Regulation and Function of Histone H3K36 Methylation in Mammalian Chromatin

项目总结 DNA和组蛋白的化学修饰是调控信息的载体，这些信息与 转录因子来控制基因组的可及性。组蛋白H3赖氨酸36(H3K36)甲基化是一种 进化上保守的染色质修饰。在酵母中，一个单一的甲基转移酶Set2催化所有 H3K36基因的甲基化状态。相比之下，H3K36甲基化状态受到更复杂的调控 哺乳动物的染色质。鉴于SETD2是催化三甲基H3K36(H3K36me3)的唯一酶，几个 包括NSD家族成员和Ash1在内的其他酶已经进化为甲基转移酶特有的 二甲基H3K36(H3K36me2)。相应地，H3K36me2的基因组分布与H3K36me2不同 H3K36me3，这意味着可能的功能分化仍然不完全清楚。 NSD和Ash11的种系突变会导致各种神经发育障碍，而躯体 这些酶的改变经常在人类癌症中发现。我们和其他人最近的工作是 发现并阐明了直接影响组蛋白H3K36的重复突变，该突变导致组蛋白H3K36的整体耗竭 H3K36me2在人类骨癌和头颈癌中的表达。因此，NSD/Ash11介导的精确调控 H3K36me2对人类发育至关重要，是肿瘤转化的关键障碍。 尽管它很重要，但人们对监管机制的了解有限 H3K36me2的建立及其在基因组调控中的作用因此，我研究的五年目标是 计划是系统地研究H3K36me2及其相关修饰物的染色质生物学。这个 概念创新建立在我们最近发现的H3K36me2和基因间DNA之间相互作用的基础上 通过招募从头DNA甲基转移酶DNMT3A进行甲基化。此外，我们还开发了 易处理的实验系统和基于CRISPR/CAS的新型基因组和表观基因组编辑平台 允许我们以多种方式剖析H3K36me2的调控。我们将确定各自的 NSD和Ash1在建立H3K36me2内含子和基因间隔区中的贡献。一种组合 染色质生物化学和功能遗传筛选方法将被用来理解 指导H3K36me2形成的监管投入。我们将进行结构-功能分析，以更好地 了解H3K36me2募集DNMT3A的分子基础，并对其进行动力学研究 与众所周知的H3K36me2和H3K27甲基化拮抗的关系。最后，我们将聘用 综合表观基因组学和表观基因组编辑工具确定H3K36me2对可及性的影响 顺式调控和重复成分。总之，这些研究将提供一个知识基础， 对发病机制和可治疗的靶点的机械洞察可以为人类疾病揭开面纱 由H3K36me2失调驱动。此外，技术工具和资源可以很容易地用于 其他染色质修饰的研究和染色质研究界的广泛兴趣。

PROJECT SUMMARY Chemical modifications of DNA and histones represent carriers of regulatory information that cooperate with transcription factors to control genome accessibility. Methylation at histone H3 lysine 36 (H3K36) is an evolutionarily conserved chromatin modification. In yeast, a single methyltransferase Set2 catalyzes all methylation states on H3K36. In contrast, H3K36 methylation states are subject to more complex regulation in mammalian chromatin. Whereas SETD2 is the sole enzyme catalyzing tri-methyl H3K36 (H3K36me3), several additional enzymes including NSD family members and ASH1L have evolved as the methyltransferases specific for di-methyl H3K36 (H3K36me2). Accordingly, the genomic distribution of H3K36me2 is distinct from that of H3K36me3, implying a possible functional divergence that remains incompletely understood. Germline mutations in NSD and ASH1L lead to various neurodevelopmental disorders, whereas somatic alterations of these enzymes are frequently found in human cancers. Recent work by us and others have identified and elucidated recurrent mutations directly affecting histone H3K36 that induced global depletion of H3K36me2 in human bone and head and neck cancers. Therefore, precise regulation of NSD/ASH1L-mediated H3K36me2 is critical for human development and represents a key barrier to neoplastic transformation. Despite its importance, there is limited understanding of the regulatory mechanisms governing H3K36me2 establishment and its function in genome regulation. Therefore, the five-year goal of my research program is to systematically examine the chromatin biology of H3K36me2 and its associated modifiers. The conceptual innovation is built on our recent findings of an interplay between H3K36me2 and intergenic DNA methylation through the recruitment of de novo DNA methyltransferase DNMT3A. In addition, we have developed tractable experimental systems and novel CRISPR/Cas-based genome- and epigenome-editing platforms that allow us to dissect the regulation of H3K36me2 in a multiplexed manner. We will determine the respective contribution of NSD and ASH1L to establishing H3K36me2 at introns and intergenic regions. A combination of chromatin biochemistry and functional genetic screening approaches will be employed to understand the regulatory input that guides the formation of H3K36me2. We will perform structural-functional analysis to better understand the molecular basis of DNMT3A recruitment by H3K36me2, and kinetic studies to delineate its relationship with the well-known antagonism between H3K36me2 and H3K27 methylation. Lastly, we will employ integrative epigenomics and epigenome-editing tools to determine the impact of H3K36me2 on the accessibility of cis-regulatory and repetitive elements. Together, these studies will provide a knowledge base upon which mechanistic insights into pathogenesis and targets amenable for therapy can be uncovered for human diseases driven by H3K36me2 dysregulation. Furthermore, the technical tools and resources could be readily applied to the study of additional chromatin modifications and of broad interest to the chromatin research community.