DEFINING REGULATORY ROLES FOR HISTONE H3 METHYLATION IN DEVELOPMENT

定义组蛋白 H3 甲基化在发育中的调控作用

基本信息

批准号：
10622583
负责人：
Justin Brumbaugh
金额：
$ 38.5万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-15 至 2026-05-31
项目状态：
未结题

项目摘要

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.

项目摘要多能干细胞具有巨大的科学和治疗潜力，因为它们具有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。但是，通过传统，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是因为哺乳动物中存在多个规范组蛋白基因的副本基因组。此外，许多组蛋白标记受几种冗余酶的调节，这些酶很难同时且在生理环境中扰动。我们研究的长期目标是解决角色在体内和组织培养系统中指导细胞命运中的组蛋白修饰。我们的方法是创新性是因为它通过利用赖氨酸到甲硫氨酸来克服现场的当前局限性（k- TO-M）组蛋白H3的突变，它们在其各自的部位起主要的甲基化负面抑制剂。该赠款的目的是表征H3K9和H3K36上甲基化的功能，这会改变在分化和发展过程中急剧。我们的中心假设是H3K9和H3K36甲基化在发展过渡中具有独特的至关重要的作用。为了检验这一假设，我们将表达突变体组蛋白，H3K9M和H3K36M，在早期胚胎和多能干细胞中。具体来说，我们将跟踪抑制H3K9和H3K36甲基化后，孕产妇至合子过渡和早期谱系决策在胚胎（项目1）中。然后，我们将应用体外细胞培养系统来研究 K-TO-M突变体对染色质和基因表达的影响（项目2）。我们方法的关键特征是突变组蛋白的表达是多西环素诱导的，它允许诱导或退出我们的组蛋白以组织和时间特异性方式的突变体。使用此工具，我们将询问单元是否能够重建组蛋白修饰以挽救突变组蛋白退出后的适当分化（项目3）。总的来说，这项工作很重要，因为它将为组蛋白的功能作用提供宝贵的见解细胞命运变化的修改。了解控制干细胞功能的调节机制是一个意识到他们巨大潜力的关键步骤。因此，我们预计拟议的工作将有对基础科学和医学的重要意义。