Structural and functional analysis of gene silencing

基因沉默的结构和功能分析

• 批准号: 10674754
• 负责人: Karim Jean Armache
• 金额: $ 36.44万
• 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/10674754
• 关键词: Acetylation; Binding; Binding Proteins; Biochemical; 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; Enzyme Interaction; Enzymes; Epigenetic Process; Evolution; Future; Gene Silencing; Genes; Genetic Recombination; 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; Visualization; Writing; X-Ray Crystallography; Yeasts; 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; reconstruction; recruit; repaired

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.

组蛋白将真核细胞核中的基因组 DNA 包装成染色质，其结构控制着所有 DNA- 基础过程，包括转录、复制、重组和修复的主要调节机制。 染色质结构基于组蛋白的翻译后修饰，组蛋白由酶沉积 或“写入器”，并被某些蛋白质的“读取器”域以高度特异性和选择性识别以介导 作者和读者之间的相互作用对于发展至关重要，并且受到干扰。 因此，在包括癌症在内的许多疾病中，染色质生物学的一个主要焦点是了解细节。 控制染色质结构的机制，这受到相对较少的高分辨率的极大阻碍 沉积和识别组蛋白修饰的染色质结合蛋白和蛋白复合物的结构。 真核基因组的大片段被包装成转录沉默的异染色质。 异染色质对于维持基因组完整性和防止染色体缺陷至关重要。 异染色质形成和 DNA 复制通常通过特定的染色质修饰来协调。 例如，组蛋白 H4 在赖氨酸 20 (H4K20) 处的不同甲基化状态调节不同的过程： H4K20me3 存在于异染色质中，而 H4K20me2 则存在于复制起点。 H4K20 由两种相关的组蛋白甲基转移酶 SUV4-20H1 和 SUV4-20H2 催化，但尚不清楚 这些酶如何被招募到染色质或如何调节它们的催化活性。 异染色质的形成和复制可以通过读者来协调，例如 Orc1， 起源识别复合体 (ORC) 使用其溴邻同源 (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的详细机制。 这些过程在很大程度上是未知的。我们将使用结构和功能方法来填补这一关键。 知识差距，AIM 1 侧重于阐明 SUV4-20H 酶的机制，AIM 2 侧重于 为了了解 Orc1 BAH 结构域的机制，我们将使用冷冻电镜来确定这些结构。 作者和读者与核小体结合，并通过体外功能实验补充结构 SUV4-20H 编写器和 Orc1 BAH 域读取器在复合体中的结构和功能研究。 与核小体的结合将揭示组蛋白沉积和识别的一般原理 我们提出的综合研究将为基本生物学提供重要的见解。 异染色质形成和复制的过程，并将提供关于如何放松管制的宝贵见解 这些复合物的数量以及由此产生的染色质结构畸变会导致疾病。

项目成果

Regulation of the Dot1 histone H3K79 methyltransferase by histone H4K16 acetylation.

• DOI: 10.1126/science.abc6663
• 发表时间: 2021-01-22
• 期刊: Science (New York, N.Y.)
• 作者: Valencia-Sánchez MI;De Ioannes P;Wang M;Truong DM;Lee R;Armache JP;Boeke JD;Armache KJ
• 通讯作者: Armache KJ