DEFINING REGULATORY ROLES FOR HISTONE H3 METHYLATION IN DEVELOPMENT

项目总结 多能干细胞具有巨大的科学和治疗潜力，因为它们有能力 分化成成人体内的任何细胞。越来越多的证据表明，差异的部分驱动因素是 表观遗传机制，如有助于建立和随后维持细胞的组蛋白修饰 身份。然而，通过传统的方法证明单个组蛋白修饰的直接作用是具有挑战性的。 突变的方法是因为哺乳动物中存在多个典型的组蛋白基因副本 基因组。此外，许多组蛋白标记受到几种多余的酶的调节，这些酶很难 同时在生理环境中不安。我们研究的长期目标是解决这一角色 在体内和组织培养系统中，组蛋白修饰在指导细胞命运中的作用。我们的方法是 创新是因为它通过利用赖氨酸到蛋氨酸(K-M)来克服目前该领域的限制 组蛋白H3上的TO-M)突变，在各自的位点上充当甲基化的显性负抑制物。 这项资助的目的是描述H3K9和H3K36上甲基化的功能，这两个基因会发生变化 在分化和发育过程中戏剧性地发生。我们的中心假设是H3K9和H3K36甲基化 在发展转型中发挥着独特而关键的作用。为了验证这一假设，我们将表达突变 组蛋白，H3K9M和H3K36M，在早期胚胎和多能干细胞中。具体地说，我们将跟踪 H3K9和H3K36甲基化抑制后母体向合子的转变和早期谱系决定 在胚胎中(项目1)。然后，我们将应用体外细胞培养系统来研究 K-M突变对染色质和基因表达的影响(项目2)。我们方法的一个关键特点是 突变的组蛋白的表达是多西环素诱导的，这允许我们的组蛋白的诱导或撤销。 以组织和时间特定的方式突变。使用此工具，我们将询问细胞是否有能力 重建组蛋白修饰以挽救突变的组蛋白退出后的正确分化(项目3)。 总体而言，这项工作意义重大，因为它将提供对组蛋白功能作用的宝贵见解 细胞命运中的修饰会改变。了解控制干细胞功能的调节机制是一个 这是实现其巨大潜力的关键一步。因此，我们预计拟议的工作将具有 对基础科学和医学都有重要意义。

PROJECT SUMMARY Pluripotent stem cells hold tremendous scientific and therapeutic potential because they have the capacity to differentiate into any cell in the adult body. Mounting evidence suggests that differentiation is driven, in part, by epigenetic mechanisms such as histone modifications that help to establish and subsequently maintain cell identity. However, demonstrating a direct role for an individual histone modification is challenging via traditional mutagenesis approaches because multiple copies of canonical histone genes are present in the mammalian genome. Moreover, many histone marks are regulated by several, redundant enzymes, which are difficult to perturb simultaneously and in a physiological context. The long-term goal of our research is to resolve the role of histone modifications in directing cell fate, both in vivo and in tissue culture systems. Our approach is innovative because it overcomes current limitations in the field by taking advantage of lysine-to-methionine (K- to-M) mutations on histone H3, which act as dominant negative inhibitors of methylation at their respective sites. The objective of this grant is to characterize the function of methylation on H3K9 and H3K36, which change dramatically during differentiation and development. Our central hypothesis is that H3K9 and H3K36 methylation have distinct and crucial roles in developmental transitions. To test this hypothesis, we will express mutant histones, H3K9M and H3K36M, in early embryos and pluripotent stem cells. Specifically, we will track the maternal to zygotic transition and early lineage decisions following suppression of H3K9 and H3K36 methylation in embryos (Project 1). We will then apply in vitro cell culture systems to investigate the molecular basis for the effects of K-to-M mutants on chromatin and gene expression (Project 2). A key feature of our approach is that expression of the mutant histones is doxycycline-inducible, which permits induction or withdrawal of our histone mutants in a tissue- and time-specific manner. Using this tool, we will ask whether cells are capable of reestablishing histone modifications to rescue proper differentiation after mutant histone withdrawal (Project 3). Collectively, this work is significant because it will provide valuable insight into the functional role of histone modifications in cell fate change. Understanding the regulatory mechanisms that control stem cell function is a crucial step in realizing their tremendous potential. We therefore anticipate that the proposed work will have important implications for both basic science and medicine.