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 甲基化在发育中的调控作用 首席研究员：Justin Brumbaugh，科罗拉多大学博尔德分校 我提交此文件是为了向我的 NIH R35 (R35GM142884) 申请行政补充 奖项标题为“定义组蛋白 H3 甲基化在发育中的调控作用”。 对 NOT-GM-22-017 (PA-20-272) 的回应。 当前资助的财务状况。我于 2021 年 7 月获得了早期研究员 R35/MIRA 资助。 目前正处于资助期的第一年，因此，我们还没有需要报告的未承付资金； 然而，根据我们目前的支出率，我们预计我们将有大约 20,000-40,000 美元 结转至第二年的未偿付资金，其中大部分是由于与新冠病毒相关的实验室限制而造成的。 使用这些资金对我们在过去几个月收集的大规模基因组样本进行测序 并正在等待分析。 家长奖励总结 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 越来越多的证据表明，具有分化成成人体内任何细胞的能力。 部分是由表观遗传机制驱动的，例如组蛋白修饰，有助于建立和 然而，随后维持细胞身份，证明了个体组蛋白修饰的直接作用。 通过传统的诱变方法是具有挑战性的，因为经典组蛋白基因的多个拷贝是 此外，许多组蛋白标记都受到多个冗余的调节。 酶，在生理背景下很难同时扰乱我们的长期目标。 研究旨在解决组蛋白修饰在体内和组织培养中指导细胞命运的作用 我们的方法是创新的，因为它通过利用优势克服了该领域当前的限制。 组蛋白 H3 上的赖氨酸到蛋氨酸 (K-to-M) 突变，作为显性失活抑制剂 此项资助的目的是表征甲基化在各自位点的功能。 残基 H3K9 和 H3K36，在分化和发育过程中发生巨大变化。 假设 H3K9 和 H3K36 甲基化在发育转变中具有独特且关键的作用。 为了检验这个假设，我们将在早期胚胎和多能性中表达突变组蛋白 H3K9M 和 H3K36M 具体来说，我们将跟踪母体到合子的转变以及随后的早期谱系决定。 抑制胚胎中的 H3K9 和 H3K36 甲基化然后我们将应用体外细胞培养系统。 研究 K-to-M 突变体对染色质和基因表达影响的分子基础。 我们方法的特点是突变组蛋白的表达是多西环素诱导的，这使得 使用此工具，我们将询问以组织和时间特异性方式诱导或撤回我们的组蛋白突变体。 突变后细胞是否能够重建组蛋白修饰以挽救正确的分化 总的来说，这项工作意义重大，因为它将提供对组蛋白撤退的宝贵见解。 了解组蛋白修饰在细胞命运改变中的功能作用。 因此，我们预计，控制干细胞功能是实现其巨大潜力的关键一步。 拟议的工作将对基础科学和医学产生重要影响。 迄今为止的简要进展（当前状态） 我们的家长补助金的主要目的是阐明具体情况。