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）： 角质形成细胞控制其对多环芳族的致癌反应 碳氢化合物（PAHS）和相关环境药物，包括 2,3,7,8-四氯迪本佐-P-二恶英（TCDD）是细胞色素的表达 P4501A1（CYP1A1）。 该建议探讨了我们的分子基础 发现自发永生的大鼠表皮细胞沉默 CYP1A1和1B1的表达，从而对PAH毒性不敏感。 他们已经确定了存在的新型负调控DNA元素（NERD） 啮齿动物CYP1A1基因的上游，该基因沉默和诱导 CYP1A1基因表达。 DNA结合分析显示核蛋白 来自大鼠，小鼠，人和其他物种的细胞中的因素，这些因素 专门结合到书呆子元素。 他们假设这种沉默 永生的角质形成细胞中的CYP1A1基因是由 书呆子结合蛋白和/或ITS的横向修饰 与这些细胞中存在的另一个因素相互作用。 存在 所有检查的细胞中类似的书呆子结合蛋白，包括几个 人的起源表明，基因表达的类似沉默可以 也发生在其他物种和组织中。 他们建议描述 书呆子元素和书呆子结合蛋白，重点是 大鼠和人类细胞。 野生型和突变的书呆子寡核苷酸将是 用于检查DNA序列决定因素的DNA结合所必需的 书呆子特定因素（使用凝胶延迟分析）和书呆子 沉默活性（使用瞬态转染分析）。 竞争性凝胶 延迟分析实验将用于确定程度 书呆子元素和先前确定的相似性或差异 消音元素。 书呆子结合蛋白将使用 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 在大鼠表皮角质形成细胞中将纯化书呆的结合蛋白 结合常规色谱和磁DNA 识别位点亲和力结合技术和纯化的蛋白质 测序。 纯化和部分纯化的书呆子结合蛋白将是 用于在小鼠中开发单克隆抗体。 结果将提供 理解消音器作用机制的基础，并促进 书呆子结合因子的未来克隆和表征，检查 它负调节基因表达的机制 组织和物种特异性表达，并确定 它调节的基因。