Molecular Analysis of Transcriptional Repression

转录抑制在发育和疾病的基因表达调控中起着关键作用 流程.为了了解动物中保守的调节过程，我们已经确定了两个 转录抑制蛋白的分子工作和生理相关性， 果蝇发育。Knirps是偶跳基因的关键调节子，作用于单独的增强子 通过短程机制。我们已经发现了Knirps和相关的阻遏物是如何通过以下途径发挥作用的： 不同的阻遏机制，涉及进化保守的CtBP辅阻遏蛋白和 组蛋白去乙酰化酶Rpd3，我们已经开发了新的见解顺式调控元件的设计 与这些抑制蛋白结合。我们已经确定了“顺式调节语法”的关键方面， 阻遏蛋白可以在确定的调控元件上发挥作用，为数学和生物学的研究奠定了基础。 内源性增强子的生物信息学分析。这项工作将使我们能够应用果蝇衍生的 为后生动物顺式调控元件设计和进化的一般分析提供了信息。 1.我们将定量绘制受转录抑制因子控制的基因的调控表面， 使用基于潜在功能的数学模型来预测新型增强子的调节特性。 2.我们将利用染色质阐明短距离和长距离阻遏物的机制， 免疫沉淀法鉴定与胚胎报告基因阻遏相关的分子事件。 3.我们将评估我们发现的与Knirps阻遏蛋白相关的辅阻遏蛋白的功能 复合物，特别是进化保守的Groucho辅阻遏物，其在短距离 镇压以前没有得到承认。 4.我们将确定CtBP的保守残基和剪接变体的生理意义 辅阻遏物，使用整体动物测定来鉴定蛋白质活性的生物学意义。 这些目标联合收割机结合经验和建模的努力，以获得“自下而上”的理解， 转录抑制我们的长期目标是了解转录控制的分子活动 蛋白质和DNA调节序列是基因表达过程的核心， 发展和疾病。

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 "cisregulatory grammar" that predicts how represser 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.