A Novel Paradigm for Aryl Hydrocarbon Receptor Signaling

芳基烃受体信号传导的新范例

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：芳基烃受体（AHR）是异种生物毒性的介体，在暴露于2,3,3,7,8-tetrachlorodibenzo-p-Dioxin（TCDD，dioxin，dioxin）和相关环境观料剂之后，最能承认有害的人类健康影响。从机械上讲，AHR被称为配体激活的转录因子，该转录因子与规范的异种生物反应元件（XRE）结合，与其异二聚化伴侣AHR核转运剂（ARNT）蛋白相关。然而，在近年来确定的AHR靶基因曲目中，许多人缺乏明确定义的XRE，强调了AHR介导的基因表达似乎涉及与涉及XRE的特征良好的过程不同的其他机制。我们最近确定了一个非愿意XRE（NC-XRE），该XRE（NC-XRE）与新型的DNA结合伴侣（Krüppel样因子6（KLF6）肿瘤抑制剂）一起募集了AHR。 DNA结合和功能研究证实，AHR-KLF6复合物的NC-XRE结合与ARNT蛋白无关。在肝脏再生研究中揭示了TCDD诱导的AHR-KLF6活性的毒理学影响。我们以前的工作表明，TCDD通过抑制G1相依赖性激酶（CDK）活性，特别是CDK2来抑制损伤后正常肝脏再生。 CDK2活性增加对于G1相细胞周期进程是必需的，并且CDK2活性的抑制会导致细胞周期停滞。新数据表明，TCDD诱导的肝脏再生抑制直接与CDK2抑制剂P21CIP1的AHR依赖性表达有关。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结合结构域的缺失并不能阻止受体与NC-XRE的结合。因此，我们假设AHR-NC-XRE结合和功能与通过XRE的信号传导根本不同。为了了解我们的长期目标以了解AHR活性的病理生理学，该提案将研究两个特定的目标，旨在将这种新型的NC-XRE调节络合物表征为旨在探索AHR-KLF6信号传导的未来研究的平台。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。具体目标2将使用ChIP-Seq和RNA-Seq提供对由AHR/KLF6转录复合物调节的功能NC-XRE位点的全局基因组评估。这种新颖的AHR复合物的识别代表了我们对AHR生物学和TCDD毒性的理解的范式转变。