CONVERGENCE OF TCDD GENE ACTIVATION WITH OTHER PATHWAYS

描述（来自申请人的摘要）：Ah受体（AHR）是配体 激活的转录因子，其结合配体， 2，3，7，8-四氯二苯并对二恶英（TCDD），被认为是介导许多 TCDD的高毒性和致癌作用。 AHR及其DNA 结合伴侣ARNT（Ah受体核转运蛋白）识别特异性 DNA序列，被称为二恶英反应元件（DREs），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 其特征在于它们能够形成同源或异源二聚体，接触DNA 通过它们的基本结构域并形成各种DNA结合物质， 识别不同的DNA序列并调节特定的基因组。 与普遍接受的理论相反， 通过DRE反应基因的调节影响，我们建议TCDD 作用包括AHR的核定位，ARNT的二聚化， 随后从其他基因的调控区中去除ARNT。 这一假设表明，ARNT，在缺乏AHR调节 这种调节受到TCDD存在的不利影响 和AHR。 我们的初步数据表明，ARNT可以形成多个 伙伴关系。 ARNT同源二聚体识别CACGTG序列， Myc/Max异源二聚体的识别位点参与调节 细胞增殖和分化。 我们将使用Myc/Max基因 作为AHR非依赖性ARNT基因调控的模型。 一是 将决定ARNT同二聚体是否识别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。然后我们将使用瞬态 转染试验以确定ARNT同二聚体的形成是否 抑制生物过程，如细胞色素P4501 A1的诱导， Myc诱导细胞转化。 为了确定为什么AHR ARNT 识别不同的DNA半位点，我们将进行定点 诱变其DNA结合区，随后进行足迹分析。 最后，我们将使用无细胞转录来鉴定相互作用的 促进AHR和ARNT基因调节的转录因子， 确定体内发现的不同AHR和ARNT比率是否会影响 AHR和ARNT异源二聚化的能力和产生的差异 基因调控 这项建议的广泛和长期目标是 了解AHR和ARNT如何调节以及决定 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.