Functional Study of Mammalian Set1A/COMPASS Methyltransferase in Stem Cells and Development

项目摘要/摘要： 高度保守的COMPASS(与Set1相关的蛋白质复合体)家族是 负责实施组蛋白H3赖氨酸4(H3K4)甲基化，与表观遗传标记相关 具有转录活性的染色质。多项全基因组测序工作报告称， COMPASS家族在许多癌症、神经系统疾病和其他疾病中经常发生突变。 因此，理解指南针函数将有助于深入了解 疾病的发病机制，这是发展有效的靶向治疗所必需的。 H3K4甲基酶Set1a是在哺乳动物中发现的六个COMPASS家族成员之一，一直是 显示涉及整个基因组的大量H3K4二甲基化和三甲基化(分别为H3K4me2/ME3)。 以往的研究表明，Set1a蛋白的缺失会导致胚胎死亡和胚胎干细胞 细胞(ESC)增殖缺陷，表明Set1A在这一过程中是必需的；然而，分子 Set1A在其中起作用的上下文主要是未知的。区分全长Set1a的角色与其 在ESCs中，Set1a的催化结构域被删除，这导致了非常令人惊讶的 发现其酶功能似乎对胚胎干细胞的生存和自我更新是必不可少的。然而， 携带催化死亡的Set1a的ESCs似乎无法正确分化，这表明 Set1a及其在胚胎干细胞自我更新和后续分化中的催化活性。总之，这些初步的 数据支持进一步研究Set1a在调节干细胞多能性和发育中的作用。 基于这些结果，这项建议的第一个目标将进一步研究Set1a的酶功能，通过 分析Set1A催化突变体在胚胎干细胞分化过程中转录和H3K4甲基化的变化 和老鼠的发育。第二个目标是确定Set1a蛋白维持的关键区域 ESC通过有条件的敲除和过度表达系统进行自我更新。第三个目标将进一步探索 胚胎干细胞中Set1a的蛋白质相互作用网络揭示Set1a功能的新机制 早期发展。这项工作将阐明Set1a在干细胞和在 这将阐明其疾病的责任，并最终促进治疗的发展。

Project Summary/Abstract: The highly conserved COMPASS (COMplex of Proteins ASsociated with Set1) family of methyltransferases is responsible for the implementation of histone H3 lysine 4 (H3K4) methylation, an epigenetic mark associated with transcriptionally active chromatin. Multiple genome-wide sequencing efforts reported that subunits of the COMPASS family are frequently mutated in large number of cancers, neurological disorders, and other diseases. Therefore, understanding COMPASS function will lend important insights into the underlying mechanisms of disease pathogenesis, which is necessary for the development of effective targeted therapeutics. The H3K4 methylase Set1A is one of six COMPASS family members identified in mammals, and has been shown to be involved in bulk H3K4 di- and trimethylation (H3K4me2/me3 respectively) across the genome. Previous studies demonstrated that the loss of Set1A protein resulted in embryonic lethality and embryonic stem cell (ESC) proliferation defects, suggesting that Set1A is required for this process; however, the molecular context in which Set1A functions is primarily unknown. To distinguish between a role for full-length Set1A vs. its catalytic domain, the catalytic domain of Set1A was deleted in ESCs, which resulted in the very surprising discovery that its enzymatic function appears to be dispensable for ESC viability and self-renewal. However, ESCs bearing catalytically dead Set1A seemed unable to properly differentiate, suggesting multiple roles for Set1A and its catalytic activity in ESC self-renewal and subsequent differentiation. Together, these preliminary data support further investigation into the role of Set1A in regulating stem cell pluripotency and development. Based on these results, the first aim of this proposal will further examine the enzymatic function of Set1A by analyzing transcriptional and H3K4 methylation changes in Set1A catalytic mutants during ESC differentiation and mouse development. The second aim will determine the critical domains of Set1A protein in maintaining ESC self-renewal via a conditional knockout and overexpression system. The third aim will further explore the protein interaction network of Set1A in ESCs to reveal novel mechanisms underlying the functions of Set1A in early development. This work will illuminate the basic functional significance of Set1A in stem cells and in development, which will elucidate its disease liability and ultimately facilitate treatment development.