The role of SAM polymerization in Polycomb-dependent chromatin structures

SAM 聚合在 Polycomb 依赖性染色质结构中的作用

• 批准号: 9030752
• 负责人: Nicole Jane Francis
• 金额: $ 26.23万
• 依托单位: MIDWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9030752
• 关键词: Address; Affect; Architecture; Binding; Biochemical; Biological Assay; Biological Process; Cell Culture Techniques; Cells; Chromatin; Chromatin Structure; Complex; Data; Development; Disease; Dissection; Drosophila genus; Epigenetic Process; Gene Expression; Gene Expression Regulation; Gene Targeting; Genomics; Goals; Growth; Histones; In Vitro; Length; Link; Macromolecular Complexes; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Membrane Proteins; Microscopy; Molecular; Multiprotein Complexes; Mutation; Nature; Nucleosomes; PRC1 Protein; Plants; Play; Polycomb; Polyhomeotic; Polymers; Proteins; Publishing; Regulation; Repression; Resolution; Role; SAM Domain; Structure; Testing; Therapeutic; Time; Work; analytical ultracentrifugation; base; biophysical techniques; cell growth; chromatin immunoprecipitation; chromatin protein; chromosome conformation capture; gene repression; genetic regulatory protein; histone modification; interest; member; molecular assembly/self assembly; mutant; new technology; next generation sequencing; novel; polymerization; protein protein interaction; protein structure; public health relevance; reconstitution; research study; restriction enzyme; stem; stoichiometry; tumor progression

 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.

 描述(由申请人提供) 问题：蛋白质相互作用模块被称为不育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，它具有不同寻常的能力，可以自结合成开放的螺旋聚合物结构。自从解决了第一个这样的SAM聚合物结构以来，十多年过去了，这表明这些聚合物可以控制染色质结构的有趣的可能性。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得到解答：1)SAM聚合物是如何调节的？2)SAM聚合物如何控制染色质结构和影响基因表达？目的：这项提案关注的是一种含有SAM的染色质相关蛋白，称为多态同源异体(Ph)。PH是表观遗传调节蛋白聚梳组(PcG)的成员，表观遗传调控蛋白在发育过程中发挥关键作用，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节；2)确定细胞中Ph低聚体的组成和化学计量比；3)确定Ph SAM聚合如何影响染色质的结合和组织。通过利用新技术，我们的目标是最终了解Ph SAM聚合物是如何控制染色质结构和基因表达的。方法：我们将利用广泛的方法来了解Ph SAM聚合物。首先，我们将使用结构导向突变和生物物理方法(包括分析超速离心法)来确定Ph SAM聚合受Ph蛋白中其他序列调控的分子基础。这一分析将使用超分辨率显微镜、体外重建和质谱学来扩展，以剖析Ph SAM聚合是如何在细胞中存在Ph的多蛋白PcG复合体中发生的。然后将结合细胞内染色质的体外重组和高分辨率分析(使用染色体构象捕获和下一代测序、限制性内切酶可及性分析和染色质免疫沉淀)来确定这些SAM依赖的聚合物对染色质结构的影响。最后，破坏或增强SAM聚合活性的Ph突变体将被测试它们对发育中的果蝇和细胞培养中的基因表达和细胞生长的影响。意义：SAMS存在于从染色质调节剂到膜蛋白的许多蛋白质中。SAM的保守结构和可能调节它的机制意味着我们对Ph SAM的解剖对于含有SAM的蛋白质以及基于调节蛋白质聚合的可能的治疗方法具有普遍的意义。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控基因表达的新机制。