SILENCING OF CYP1A1 GENE EXPRESSION IN KERATINOCYTES

角质形成细胞中 CYP1A1 基因表达的沉默

• 批准号: 2713581
• 负责人: MICHAEL STEVEN DENISON
• 金额: $ 17.65万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2713581
• 关键词: DNA binding protein; DNA footprinting; carbopolycyclic compound; cell transformation; crosslink; cytochrome P450; dioxins; environmental toxicology; gene expression; gene induction /repression; genetic regulatory element; keratinocyte; laboratory mouse; laboratory rat; monoclonal antibody; protein purification; southern blotting; western blottings

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.

描述（改编自申请人的摘要）： 的主要特征 角质形成细胞控制其对多环芳香族化合物的致癌反应 碳氢化合物 (PAH) 和相关环境物质，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（CYP1A1）。 该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 CYP1A1 和 1B1 的表达，从而对 PAH 毒性变得不敏感。 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，沉默组成型和诱导型 CYP1A1 基因表达。 DNA结合分析揭示了核蛋白 存在于大鼠、小鼠、人类和其他物种细胞中的因子 特异性结合 NeRD 元件。 他们假设沉默 永生化角质形成细胞中的 CYP1A1 基因由以下因素介导 NeRD 结合蛋白和/或其翻译后修饰 与这些细胞中存在的另一种因子相互作用。 的存在 所有检查的细胞中都有类似的 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以 也发生在其他物种和组织中。 他们建议表征 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 用于检查 DNA 结合所需的 DNA 序列决定因素 NeRD 特定因素（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 结合蛋白将使用以下方法进行表征 DNA 足迹分析、核苷酸修饰的组合 技术、紫外线和化学交联以及西南印迹。 这 NeRD 结合蛋白将从大鼠表皮角质形成细胞中纯化 结合使用传统色谱和磁性 DNA 识别位点亲和结合技术和纯化的蛋白质 已测序。 纯化和部分纯化的 NeRD 结合蛋白将 用于开发小鼠单克隆抗体。 结果将提供一个 为理解消音器作用机制奠定基础，并促进 NeRD 结合因子的未来克隆和表征、检查 它负向调节基因表达的机制， 组织和物种特异性表达并确定其谱 它所调节的基因。