DEFINING REGULATORY ROLES FOR HISTONE H3 METHYLATION IN DEVELOPMENT

项目摘要 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，差异化在一定程度上是由 表观遗传机制，如组蛋白修饰，有助于建立和随后维持细胞 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。 诱变方法，因为在哺乳动物中存在多个拷贝的典型组蛋白基因， 基因组此外，许多组蛋白标记物由几种多余的酶调节，这些酶很难被识别。 同时在生理环境中受到干扰。我们研究的长期目标是解决 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。我们的做法是 创新，因为它通过利用赖氨酸到甲硫氨酸（K-Met）克服了该领域的当前限制。 to-M）突变，其在其各自位点作为甲基化的显性负抑制剂。 这项资助的目的是表征H3 K9和H3 K36甲基化的功能， 在分化和发育过程中，我们的中心假设是H3 K9和H3 K36甲基化 在发展转型中具有独特而关键的作用。为了验证这一假设，我们将表达突变体 组蛋白，H3 K9 M和H3 K36 M，在早期胚胎和多能干细胞。具体来说，我们将跟踪 H3 K9和H3 K36甲基化抑制后母体向合子的转变和早期谱系决定 胚胎（1）。然后，我们将应用体外细胞培养系统来研究细胞凋亡的分子基础。 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强力霉素诱导的，这允许我们的组蛋白的诱导或撤回。 以组织和时间特异性的方式进行突变。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回后挽救适当的分化（项目3）。 总的来说，这项工作是有意义的，因为它将提供有价值的洞察组蛋白的功能作用 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 实现其巨大潜力的关键一步。因此，我们预计拟议的工作将有 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.