Molecular Analysis of Transcriptional Repression

描述(由申请人提供)：转录抑制在发育和疾病过程中对基因表达的调节起着关键作用。为了了解动物中保守的调控过程，我们确定了转录抑制蛋白的分子工作方式和生理相关性，这些蛋白在果蝇的发育中发挥关键作用。Knirps是偶数跳过基因的关键调控因子，通过短距离机制作用于不同的增强子元件。我们已经发现Knirps和相关的抑制因子是如何通过不同的抑制机制发挥作用的，涉及进化保守的CtBP辅阻遏子蛋白和组蛋白去乙酰基酶Rpd3，我们对结合这些抑制因子的顺式调控元件的设计有了新的见解。我们已经确定了“顺式调控语法”的关键方面，该语法预测了抑制蛋白如何在定义的调控元件上发挥作用，为内源性增强子的数学和生物信息学分析奠定了基础。这项工作将使我们能够将来自果蝇的信息应用于后生动物顺式调控元件的设计和进化的一般分析。1.我们将定量地绘制由转录抑制因子控制的基因的调控表面，并使用基于势函数的数学模型来预测新型增强子的调控特性。2.我们将利用染色质免疫沉淀来阐明短程和长程抑制基因的机制，以确定与胚胎报告基因抑制相关的分子事件。3.我们将评估我们发现的与Knirps抑制因子复合体相关的辅助抑制因子的功能，特别是进化保守的Groucho辅助抑制因子，它在短程抑制中的作用以前没有被认识到。4.我们将通过全动物实验确定CtBP辅阻遏子的保守残基和剪接变异体的生理意义，以确定这些蛋白活性的生物学意义。这些目标结合了经验和模型的努力，以获得对转录抑制的“自下而上”的理解。我们的长期目标是了解转录控制蛋白和DNA调控序列的分子活性，它们是发育和疾病中重要的基因表达过程的核心。

DESCRIPTION (provided by applicant): Transcriptional repression plays a key role in regulation of gene expression in development and in disease processes. To understand conserved regulatory processes in animals, we have determined both the molecular workings and physiological relevance of transcriptional represser proteins that play key roles in Drosophila development. Knirps is a key regulator of the even-skipped gene, acting on separate enhancer elements via a short-range mechanism. We have discovered how Knirps and related repressers function via distinct repression mechanisms, involving the evolutionary conserved CtBP corepressor protein and a histone deacetylase Rpd3, and we have developed novel insights into the design of cis regulatory elements that bind these repressers. We have identified key aspects of a "cis regulatory grammar" that predicts how repressor proteins can function on defined regulatory elements, laying the groundwork for mathematical and bioinformatic analysis of endogenous enhancers. This work will allow us to apply Drosophila-derived information to general analysis of metazoan cis regulatory element design and evolution. 1. We will quantitatively "map" regulatory surfaces of genes controlled by transcriptional repressors, and use potential function-based mathematical models to predict the regulatory properties of novel enhancers. 2. We will elucidate of mechanisms of short- and long-range repressors using chromatin immunoprecipitation to identify molecular events associated with repression of embryonic reporter genes. 3. We will assess the function of corepressors we discovered associated with the Knirps represser complex, in particular the evolutionary conserved Groucho corepressor, whose role in short-range repression was previously unrecognized. 4. We will identify the physiological significance of conserved residues and splice-variants of the CtBP corepressor, using whole-animal assays to identify the biological significance of the proteins' activities. These aims combine empirical and modeling efforts to obtain a "bottoms up" understanding of transcriptional repression. Our long-term goal is to understand molecular activities of transcriptional control proteins and DNA regulatory sequences that are central to gene expression processes important in development and disease.