MOLECULAR ANALYSIS OF TRANSCRIPTIONAL REPRESSION

转录抑制的分子分析

• 批准号: 2857346
• 负责人: David N Arnosti
• 金额: $ 23.56万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2857346
• 关键词: Drosophilidae; arthropod genetics; gene induction /repression; genetic transcription; in situ hybridization; protein binding; protein purification; protein structure function; site directed mutagenesis; transcription factor; yeast two hybrid system

The long-term objective of this project is to characterize the molecular mechanisms by which eukaryotic transcriptional repressors control gene expression. Transcriptional regulation underlies crucial aspects of many biological processes, including development, immune responses, pathological states such as cancer, and the physiology of homeostasis and adaptation. While transcriptional activation in eukaryotes is beginning to be understood in mechanistic terms, negative regulation of genes by repressors is less well understood. This study will use complementary biochemical and genetic approaches to determine the mechanism by which the Drosophila knirps repressor protein regulates transcription. Drosophila is a good model system for such studies because of the extensive genetics, the sophisticated tools available for generating transgenic animals, and the established systems for in vitro transcription. The knirps repressor, a member of the nuclear receptor superfamily of transcription factors, plays an important role in early embryogenesis. Initial work indicates that knirps can repress enhancers and basal promoters from a short range. This activity is characteristic of an important family of repressors in Drosophila, and likely represents a common mode of repression in other eukaryotes. We will define regions of knirps protein vital for repression in vivo using two novel Drosophila embryo assays. This genetic information will complement in vitro studies, including in vitro transcriptional assays, which will provide mechanistic insights into knirps activity, and direct binding studies, which will identify proteins which may interact with knirps to mediate repression. We will also identify knirps-interacting proteins using a yeast two-hybrid screen, and evaluate candidate target proteins using genetic and biochemical information developed from the other assays. It is clear that the basic circuitry regulating genes in Drosophila and vertebrates is highly conserved. Analysis of repression is crucial for understanding biological processes ranging from development to cancer. For instance, transcriptional repressors such as the retinoblastoma, Wilm's tumor, and MDM2 proteins are important factors in human cancers. This project will employ a multidisciplinary approach to provide detailed mechanistic information about this important form of gene regulation.

该项目的长期目标是表征 真核转录抑制因子调控基因的机制 表情 转录调控是生物学的关键方面， 许多生物过程，包括发育，免疫反应， 病理状态，如癌症，以及体内平衡的生理学 和适应。 虽然真核生物中的转录激活是 开始被机械地理解， 基因被阻遏物抑制的机制还不太清楚。 这项研究将使用互补的生化和遗传方法， 确定果蝇knirps阻遏蛋白的机制 调节转录。 果蝇是一个很好的模型系统， 因为广泛的遗传学，复杂的工具， 可用于产生转基因动物，并且已建立的系统 用于体外转录。 knirps阻遏物， 核受体超家族转录因子，发挥作用， 在早期胚胎发育中的重要作用。 初步工作表明， knirp可以从短范围抑制增强子和基础启动子。 这种活性是一个重要的阻遏物家族的特征， 果蝇，并可能代表了一种共同的模式，抑制在其他 真核生物 我们将定义knirps蛋白在体内抑制的重要区域 使用两种新的果蝇胚胎试验。 这些基因信息将 补充体外研究，包括体外转录测定， 这将提供对knirps活动的机械见解，并直接 结合研究，这将确定蛋白质可能相互作用， 以调解镇压。 我们还将识别knirps-互动 利用酵母双杂交筛选蛋白质，并评估候选靶标 蛋白质利用遗传和生物化学信息， 其他化验。 很明显，果蝇和果蝇中调节基因的基本回路 脊椎动物是高度保守的。 对压抑的分析对于 了解从发育到癌症的生物学过程。 例如，转录抑制因子如视网膜母细胞瘤， Wilm肿瘤和MDM 2蛋白是人类癌症的重要因素。 该项目将采用多学科方法， 关于这种重要基因形式的详细机制信息 调控