证实，AhR-KLF6复合体与NC-XRE的结合不依赖于Arnt蛋白。肝再生研究揭示了TCDD诱导AhR-KLF6活性的毒理学分支。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)的活性，特别是CDK2，来抑制损伤后的正常肝再生。CDK2活性的增加是G1期细胞周期进程所必需的，抑制CDK2活性会导致细胞周期停滞。新的数据表明，TCDD诱导的肝再生抑制与依赖于AhR的CDK2抑制物p21Cip1的表达直接相关。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蛋白与DNA相互作用的初步分析表明，XRE结合所需的AhR的DNA结合域的缺失并不阻止受体与NC-XRE的结合。因此，我们假设AhR-NC-XRE的结合和功能与通过XRE的信号有根本的不同。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，这项提案将检查两个特定的目标，旨在将这种新的NC-XRE调节复合体描述为未来探索AhR-KLF6信号的研究平台。具体目的1描述了NC-XRE蛋白-DNA复合体的详细表征和功能分析。特定目标2将使用CHIP-SEQ和RNA-SEQ来提供由AhR/KLF6转录复合体调控的功能性NC-XRE位点的全球基因组评估。这一新的AhR复合体的发现代表着我们对AhR生物学和TCDD毒性的理解的范式转变。