NGF-INDUCED DEREPRESSION OF PERIPHERIN GENE EXPRESSION

神经生长诱导PC12细胞分化的实验研究 因子(NGF)是研究外部刺激如何引起的模型系统 终末分化的细胞。为了了解这一机制 通过NGF触发神经元分化，我们正在研究 神经特异性中间丝--外周蛋白基因的调控 当NGF时转录诱导的蛋白质具有延迟的时间进程 刺激PC12细胞分化为神经元。以前我们有过 外周蛋白启动子中明确的近端和远端阳性元件 它们是NGF诱导所必需的，也是负调控的 具有抑制外周血凝素活性功能的元素(NRE) 在未分化和非神经细胞中。DNA迁移率变化分析 证明与NRE结合的蛋白质复合体在 神经生长因子诱导神经元分化。我们已经提出了一个两步模型 NGF对外周蛋白的诱导，其中抑制物从 NRE上的蛋白质复合体，加上来自NRE的积极信号 远端正向元件，导致基因的去阻遏。这 提案将重点关注抑制物对外周蛋白的负调控 与NRE结合的蛋白质。一个主要目标将是了解如何 NGF介导的机制缓解分化中外周蛋白的抑制 PC12细胞。阻遏蛋白从细胞内释放出来的假说 NGR介导的分化过程中的NRE将直接由DNA进行测试 亲和沉淀法。阻遏蛋白将被提纯 和/或克隆以确定NGF如何修改其表达和 活动。此外，拥有克隆的阻遏蛋白将 促进使用体外转录来定义水平的研究 阻遏蛋白的作用，以及结构研究识别 蛋白质的抑制子结构域。这些实验将进一步推动我们的 对负转录调控机制的理解。最后， NRE突变对外周血细胞表达模式的影响 转基因小鼠中的启动子-LacZ融合基因将预示体内的 NRE在神经元特异性外周控制中的重要性 表情。了解NGF诱导的机制 PC12细胞中神经特异性基因的表达可能提示 触发相关肿瘤细胞的末端分化，如 神经母细胞瘤。

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.