AHR Signaling in Mammalian and Non-Mammalian Models

说明(申请人提供)：2，3，7，8-四氯二苯并-对二恶英(TCDD)和多环芳烃是普遍存在的环境污染物，对人体健康有不良影响。这些化合物通过激活芳烃受体(AHR)而产生毒性。AHR还具有调节血管发育、免疫功能和细胞生长的生理作用，这表明它在人类疾病中发挥了作用。要了解这些不同的功能和AHR在人类疾病中的可能作用，重要的是确定AHR信号是如何调节的。AHR信号的负调控机制还知之甚少。AHR转录激活功能的抑制因子--AHR抑制因子(AHRR)已经被发现，但它在调节AHR信号转导中的作用仍然是个谜，AHR途径之外的可能功能几乎被忽略了。最近的流行病学研究发现，AHRR基因Pro185和Ala185的多态与人类生殖功能障碍有关，AHRR基因可能是人类的肿瘤抑制基因。然而，有关AHRR及其变体的生化和功能特征的基本问题仍然没有解决，阻碍了对其在人类疾病中的作用的充分了解。这些研究将利用已建立的脊椎动物模型系统(人类细胞和斑马鱼胚胎)来确定AHRR及其多态变体的转录因子特异性和基因选择性，AHRR抑制AHR和低氧诱导因子(HIF)的机制，以及AHRR在调节胚胎发育和体内对TCDD和低氧的反应中的作用。中心假说是AHRR通过转录抑制机制来调节几种转录因子的转录活性。在目标1中，我们将检验人类AHRR可以抑制多种结构性活性和条件性转录因子的假设。我们还将在人类细胞系中进行功能获得(Tet-on)和功能丧失(SiRNA)实验，以确定AHRR抑制AHR和HIF靶基因的特异性。在目标2中，我们将使用AHRR突变体、共免疫共沉淀法和染色质免疫共沉淀法以及Arnt缺陷细胞来验证几个假设：a)人类AHRR及其变体通过转录抑制机制发挥作用；b)AHRR抑制是Arnt依赖的；以及c)AHRR通过与AHR和HIF或它们的转录复合体结合来抑制。在目标3中，我们将在强大的斑马鱼胚胎模型系统中研究AHRR的体内功能。我们将为两个斑马鱼AHRR近亲各自产生胚系零突变体。通过这些AHRR缺失的FISH结合AHRR过表达的实验，我们将评估AHRR的体内转录因子和基因靶标特异性，反式抑制在作用机制中的作用，以及AHRR在胚胎发育和对AHR和HIF激活剂的反应中的作用。

DESCRIPTION (provided by applicant): 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polynuclear aromatic hydrocarbons are ubiquitous environmental contaminants with adverse effects on human health. These compounds cause toxicity by activating the aryl hydrocarbon receptor (AHR). The AHR also has physiological roles regulating vascular development, immune function, and cell growth, suggesting a role in human disease. To understand these diverse functions and the possible role of AHR in human disease, it is important to determine how AHR signaling is regulated. The negative regulation of AHR signaling is poorly understood. An inhibitor of AHR transcriptional activation function, AHR repressor (AHRR), has been identified, but its role in regulating AHR signaling remains enigmatic, and possible functions beyond the AHR pathway have been virtually ignored. Recent epidemiological studies have linked AHRR Pro185 and Ala185 polymorphisms to human reproductive disorders and AHRR has been identified as a likely tumor suppressor gene in humans. However, fundamental questions concerning the biochemical and functional characteristics of the AHRR and its variants remain unresolved, preventing a full understanding of its roles in human disease. The studies proposed here will utilize established vertebrate model systems (human cells and zebrafish embryos) to determine the transcription factor specificity and gene selectivity of AHRR and its polymorphic variants, the mechanism by which AHRR represses AHR and hypoxia inducible factors (HIFs), and the role of AHRR in regulating embryonic development and the response to TCDD and hypoxia in vivo. The central hypothesis is that AHRR acts through a transrepression mechanism to regulate the transcriptional activity of several transcription factors. In Aim 1, we will test the hypothesis that human AHRR can repress a variety of constitutively active and conditional transcription factors. We will also use gain-of-function (Tet-On) and loss-of- function (siRNA) experiments in human cell lines to determine the AHR and HIF target gene specificity of repression by AHRR. In Aim 2, we will use AHRR mutants, co-immunoprecipitation and chromatin immunoprecipitation assays, and ARNT-deficient cells to test several hypotheses: a) that the human AHRR and its variants act by a transrepression mechanism; b) that AHRR repression is ARNT-dependent; and c) that AHRR represses by binding to AHR and HIF or their transcription complexes. In Aim 3, we will investigate the in vivo function of AHRR in the powerful zebrafish embryo model system. We will generate germ-line null mutants for each of the two zebrafish AHRR paralogs. With these AHRR-null fish combined with AHRR overexpression experiments, we will assess the in vivo transcription factor and gene target specificity of AHRRs, the role of transrepression in the mechanism of action, and the roles of AHRR in embryonic development and the response to activators of AHR and HIF.