Molecular Analysis of Transcriptional Repression

描述（由申请人提供）：该项目的长期目标是确定短距离转录抑制因子的分子机制。在许多生物学过程中，阻遏物在基因表达的控制中起着核心作用。虽然镇压已成为一个激烈的调查主题，我们缺乏一个机械的了解如何镇压是利用体内，特别是在后生动物，其发展计划涉及相当复杂的转录调控。该项目将利用果蝇的独特优势，应用生物化学和遗传学方法，从分子上了解胚胎中短程阻遏物的作用。 首先，我们将描述的CtBP依赖和独立的抑制活动的果蝇Knirps因子使用转基因胚胎试验，研究在生理背景下的镇压，并延长这项工作与细胞培养试验。其次，我们将在胚胎和细胞培养试验中表征野生型CtBP辅因子的阻遏活性，以鉴定活性所需的残基，并确定启动子背景对活性的影响。第三，我们将分析胚胎中含有明确的激活子和阻遏子位点的转录开关，以确定内源性Knirps和Giant阻遏子有效阻遏的要求。第四，我们将通过从胚胎提取物中纯化这些蛋白质来鉴定Knirps和CtBP相关因子，并测试组蛋白脱乙酰酶的存在。第五，我们将使用染色质免疫沉淀技术，研究胚胎和细胞培养物中基因蛋白质复合物在阻遏前后的性质。 转录阻遏物参与与人类疾病如癌症和发育异常相关的许多调节过程，因此阻遏物活性的知识将有助于治疗干预措施的设计。生物信息学的解释推定的调控区域也将受益于更深入地了解影响阻遏物活性的因素。

DESCRIPTION (provided by applicant): This project's long-term aim is to identify the molecular mechanisms of short-range transcriptional repressors. Repressors play a central role in the control of gene expression in many biological processes. While repression has become a topic of intense investigation, we lack a mechanistic understanding of how repression is utilized in vivo, especially in metazoans, whose developmental programs involve transcriptional regulation of considerable complexity. The project will use the unique advantages of Drosophila to apply biochemical and genetic approaches to gain a molecular understanding of the action of short-range repressors in the embryo. First, we will characterize the CtBP-dependent and -independent repression activities of the Drosophila Knirps factor using transgenic embryo assays to study repression in the physiological context, and extend this work with cell culture assays. Second, we will characterize the repression activity of wild-type CtBP cofactor in embryo and ceil-culture assays to identify residues required for activity, and to determine the effect of promoter context on activity. Third, we will analyze in the embryo transcriptional switches containing well-defined activator and repressor sites to identify the requirements for effective repression by endogenous Knirps and Giant repressors. Fourth, we will identify Knirps- and CtBP-associated factors by purifying these proteins from embryo extracts, and test for the presence of histone deacetylases. Fifth, we will examine the nature of protein complexes at genes in embryos and cell culture before and after repression, using chromatin immunoprecipitation techniques. Transcriptional repressors are involved in host of regulatory processes linked to human diseases such as cancer and developmental abnormalities, therefore knowledge of repressor activity will facilitate design of therapeutic interventions. Bioinformatic interpretation of putative regulatory regions will also benefit from a deeper understanding of factors affecting repressor activity.