Exploring second-tier post-translational modifications in chromatin biology: A new class of histone modifications with undetermined function

探索染色质生物学中的二级翻译后修饰：功能未定的新型组蛋白修饰

• 批准号: 9328461
• 负责人: John David Bagert
• 金额: $ 5.71万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9328461
• 关键词: Acetylation; Affect; Affinity Chromatography; Amber; Antibodies; Biochemical Genetics; Biology; C-terminal; CRISPR/Cas technology; Cancer cell line; Cell Cycle; Cell physiology; Cellular Stress; ChIP-seq; Chemicals; Chromatin; Chromatin Structure; DNA Repair; Deubiquitination; Environment; Enzymes; Epigenetic Process; Excision; Future; Gene Activation; Gene Expression Regulation; Genes; Goals; Hereditary Disease; High-Throughput Nucleotide Sequencing; Histone H2A; Histone H2B; Histones; Human Biology; Hydrolase; Immunoassay; Incubated; Knock-out; Knowledge; Lysine; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Methionine; Methodology; Methods; Methylation; Methyltransferase; Modification; Molecular; Monoubiquitination; N-terminal; Nuclear; Nuclear Extract; Nuclear Proteins; Nucleosomes; Output; Peptides; Physiological; Post-Translational Protein Processing; Process; Protein Engineering; Proteins; Proteomics; Research; Role; Side; Techniques; Testing; Trans-Splicing; Transcriptional Regulation; Ubiquitin; Ubiquitination; Work; amino group; chromatin immunoprecipitation; chromatin modification; crosslink; design; epigenome; experimental study; gene repression; genome editing; histone acetyltransferase; histone methyltransferase; histone modification; in vitro Assay; in vitro activity; in vivo; intein; professor; reconstitution; tool; ubiquitinated H2A

PROJECT SUMMARY Histone post-translational modifications (PTMs) affect a variety of nuclear processes, including gene regulation, DNA repair, and maintenance of chromatin structure. Misregulation of these chemical modifications has broad implications for human biology and drives a myriad of genetic diseases. While most PTMs are small chemical moieties (e.g. methylation and acetylation), monoubiquitination entails the covalent attachment of the bulky 8.5 kDa protein ubiquitin to the side-chain amino group of target lysines. Ubiquitination of histone proteins H2A (H2Aub) and H2B (H2Bub) serves important roles in DNA damage repair and transcriptional regulation. Indeed, H2Aub and H2Bub, along with the enzymes associated with the addition and removal of ubiquitin, have been implicated in various cancers. Preliminary experiments show strong evidence that the ubiquitin moiety itself on both H2Aub and H2Bub contains numerous methylation and acetylation PTMs in vivo. Hereafter, these modifications are called “second-tier” PTMs to distinguish them from the many examples of PTMs that are directly conjugated to histone proteins. This proposed work pursues the hypothesis that second-tier PTMs contribute to and alter the functional output of chromatin ubiquitination of histones H2A and H2B, ultimately affecting chromatin structure, gene activation, and gene repression. The goals of the proposed research are to (1) validate the second-tier PTMs on H2Aub and H2Bub identified in preliminary experiments using alternative approaches, (2) determine where these modifications are distributed over the epigenome and whether these modifications are altered as a function of cell state and type, (3) identify the enzymes that install these PTMs, and (4) investigate the functional role of these modifications in chromatin biology. Central to these endeavors will be the core expertise of Professor Tom Muir’s lab in producing “designer chromatin,” a process that uses chemical biology, protein engineering, and chromatin reconstitution techniques to create chemically-defined nucleosomes. Designer chromatin will be used in combination with biochemical, genetic, and proteomics tools to accomplish the proposed research goals. These experiments will elucidate the functional role of this unexplored level of chromatin modification and deepen our fundamental understanding of histone ubiquitination in chromatin. In addition, the proposed research will develop a methodological and experimental framework for studying second-tier PTMs, which will guide future efforts to study these modifications, whether in the context of chromatin or other cytosolic processes.

项目摘要 组蛋白翻译后修饰（PTM）影响多种核过程，包括基因调控， DNA修复和染色质结构的维持。这些化学修饰的失调具有广泛的 对人类生物学的影响，并驱动无数的遗传疾病。虽然大多数PTM是小化学品， 由于大部分的结构（例如甲基化和乙酰化），单泛素化需要共价连接大的8.5-羟基-N-甲基-N- kDa蛋白质泛素与靶赖氨酸的侧链氨基连接。组蛋白H2 A的泛素化 H2 Aub和H2 B（H2 Bub）在DNA损伤修复和转录调控中发挥重要作用。的确， H2 Aub和H2 Bub，沿着与泛素的添加和去除相关的酶一起，已经被研究， 与多种癌症有关初步的实验显示，强有力的证据表明，泛素部分本身对 H2 Aub和H2 Bub在体内均含有大量的甲基化和乙酰化PTM。此后，这些 修改被称为“第二层”PTM，以将它们与直接被修改的PTM的许多示例区分开。 与组蛋白结合。 这项拟议的工作追求的假设，即第二层PTM有助于并改变功能 组蛋白H2 A和H2 B的染色质遍在化输出，最终影响染色质结构、基因 激活和基因抑制。本研究的目标是：（1）验证第二层PTM， H2 Aub和H2 Bub在使用替代方法的初步实验中鉴定，（2）确定这些 修饰分布在表观基因组上，以及这些修饰是否作为 细胞状态和类型，（3）确定安装这些PTM的酶，以及（4）研究 染色质生物学中的这些修饰。这些努力的核心将是汤姆教授的核心专业知识 缪尔的实验室正在生产“设计染色质”，这是一个利用化学生物学、蛋白质工程和 染色质重建技术，以创建化学定义的核小体。将使用设计师染色质 结合生物化学，遗传学和蛋白质组学工具来实现拟议的研究目标。 这些实验将阐明这一未被探索的染色质修饰水平的功能作用，并加深对染色质修饰的认识。 我们对染色质中组蛋白泛素化的基本理解。此外，拟议的研究将 为研究第二层PTM制定一个方法和实验框架，这将指导未来 无论是在染色质还是其他胞质过程中，研究这些修饰的努力。