Molecular Basis of the Selective Assembly of Functionally Distinct PRC1s

项目摘要/摘要 问题：多梳抑制复合体1(Prc1)是一种多蛋白组装，它从表观上调节 染色质，如果调控不当，就会导致癌症。最初在果蝇中被鉴定为四种成分 由于复杂，PRC1扩大了其成员和进化方面的功能。一个重要的 关于人类PRC1的悬而未决的问题是，某些特定的PRC1蛋白组合是如何组装的 其他的则不是，以及在细胞分化过程中形成的PRC1的类型是如何变化的。PRC1程序集是 目前已知通过一系列1：1的蛋白质-蛋白质相互作用发生。不幸的是，这种观点， 没有解释一个PRC1结构域的蛋白质-蛋白质相互作用如何以某种方式影响结合 另一种看似无关的蛋白质-蛋白质相互作用的选择性。我们已经确定了新的相互作用 次于已知的1：1直接相互作用，为包括特定蛋白质提供选择性检查点 从而允许选择性的PRC1组件的组合。目标：我们将系统地描述 这些次级蛋白质-蛋白质相互作用然后研究它在组装和功能不同的 PRC1s。方法：我们将使用生物物理方法(X射线结晶学、分析性超速离心法、 生物层干涉法)来剖析这些选择性组装的分子基础。功能界别 形成这些组件的二次相互作用的后果将使用一种新的FRET来评估- 基于组蛋白修饰实验。意义：基因调控由许多不同的蛋白质执行， 所有这些都可以在多组件系统中运行。当前研究基因的基因组学方法 调控，虽然信息量很大，但缺乏对蛋白质-蛋白质相互作用的分子观点 对这些系统有全面的了解。我们建议通过确定具体的PRC1如何来填补这一空白 装配和运行。通过定义作为PRC1组装基础的分子编排，我们将提供 这些复合体在基因组调控中的调控和功能的新视角。我们的结果可能 也可广泛应用于理解其他基因调控系统，其中许多系统利用多个 具有不同同源组合的组件系统。

PROJECT SUMMARY/ABSTRACT PROBLEM: Polycomb repressive complex 1 (PRC1) is a multi-protein assembly that epigenetically regulates chromatin, which when misregulated, results in cancer. Originally identified in Drosophila as a four-component complex, PRC1 has expanded its membership and functional repertoire over evolution. An important unanswered question concerning human PRC1 is how certain combinations of PRC1 proteins assemble while others do not, and how the types of PRC1s formed change during cellular differentiation. PRC1 assembly is currently understood to occur through a series of 1:1 protein-protein interactions. This view, unfortunately, does not explain how the protein-protein interaction of one PRC1 domain can somehow influence the binding selectivity of another, seemingly unrelated protein-protein interaction. We have identified novel interactions secondary to the known 1:1 direct interactions that provide selective checkpoints for including specific protein combinations thereby allowing selective PRC1 assemblies. OBJECTIVE: We will systematically characterize these secondary protein-protein interactions then investigate its role in the assembly and function of the distinct PRC1s. METHODS: We will use biophysical methods (X-ray crystallography, analytical ultracentrifugation, biolayer interferometry) to dissect the molecular basis of these selective assemblies. The functional consequences of the secondary interactions that form these assemblies will be assessed using a novel FRET- based histone modification assay. SIGNIFICANCE: Gene regulation is performed by many different proteins, all of which work within multi-component systems. Current genomic approaches to investigating gene regulation, while informative, lack a molecular perspective of protein-protein interactions necessary for a complete understanding of these systems. We propose to fill this gap by determining how specific PRC1s assemble and function. By defining the molecular choreography underlying PRC1 assembly, we will provide a new perspective on the regulation and function of these complexes in genome regulation. Our results may also be broadly applicable for understanding other gene regulatory systems, many of which utilize multi- component systems with different homolog combinations.