SILENCING OF CYP1A1 GENE EXPRESSION IN KERATINOCYTES

描述（摘自申请人摘要）： 角质形成细胞对多环芳烃致癌反应的调控 碳氢化合物（PAHs）和相关环境因子，包括 2，3，7，8-四氯二苯并对二恶英（TCDD），是细胞色素 P4501A1（CYP1A1）。 这项提案探讨了我们的分子基础， 发现自发永生化的大鼠表皮细胞沉默 CYP 1A 1和1B 1的表达，从而变得对PAH毒性不敏感。 他们发现了一种新的负调控DNA元件（NeRD）， 啮齿动物CYP 1A 1基因上游，该基因沉默组成型和诱导型 CYP 1A 1基因表达。 DNA结合分析显示， 存在于来自大鼠、小鼠、人和其他物种的细胞中的因子， 特异性结合NeRD元件。 他们假设，沉默的 永生化角质形成细胞中的CYP 1A 1基因由 NeRD结合蛋白的翻译后修饰和/或其 与这些细胞中存在的另一种因子相互作用。 的存在 在所有检查的细胞中，NeRD结合蛋白相似，包括几种 人类起源，表明类似的基因表达沉默可能 也发生在其他物种和组织中。 他们建议将 NeRD元件和NeRD结合蛋白，重点关注来自 大鼠和人类细胞。 野生型和突变型NeRD寡核苷酸将被 用于检查DNA结合所需的DNA序列决定簇， NeRD特异性因子（使用凝胶阻滞分析）以及NeRD 沉默活性（使用瞬时转染分析）。 竞争凝胶 延迟分析实验将用于确定 NeRD元件与先前识别的元件之间的相似性或差异 消音器元件 NeRD结合蛋白将使用 结合DNA足迹分析、核苷酸修饰 技术，UV和化学交联和西南印迹。 的 NeRD结合蛋白将从大鼠表皮角质形成细胞中纯化 使用常规层析和磁性DNA的组合， 识别位点亲和结合技术和纯化的蛋白 测序 纯化和部分纯化的NeRD结合蛋白将被纯化。 用于在小鼠中开发单克隆抗体。 结果将提供一个 了解消声器作用机理的基础， NeRD结合因子的未来克隆和表征，检查 在它负调节基因表达的机制中，它的 组织和物种特异性表达，并确定 它调控的基因

DESCRIPTION (Adapted from APPLICANT'S ABSTRACT): A principal feature of keratinocytes governing their carcinogenic response to polycyclic aromatic hydrocarbons (PAHs) and related environmental agents, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is the expression of cytochrome P4501A1 (CYP1A1). This proposal explores the molecular basis for our finding that spontaneously immortalized rat epidermal cells silence the expression of CYP1A1 and 1B1 and thereby become insensitive to PAH toxicity. They have identified a novel negative regulatory DNA element (NeRD) present upstream of the rodent CYP1A1 gene which silences constitutive and inducible CYP1A1 gene expression. DNA binding analysis revealed a nuclear protein factor present in cells from rat, mouse, human and other species which specifically binds to the NeRD element. They hypothesize that silencing of the CYP1A1 gene in immortalized keratinocytes is mediated by postranslational modification of the NeRD binding protein and/or its interaction with another factor present in these cells. The presence of similar NeRD-binding proteins in all cells examined, including several of human origin, suggests that a similar silencing of gene expression could occur in other species and tissues as well. They propose to characterize the NeRD element and NeRD-binding proteins, focusing on those proteins from rat and human cells. Wild-type and mutant NeRD oligonucleotides will be used to examine the DNA sequence determinants necessary for DNA binding of the NeRD specific factor (using gel retardation analysis) as well as NeRD silencing activity (using transient transfection analysis). Competitive gel retardation analysis experiments will be used to determine the degree of similarity or difference between the NeRD element and previously identified silencer elements. The NeRD binding protein(s) will be characterized using a combination of DNA footprinting analysis, nucleotide modification techniques, UV and chemical cross-linking and southwestern blotting. The NeRD binding protein(s) will be purified from rat epidermal keratinocytes using a combination of conventional chromatographic and magnetic DNA recognition site affinity binding techniques and the purified proteins sequenced. Purified and partially purified NeRD-binding proteins will be used to develop monoclonal antibodies in mice. The results will provide a basis for understanding mechanism of silencer action and facilitate the future cloning and characterization of the NeRD-binding factor, examination of the mechanisms by which it negatively regulates gene expression, its tissue- and species-specific expression and determining the spectrum of genes it regulates.