CONVERGENCE OF TCDD GENE ACTIVATION WITH OTHER PATHWAYS

描述(摘自申请者摘要)：Ah受体(AHR)是一种配体 激活的转录因子，结合配体，如 2，3，7，8-四氯二苯并对二恶英(TCDD)，被认为介导了许多 TCDD的剧毒和致癌作用。AHR及其DNA 结合伙伴ARNT(ah受体核转运体)识别特异性 DNA序列，称为二恶英反应元件(DRE)，导致 基因转录的激活。AHR和ARNT都是 碱性/螺旋-环-螺旋蛋白。碱性/螺旋-环-螺旋蛋白是 以形成同源或异源二聚体的能力为特征，接触DNA 通过它们的基本结构域并形成各种DNA结合物种，这些物种 识别不同的DNA序列并调节特定的基因集合。 与普遍接受的TCDD发挥其不利作用的理论相反 通过对DRE反应基因的调控，我们认为TCDD 行动包括AHR的核本地化，ARNT的二聚化和 随后将Arnt从其他基因的调节区中移除。 这一假说表明，在缺乏AHR的情况下，ARNT调节 TCDD的存在会对这种调节产生不利影响 和AHR。我们的初步数据表明，Arnt可以形成多个 合伙企业。Arnt同源二聚体识别CACGTG序列， 参与调控的Myc/Max异源二聚体的识别位点 细胞的增殖和分化。我们将使用Myc/Max基因 调控作为AHR非依赖性ARNT基因调控的模型。首先，我们 将确定Arnt同源二聚体是否识别CACGTG位点 具有生物学意义所需的高亲和力 与Myc和Max的结合亲和力。然后我们将使用瞬变 转染试验确定ARNT同源二聚体的形成是否可以 抑制生物过程，如诱导细胞色素P4501A1和 MYC诱导细胞转化。要确定为什么AHR ARNT 识别不同的DNA半位点，我们将进行位点定向 对它们的DNA结合区进行突变，然后进行足迹分析。 最后，我们将使用无细胞转录来鉴定相互作用 促进AHR和ARNT基因调控的转录因子 确定体内发现的不同AHR和ARNT比率是否会影响 AHR和ARNT的异二聚化能力及其差异 基因调控。这项提议的广泛、长期目标是 了解AHR和ARNT如何调节以及规定 TCDD毒性和致癌性的组织特异性效应。

DESCRIPTION (from Applicant's Abstract): The Ah receptor (AHR) is a ligand activated transcription factor that binds ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and is thought to mediate many of the highly toxic and carcinogenic actions of TCDD. The AHR and its DNA binding partner ARNT (Ah receptor nuclear translocator) recognize specific DNA sequences, referred to as dioxin responsive element (DREs) resulting in the activation of gene transcription. Both the AHR and ARNT are basic/helix-loop-helix proteins. Basic/helix-loop-helix proteins are characterized by their ability to form homo- or hetero-dimers, contact DNA via their basic domains and form a variety of DNA binding species that recognize distinct DNA sequences and regulate specific sets of genes. Contrary to the generally accepted theory that TCDD exerts its adverse affects through the regulation of DRE responsive genes, we propose that TCDD actions include nuclear localization of the AHR, dimerization of ARNT and the subsequent removal of ARNT from the regulatory regions of other genes. This hypothesis suggests that ARNT, in the absence of the AHR regulates genes and that this regulation is adversely affected by the presence of TCDD and the AHR. Our preliminary data has shown that ARNT can form multiple partnerships. The ARNT homodimer recognizes the CACGTG sequence, the recognization site of the Myc/Max heterodimer involved in regulating cellular proliferation and differentiation. We will use Myc/Max gene regulation as a model of AHR-independent ARNT gene regulation. First, we will determine whether the ARNT homodimer will recognize the CACGTG site with high affinity required for biological significance and compare this binding affinity to that of Myc and Max. Then we will use transient transfection assays to determine whether theARNT homodimer formation may repress biological processes such as the induction of cytochrome P4501A1 and Myc induced cellular transformation. To determine why the AHR ARNT recognize different DNA half-sites, we will perform site-directed mutagenesis of their DNA binding regions followed by footprinting analysis. Finally, we will use cell-free transcription to identify the interacting transcription factors that facilitate AHR and ARNT gene regulation and determine whether different AHR and ARNT ratios found in vivo may affect the ability of the AHR and ARNT to heterodimerize and resulting differential gene regulation. The broad, long-term objectives of this proposal are to understand how the AHR and ARNT regulate and the mechanisms that dictate the tissue-specific effects of TCDD toxicity and carcinogenicity.