A Global Characterization of the Interaction Between the AhR and KLF-6

描述（由申请人提供）：芳烃受体（AhR）是一种碱性螺旋-环-螺旋转录因子，可由多种多环芳烃激活，包括原型配体和持久性环境污染物2，3，7，8-四氯二苯并-对-二恶英（二恶英，TCDD）。典型地，配体激活的AhR易位到细胞核，从分子伴侣解离，并与芳烃受体核转位器（Arnt）异二聚化，以通过顺式DNA基序GCGTG（称为异生素反应元件（XRE））转录调节靶基因。最近的研究表明，一些二恶英响应AhR靶基因缺乏XRE。我们最近发现AhR通过与Kruppel样因子6（KLF 6）的相互作用形成一种新的蛋白质复合物，KLF 6通过一种新的非共有XRE（NC-XRE）结合DNA。我们随后表明，KLF 6/AhR复合物调节一组新的AhR基因的表达，响应二恶英，通过结合NC-XRE。因此，我假设KLF 6/AhR异二聚体代表了一种新的调控复合物，通过与NC-XRE的结合对新的靶基因进行转录控制。为了研究这种新的蛋白质相互作用，我将使用体内染色质免疫沉淀下一代测序（ChIP-seq）实验，以确定该复合物在小鼠肝脏中的基因靶点，再加上全转录组鸟枪测序，以锚DNA结合KLF 6/AhR转录因子复合物的基因表达变化。这种方法将使我们能够描绘KLF 6/AhR靶基因中的功能性与非功能性NC-XRE。我将研究KLF 6/AhR蛋白质-蛋白质相互作用，并确定相互作用所需的蛋白质元件，并表征KLF 6/AhR结合所需的关键NC-XRE DNA基序元件。人们普遍认为，AhR是暴露于二恶英后产生毒性所必需的。然而，关于AhR驱动这种毒性发展的机制知之甚少。由于我们的数据表明NC-XRE代表了二恶英暴露后AhR的一种新型信号级联反应，这项工作将为AhR的二恶英毒性机制提供新的见解，同时为识别和治疗有害疾病提供新的预后和诊断工具。与TCDD暴露相关的健康影响。

DESCRIPTION (provided by applicant): The Aryl Hydrocarbon Receptor (AhR) is a basic helix-loop-helix transcription factor activated by diverse polycyclic aromatic hydrocarbons, including the prototypical ligand and persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin,TCDD). Canonically, the ligand activated AhR translocates to the nucleus, dissociates from chaperones, and heterodimerizes with the Aryl Hydrocarbon Receptor Nuclear Translocator (Arnt) to transcriptionally regulate target genes via the cis DNA motif GCGTG, termed the xenobiotic response element (XRE). Recent work has shown a number of dioxin responsive AhR target genes lack an XRE. We have recently discovered the AhR to form a novel protein complex through an interaction with Kruppel-like Factor 6 (KLF6), which binds DNA via a novel non-consensus XRE (NC-XRE). We have subsequently shown that the KLF6/AhR complex regulates expression of a novel set of AhR genes, in response to dioxin, via binding to the NC-XRE. In this light, I hypothesize the KLF6/AhR heterodimer represents a novel regulatory complex conferring transcriptional control on novel target genes via binding to the NC-XRE. To investigate this novel protein interaction I will use in vivo chromatin immunoprecipitation next generation sequencing (ChIP-seq) experiments to identify the gene targets of this complex in the mouse liver coupled with whole transcriptome shotgun sequencing to anchor DNA binding of the KLF6/AhR transcription factor complex to changes in gene expression. This method will allow us to delineate functional vs. non-functional NC-XREs in KLF6/AhR target genes. I will examine the KLF6/AhR protein-protein interaction and identify the protein elements necessary for the interaction and characterize the key NC-XRE DNA motif elements necessary for KLF6/AhR binding. It is widely accepted that the AhR is required for the development of toxicity following exposure to dioxin. However, little is known about the mechanisms of the AhR driving the development of this toxicity. As our data suggest the NC-XRE represents a novel signaling cascade for the AhR following dioxin exposure, this work will provide novel insight on the mechanisms of dioxin toxicity with respect to the AhR, while shedding light on novel prognostic and diagnostics tools for identifying and treating the deleterious health effects associated with TCDD exposure.