DEFINING REGULATORY ROLES FOR HISTONE H3 METHYLATION IN DEVELOPMENT

项目概要 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，差异化部分是由 表观遗传机制，例如组蛋白修饰，有助于建立并随后维持细胞 身份。然而，通过传统方法证明单个组蛋白修饰的直接作用具有挑战性。 诱变方法是因为哺乳动物中存在多个典型组蛋白基因拷贝 基因组。此外，许多组蛋白标记是由几种多余的酶调节的，而这些酶很难被调节。 同时在生理背景下扰乱。我们研究的长期目标是解决这个问题 组蛋白修饰在体内和组织培养系统中指导细胞命运的研究。我们的方法是 创新性在于它利用赖氨酸转蛋氨酸（K- to-M) 组蛋白 H3 上的突变，其在各自位点充当甲基化的显性负性抑制剂。 本次资助的目的是表征 H3K9 和 H3K36 甲基化的功能，这会改变 在分化和发育过程中显着。我们的中心假设是 H3K9 和 H3K36 甲基化 在发展转型中具有独特且关键的作用。为了检验这个假设，我们将表达突变体 早期胚胎和多能干细胞中的组蛋白 H3K9M 和 H3K36M。具体来说，我们将跟踪 抑制 H3K9 和 H3K36 甲基化后母体到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其分子基础。 K-to-M 突变体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征是 突变组蛋白的表达是多西环素诱导的，这允许诱导或撤回我们的组蛋白 以组织和时间特异性方式产生突变体。使用这个工具，我们将询问细胞是否能够 重建组蛋白修饰以在突变组蛋白撤除后挽救正确的分化（项目 3）。 总的来说，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 细胞命运改变的修饰。了解控制干细胞功能的调节机制是一个关键 实现其巨大潜力的关键一步。因此，我们预计拟议的工作将 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.