The role of SAM polymerization in Polycomb-dependent chromatin structures

 描述（由申请人提供） 问题：蛋白质相互作用模块称为无菌α基序（SAM），它存在于许多蛋白质中，包括那些参与基因调控的蛋白质，具有不寻常的能力，自缔合成一个开放的螺旋聚合物结构。自从解决第一个这样的SAM聚合物结构以来，已经过去了十多年，这表明这些聚合物可以控制染色质结构的有趣可能性。部分由于技术障碍，关于SAM聚合物功能的关键机制问题仍然没有答案：1）SAM聚合物如何调节？2)SAM聚合物如何控制染色质结构和影响基因表达？目的：本研究的重点是一种含SAM的染色质相关蛋白，称为多同源异型蛋白（Ph）。Ph是表观遗传调节蛋白的Polycomb组（PcG）的成员，其在发育中起关键作用，并与癌症进展有关。我们的目标是：1）剖析Ph SAM聚合活性的机制和调控; 2）确定细胞中Ph寡聚体的组成和化学计量; 3）确定Ph SAM聚合如何影响染色质结合和组织。通过利用新技术，我们的目标是最终了解Ph SAM聚合物如何控制染色质结构和基因表达。方法：我们将利用广泛的方法来了解Ph SAM聚合物。首先，我们将使用结构指导突变和生物物理方法（包括分析超离心），以确定Ph SAM聚合的分子基础是由Ph蛋白中的其他序列调节。这种分析将扩展使用超分辨率显微镜，体外重建，和质谱分析如何Ph SAM聚合可以发生在多蛋白PcG复合物中，其中Ph存在于细胞中。这些SAM依赖性聚合物如何影响染色质结构，然后将使用体外重建和细胞中染色质的高分辨率分析（使用染色体构象捕获和下一代测序，限制性酶可及性测定和染色质免疫沉淀）的组合来确定。最后，将测试具有破坏或增强的SAM聚合活性的Ph突变体对发育中的果蝇和细胞培养物中的基因表达和细胞生长的影响。意义：SAM存在于从染色质调节因子到膜蛋白的许多蛋白质中。SAM的保守架构和可能的机制，调节它意味着我们的解剖Ph SAM具有一般意义的SAM含蛋白质，并为可能的治疗方法的基础上调节蛋白质聚合。关于PcG蛋白和其他染色质调节剂的大部分工作都集中在组蛋白修饰上。我们对Ph SAM聚合活性的研究揭示了通过控制蛋白质和染色质结构来调节基因表达的新机制。

 DESCRIPTION (provided by applicant) PROBLEM: The protein interaction module called the Sterile Alpha Motif (SAM), which is found in many proteins including those involved in gene regulation, possesses the unusual ability to self-associate into an open-ended helical polymer architecture. Over a decade has passed since solving the first such SAM polymer structure suggested the intriguing possibility that these polymers could control chromatin architecture. In part due to technical barriers, key mechanistic questions regarding the function of SAM polymers have remained unanswered: 1) how are SAM polymers regulated? 2) how do SAM polymers control chromatin structure and influence gene expression? OBJECTIVE: This proposal focuses on a SAM-containing, chromatin-associated protein called Polyhomeotic (Ph). Ph is a member of the Polycomb group (PcG) of epigenetic regulatory proteins which play a critical role in development and have been implicated in cancer progression. Our aims are: 1) dissect the mechanism and regulation of Ph SAM polymerization activity; 2) determine the composition and stoichiometry of Ph oligomers in cells; 3) determine how Ph SAM polymerization affects chromatin binding and organization. By utilizing new technologies, our goal is to finally understand how the Ph SAM polymer controls chromatin architecture and gene expression. METHODS: We will utilize a broad spectrum of approaches to understand Ph SAM polymers. First, we will use structure guided mutations and biophysical methods (including analytical ultracentrifugation) to determine the molecular basis by which Ph SAM polymerization is regulated by other sequences in the Ph protein. This analysis will be extended using super-resolution microscopy, in vitro reconstitution, and mass spectrometry to dissect how Ph SAM polymerization can occur in the multi-protein PcG complexes in which Ph exists in cells. How these SAM-dependent polymers affect chromatin structure will then be determined using a combination of in vitro reconstitution and high resolution analysis of chromatin in cells (using chromosome conformation capture and next generation sequencing, restriction enzyme accessibility assays, and chromatin immunoprecipitation). Finally, Ph mutants with disrupted or enhanced SAM polymerization activity will be tested for their effect on gene expression and cell growth in developing Drosophila and cell culture. SIGNIFICANCE: SAMs are found in many proteins spanning from chromatin regulators to membrane proteins. The conserved architecture of the SAM and possibly of the mechanisms that regulate it mean that our dissection of Ph SAM has general implications for SAM-containing proteins, and for possible therapeutic approaches based on modulating protein polymerization. Much of the work on PcG proteins and other chromatin regulators has focused on histone modifications. Our work on Ph SAM polymerization activity is revealing new mechanisms for regulating gene expression by controlling protein and chromatin architecture.