NGF-INDUCED DEREPRESSION OF PERIPHERIN GENE EXPRESSION

NGF 诱导的外周蛋白基因表达去抑制

• 批准号: 3417893
• 负责人: MARY A THOMPSON
• 金额: $ 22.95万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3417893
• 关键词: PC12 cells; binding proteins; cell differentiation; gene expression; genetic promoter element; genetic regulation; genetic transcription; genetically modified animals; immunoprecipitation; laboratory mouse; laboratory rat; molecular cloning; nerve /myelin protein; neurotrophic factors; protein structure function

The induction of neuronal differentiation in PC12 cells by nerve growth factor (NGF) is a model system for studying how an external stimulus causes a cell to terminally differentiate. In order to understand the mechanism by which NGF triggers neuronal differentiation, we are studying the regulation of the peripherin gene, a neural specific intermediate filament protein which is transcriptionally induced with a late time course when NGF stimulates PC12 cells to differentiate into neurons. Previously we have defined proximal and distal positive elements in the peripherin promoter which are necessary for induction by NGF, as well as a negative regulatory element (NRE) which has a functional role in repressing peripherin activity in undifferentiated and non-neuronal cells. DNA mobility shift assays demonstrate that the protein complex binding to the NRE is altered during NGF-induced neuronal differentiation. We have proposed a two-step model of induction of peripherin-by NGF in which dissociation of a repressor from the protein complex at the NRE, coupled with a positive signal from the distal positive element, results in derepression of the gene. This proposal will focus on the negative regulation of peripherin by repressor proteins binding to the NRE. A major goal will be to understand how NGF-mediated mechanisms relieve repression of peripherin in differentiated PC12 cells. The hypothesis that a repressor protein is released from the NRE during NGR-mediated differentiation will be directly tested by the DNA affinity precipitation assay. The repressor protein will be purified and/or cloned in order to determine how NGF modifies its expression and activity. In addition, possession of the cloned repressor protein will facilitate studies using in vitro transcription to define the level of action of the repressor protein, as well as structural studies identifying the repressor domain of the protein. These experiments will further our understanding of mechanisms of negative transcriptional control. Finally, the effect of mutation at the NRE on the expression pattern of a peripherin promoter-lacZ fusion gene in transgenic mice will indicate the in vivo importance of the NRE for the neuronal-specific control of peripherin expression. Understanding the mechanism by which NGF induces neural-specific gene expression in PC12 cells may suggest approaches to trigger terminal differentiation in related tumor cells such as neuroblastoma.

神经生长对PC 12细胞神经元分化的诱导作用 神经生长因子（NGF）是研究外部刺激如何引起 一个细胞最终分化。 为了理解这个机制 神经生长因子触发神经元分化的机制，我们正在研究 神经特异性中间丝外周蛋白基因的调控 一种蛋白质，当NGF 刺激PC 12细胞分化为神经元。 以前我们有 外周蛋白启动子中定义的近端和远端阳性元件 这是必要的诱导神经生长因子，以及负调节 在抑制外周蛋白活性中具有功能作用的元件（NRE 在未分化和非神经元细胞中。 DNA迁移率变动分析 证明与NRE结合的蛋白质复合物在 NGF诱导的神经元分化。 我们提出了两步模型， 通过NGF诱导外周蛋白，其中阻遏物从 NRE上的蛋白质复合物，再加上来自NRE的阳性信号， 远端阳性元件，导致基因的去阻遏。 这 一项提案将集中在阻遏物对外周蛋白的负调节上 与NRE结合的蛋白质。 一个主要的目标将是了解如何 神经生长因子介导的机制缓解了分化型乳腺癌外周蛋白的抑制 PC 12细胞。 阻遏蛋白从细胞中释放出来的假说， NGR介导的分化过程中的NRE将通过DNA直接测试， 亲和沉淀测定。 将纯化阻遏蛋白 和/或克隆以确定NGF如何修饰其表达， 活动 此外，拥有克隆的阻遏蛋白将 促进使用体外转录的研究，以确定 阻遏蛋白的作用，以及结构研究， 蛋白质的阻遏物结构域。 这些实验将进一步促进我们的 负转录控制机制的理解。 最后， NRE突变对外周蛋白表达模式的影响 启动子-lacZ融合基因在转基因小鼠中的表达将表明在体内 NRE对外周蛋白神经元特异性调控的重要性 表情 了解NGF诱导的机制 PC 12细胞中神经特异性基因表达可能提示了 引发相关肿瘤细胞的终末分化， 神经母细胞瘤