AHR signaling in Mammalian and Non-Mammalian Models

描述（由申请人提供）：芳烃受体（AHR）是碱基-螺旋-环螺旋/Per-ARNT-Sim （bHLH-PAS）家族中的配体激活转录因子。卤代芳烃，如2,3,7,8-四氯二苯并-对二恶英（TCDD）通过AHR作用，导致哺乳动物、鱼类和其他脊椎动物体内外生代谢酶的表达改变和其他导致毒性的变化。然而，人们对AHR的正常功能以及TCDD和相关化学物质通过AHR引起毒性的确切机制知之甚少。我们和其他人最近鉴定了脊椎动物AHR家族的三个成员：AHR1， AHR2和AHR阻遏因子（AHRR），我们发现鱼类中还有额外的AHR多样性，每个物种多达5个基因。我们建议在脊椎动物模型系统（鱼、小鼠细胞、人类细胞）中进行一套综合研究，利用每种模型的独特特征，更好地了解AHR和AHRR蛋白的功能及其在二恶英毒性和正常发育中的作用。1)我们将在斑马鱼（Danio rerio）胚胎中使用基于morpholino反义技术的RNA敲除策略来验证AHR1， AHR2和AHRR在发育和二恶英毒性中具有不同作用的假设。这些研究将利用斑马鱼的外部发育和透明胚胎。阻断ahr1、AHR2和AHRR表达对二恶英发育毒性敏感性和基因表达的影响也将被确定。2)我们将检验来自鱼类的多个AHR经历了亚功能化的假设，因此可以用来区分人类AHR的多种功能。研究人员将在体外对medaka （Oryzias latipes）和河豚（Fugu rubripes）的多个AHR进行表征，并在AHR缺陷的小鼠细胞TCDD中表达，以确定单个鱼类AHR是否调节由小鼠AHR控制的不同基因亚群。利用河豚紧凑的基因组，我们还将利用表达GFP报告结构的斑马鱼胚胎进行AHR启动子和调控元件的体内定位。3)我们将描述人类AHRR的功能、表达和调控，检查AHR激动剂的诱导性、结构-活性和剂量-反应关系，以及涉及AHRR诱导的调控元件。我们将检验AHR抑制AHR功能是通过竞争ARNT和/或AHR反应元件发生的假设。这些研究将更好地了解人类AHRR在调节二恶英效应中的可能作用。

DESCRIPTION (provided by applicant): The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor in the basic-helix-loophelix/Per-ARNT-Sim (bHLH-PAS) family. Halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) act through the AHR to cause altered expression of xenobiotic-metabolizing enzymes and other changes leading to toxicity in mammals, fish, and other vertebrate animals. However, the normal functions of the AHR and the exact mechanisms by which TCDD and related chemicals act through the AHR to cause toxicity are poorly understood. We and others have recently characterized three members of the vertebrate AHR family: AHR1, AHR2, and AHR Repressor (AHRR), and we have found that there is additional AHR diversity in fish, with up to 5 genes per species. We propose an integrated set of studies in vertebrate model systems (fish, mouse cells, human cells) that will take advantage of the unique features of each model to better understand the function of AHR and AHRR proteins and their roles in dioxin toxicity and normal development. 1) We will use an RNA knock-down strategy employing morpholino anti-sense technology in zebrafish (Danio rerio) embryos to test the hypothesis that AHR1, AHR2, and AHRR have distinct roles during development and in dioxin toxicity. These studies will take advantage of the external development and transparent embryos of zebrafish. The effect of blocking AHR 1, AHR2, and AHRR expression on sensitivity to dioxin developmental toxicity and gene expression will also be determined. 2) We will test the hypothesis that multiple AHRs from fish have undergone subfunctionalization and can therefore be used to distinguish multiple functions of the human AHR. Multiple AHRs from medaka (Oryzias latipes) and pufferfish (Fugu rubripes) will be characterized in vitro and expressed in AHR-deficient mouse cells ¿ TCDD to determine whether individual fish AHRs regulate distinct subsets of genes controlled by the murine AHR. Taking advantage of the compact genome of pufferfish, we will also conduct in vivo mapping of AHR promoters and regulatory elements using zebrafish embryos expressing GFP reporter constructs. 3) We will characterize the function, expression, and regulation of the human AHRR, examining inducibility by AHR agonists, structure-activity and dose-response relationships, and regulatory elements involved in AHRR induction. We will test the hypotheses that AHRR inhibition of AHR function occurs through competition for ARNT and/or AHR response elements. These studies will provide a better understanding of the possible role of human AHRR in modulating dioxin effects.