Structural and functional analysis of gene silencing

基因沉默的结构和功能分析

• 批准号: 10387566
• 负责人: Karim Jean Armache
• 金额: $ 4.82万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-17 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10387566
• 关键词: Acetylation; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biological Process; Biology; Catalysis; Cell Nucleus; Cell physiology; Cells; Chemicals; Chromatin; Chromatin Structure; Chromosome abnormality; Complement; Complex; Cryoelectron Microscopy; DNA; DNA biosynthesis; Data; Deposition; Development; Disease; Enzymes; Epigenetic Process; Evolution; Future; Gene Silencing; Genes; Genetic Transcription; Genome; Genome Stability; Genomic DNA; Goals; Heterochromatin; Histone H4; Histones; Human; In Vitro; Kluyveromyces; Knowledge; Lysine; Macromolecular Complexes; Malignant Neoplasms; Mammals; Mediating; Methods; Methylation; Modification; Molecular; Molecular Evolution; Nature; Nucleosomes; ORC1L gene; Play; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Reader; Regulation; Replication Origin; Resolution; Role; Saccharomyces cerevisiae Proteins; Signal Transduction; Specificity; Structure; System; Testing; Ubiquitination; Variant; Writing; X-Ray Crystallography; Yeasts; base; chromatin modification; comparative; design; experimental study; genome integrity; heterochromatin-specific nonhistone chromosomal protein HP-1; histone methyltransferase; histone modification; improved; in vivo; insight; intermolecular interaction; novel; origin recognition complex; prevent; protein complex; recombinational repair; reconstruction; recruit

Histones package genomic DNA in the eukaryotic nucleus into chromatin, whose structure controls all DNA- based processes, including transcription, replication, recombination, and repair. A major mechanism regulating chromatin structure is based on post-translational modifications of the histones, which are deposited by enzymes or “writers” and recognized with high specificity and selectivity by “reader” domains of certain proteins to mediate downstream functions. The interplay between writers and readers is essential for development and is perturbed in many diseases, including cancer. A major focus in chromatin biology is thus to understand the detailed mechanisms that control chromatin structure, which is greatly hampered by the relatively few high-resolution structures of chromatin-bound proteins and protein complexes that deposit and recognize histone modifications. Large segments of the eukaryotic genome are packaged into transcriptionally silent heterochromatin. Heterochromatin is critical to maintain genome integrity and to prevent chromosomal defects. Interestingly, heterochromatin formation and DNA replication are often coordinated through specific chromatin modifications. For example, different methylation states of histone H4 at lysine 20 (H4K20) regulate distinct processes: H4K20me3 is found in heterochromatin, whereas H4K20me2 is found at the origins of replication. Methylation of H4K20 is catalyzed by two related histone methyltransferases, SUV4-20H1 and SUV4-20H2, but it is not known how these enzymes are recruited to chromatin or how their catalytic activity is regulated. In addition, heterochromatin formation and replication can be coordinated through readers, such as Orc1, a subunit of the Origin Recognition Complex (ORC). Orc1 binds to chromatin using its bromo-adjacent homology (BAH) domain, which interacts with histone modifications and heterochromatin proteins. The detailed mechanisms involved in these processes are largely unknown. We will use structural and functional approaches to fill this critical knowledge gap, with AIM 1 focusing on elucidating the mechanisms of SUV4-20H enzymes and AIM 2 focusing on understanding the mechanisms of Orc1 BAH domains. We will use cryo-EM to determine structures of these writers and readers bound to nucleosomes and complement the structures with functional experiments in vitro and in vivo. The structural and functional studies of SUV4-20H writers and Orc1 BAH domain readers in complex with nucleosomes will uncover general principles that underlie the deposition and recognition of histone modifications. Our proposed comprehensive studies will provide crucial insights into the fundamental biological processes of heterochromatin formation and replication and will provide invaluable insights into how deregulation of these complexes and resulting aberrations in chromatin structure contribute to diseases.

组蛋白在真核核中包装基因组DNA成染色质，其结构控制所有DNA- 基于转录，复制，重组和修复的过程。调节的主要机制 染色质结构基于组蛋白的翻译后修饰，这些修饰由酶沉积 或“作家”，并以某些蛋白质的“读取器”领域具有高特异性和选择性认可 下游功能。作家和读者之间的相互作用对于发展至关重要，并且受到干扰 在包括癌症在内的许多疾病中。因此，染色质生物学的主要重点是了解详细的 控制染色质结构的机制，该机制受到相对较少的高分辨率极大地阻碍 结合染色质蛋白质和蛋白质复合物的结构，这些结构沉积并识别组蛋白修饰。 真核基因组的大片段被包装到转录静音异染色质中。 异染色质对于维持基因组完整性和预防染色体缺陷至关重要。有趣的是， 异染色质形成和DNA复制通常通过特定的染色质修饰进行协调。 例如，赖氨酸20（H4K20）的组蛋白H4的不同甲基化状态调节不同的过程： H4K20me3在异染色质中发现，而H4K20me2在复制的起源处。甲基化 H4K20由两个相关的组蛋白甲基转移酶SUV4-20H1和SUV4-20H2催化，但尚不清楚 如何募集这些酶为染色质或如何调节其催化活性。此外， 可以通过读者（例如ORC1）来协调异染色质的形成和复制 原点识别综合体（ORC）。 ORC1使用其Bromo-Adjacent同源（BAH）结构域与染色质结合， 与组蛋白修饰和异染色质蛋白相互作用。涉及的详细机制 这些过程在很大程度上未知。我们将使用结构和功能的方法来填补这一关键 知识差距，AIM 1专注于阐明SUV4-20H酶的机制和AIM 2聚焦 了解ORC1 BAH域的机制。我们将使用Cryo-EM确定这些结构 作家和读者与核小体结合并通过体外功能实验完成结构 和体内。 SUV4-20H作家和ORC1 BAH域读取器的结构和功能研究 用核小体将发现基于组蛋白沉积和识别的一般原理 修改。我们提出的综合研究将为基本生物学提供关键的见解 异染色质形成和复制的过程，并将提供无价的见解 这些复合物和染色质结构的畸变有助于疾病。