SILENCING OF CYP1A1 GENE EXPRESSION IN KERATINOCYTES

描述(改编自申请人的摘要)：主要特征 角质形成细胞调控其对多环芳烃的致癌反应 碳氢化合物(多环芳烃)和相关环境制剂，包括 2，3，7，8-四氯二苯并对二恶英(TCDD)，是细胞色素的表达 P4501A1(细胞色素P1A1)。这项提案探索了我们的 发现自发永生化的大鼠表皮细胞沉默了 表达CYP1A1和1b1，从而对多环芳烃的毒性不敏感。 他们发现了一种新的负调控DNA元件(NERD) 啮齿动物CYP1A1基因的上游，它使结构性和诱导性沉默 细胞色素P1A1基因的表达。DNA结合分析揭示了一种核蛋白 在大鼠、小鼠、人类和其他物种的细胞中存在的因子 专门绑定到书呆子元素。他们假设沉默的人 永生化角质形成细胞中的细胞色素P1A1基因由 NERD结合蛋白和/或其基因的翻译后修饰 与这些细胞中存在的另一种因子相互作用。.的存在 所有细胞中类似的NERD结合蛋白，包括几个 人类起源，表明类似的基因表达沉默可能 在其他物种和组织中也存在。他们建议将 NERD元件和NERD结合蛋白，重点是来自 老鼠和人类细胞。野生型和突变型NERD寡核苷酸将 用于检查DNA结合所需的DNA序列决定因素 NERD特定因子(使用凝胶延迟分析)以及NERD 沉默活性(使用瞬时转染分析)。竞争性凝胶 迟滞分析实验将被用来确定 书呆子元素与之前确定的元素之间的相似或不同 消音器元件。书呆子结合蛋白(S)将用 DNA足迹分析、核苷酸修饰的组合 技术，紫外线和化学交联和西南印迹。这个 NERD结合蛋白(S)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及其纯化蛋白 已排序。纯化和部分纯化的NERD结合蛋白将是 用于在小鼠身上开发克隆抗体。结果将提供一个 了解消声器作用机理的基础，并有助于 未来NERD结合因子的克隆和鉴定 在它负性调节基因表达的机制中，它的 组织和物种特异性表达和测定光谱 它所调控的基因。

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.