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

项目总结 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。对这些化学修饰的错误调控是广泛的 对人类生物学的影响，并导致无数的遗传疾病。虽然大多数PTM是小的化学物质 部分(如甲基化和乙酰化)，单一泛素化需要笨重的8.5 KDA蛋白泛素到靶标赖氨酸的侧链氨基。组蛋白H_2A的泛素化 (H_2Aub)和H_2B(H_2Bub)在DNA损伤修复和转录调控中发挥重要作用。的确， H_2Aub和H_2Bub，以及与泛素的添加和移除相关的酶，已经被 与多种癌症有关。初步实验显示，强有力的证据表明，泛素部分本身就在 在体内，H_2Aub和H_2Bub都含有大量的甲基化和乙酰化的PTM。此后，这些 修改被称为“第二层”的PTM，以区别于许多直接 结合到组蛋白蛋白上。 这项拟议的工作追求的假设是，第二层PTM有助于并改变功能 组蛋白H_2A和H_2B染色质泛素化的产物，最终影响染色质结构，基因 激活和基因抑制。拟议研究的目标是：(1)验证第二级PTMS的有效性 H2Aub和H2Bub在使用替代方法的初步实验中确定，(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PTM的功能作用 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 Muir的实验室生产“设计染色质”，这是一种使用化学生物学、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 与生化、遗传和蛋白质组学工具相结合，以实现拟议的研究目标。 这些实验将阐明这一未知水平的染色质修饰的功能作用并深化 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 建立一个研究第二级技术管理的方法学和实验框架，这将指导未来 努力研究这些修饰，无论是在染色质还是在其他胞质过程中。

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.