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

• 批准号: 10577382
• 负责人: Justin Brumbaugh
• 金额: $ 3.37万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/10577382
• 关键词: Administrative Supplement; Adult; Award; Basic Science; Cell Culture System; Cells; Chromatin; Colorado; Development; Disease model; Dominant-Negative Mutation; Doxycycline; Embryo; Enzymes; Epigenetic Process; Funding; Gene Expression; Genes; Genomics; Goals; Grant; Histone H3; Histones; In Vitro; Individual; Lysine; Medicine; Methionine; Methylation; Molecular; Mutagenesis; Mutation; Parents; Physiological; Pluripotent Stem Cells; Reporting; Research; Research Personnel; Role; Sampling; Site; System; Testing; Therapeutic; Time; Tissues; Transplant-Related Disorder; United States National Institutes of Health; Universities; Withdrawal; Work; base; cell type; coronavirus disease; directed differentiation; drug testing; histone modification; in vivo; inhibitor; innovation; insight; mammalian genome; mutant; parent grant; response; stem cell function; stem cells; tissue culture; tool

Project Title: Defining regulatory roles for histone H3 methylation in development Principle Investigator: Justin Brumbaugh, University of Colorado Boulder I am submitting this document is to apply for an administrative supplement to my NIH R35 (R35GM142884) award entitled, “Defining regulatory roles for histone H3 methylation in development.” This request is in response to NOT-GM-22-017 (PA-20-272). Financial status of current grant. I was awarded an early-stage investigator R35/MIRA grant in July 2021. I am currently in the first year of the funding period. As a result, we do not yet have unobligated funds to report; however, based on our current spending rate, we anticipate that we will have approximately $20,000-$40,000 of unobligated funds to carry into year 2, most of which are a result of COVID-related lab restrictions. We plan to use these funds to sequence large-scale genomics samples that we have collected over the past months and are awaiting analysis. Parent Award Summary 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 residues 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. 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. 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. 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. Brief progress to date (current status). The primary aims of our parent grant are to clarify how particular

项目名称：定义组蛋白H3甲基化的调节作用 主要调查员：科罗拉多大学博尔德大学贾斯汀·布鲁姆布 我提交的文件是向我的NIH R35（R35GM142884）申请管理补充 奖项题为“定义组蛋白H3甲基化的监管作用”。这个请求在 对NOT-GM-22-017（PA-20-272）的响应。 当前赠款的财务状况。我于2021年7月被授予早期调查员R35/Mira Grant。 目前在资助期的第一年。结果，我们尚未有未划定的资金来报告； 但是，根据我们目前的支出率，我们预计我们将拥有约20,000至40,000美元 在未受损害的资金中载入了2年级，其中大多数是与共同相关的实验室限制的结果。我们计划 使用这些资金对过去几个月中收集的大规模基因组样本进行测序 并正在等待分析。 家长奖摘要 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 具有分化成人体内任何细胞的能力。越来越多的证据表明差异化 部分地是由表观遗传机制（例如组蛋白修饰）驱动的，这些机制有助于建立和 随后保持细胞身份。但是，展示了单个Hisstone修改的直接作用 通过传统诱变方法具有挑战性，因为规范组蛋白基因的多个副本是 存在于哺乳动物基因组中。此外，许多Hisstone标记受几种多余的调节 酶，在物理环境中很难干扰。我们的长期目标 研究是在体内和组织培养中解决组蛋白修饰在指导细胞命运中的作用 系统。我们的方法是创新的，因为它通过利用了现场的当前限制 组蛋白H3上的赖氨酸到甲硫氨酸（K-TO-M）突变，它们是主要的负阴性抑制剂 甲基化各自的位置。该赠款的目的是表征甲基化的功能 残基H3K9和H3K36，它们在分化和发育过程中发生巨大变化。我们的中心 假设H3K9和H3K36甲基化在发育过渡中具有不同的至关重要的作用。到 检验该假设，我们将在早期胚胎和多能中表达突变组蛋白，H3K9M和H3K36M 干细胞。具体而言，我们将跟踪孕产妇至合子过渡和早期谱系决策。 胚胎中H3K9和H3K36甲基化的抑制。然后，我们将在体外细胞培养系统上应用于 研究K-TO-M突变体对染色质和基因表达的影响的分子基础。钥匙 我们方法的特征是突变组蛋白的表达是多西环素诱导的，允许 以组织和时间特异性的方式诱导或退出我们的Hisstone突变体。使用此工具，我们会问 细胞是否能够重新建立组蛋​​白修饰以挽救突变体后的适当分化 Hisstone退出。总的来说，这项工作是重要的，因为它将为您提供宝贵的见解 组蛋白修饰在细胞命运变化中的功能作用。了解监管机制 控制干细胞功能是实现其巨大潜力的关键步骤。因此，我们预计 拟议的工作将对基础科学和医学具有重要意义。 简短的进度到日期（当前状态）。我们父母赠款的主要目的是阐明