DEFINING REGULATORY ROLES FOR HISTONE H3 METHYLATION IN DEVELOPMENT

项目名称：确定组蛋白H3甲基化在发育中的调控作用 主要研究者：Justin Brumbaugh，科罗拉多大学博尔德分校 我提交这份文件是为了申请对我的NIH R35（R35GM142884）的行政补充 该奖项名为“定义组蛋白H3甲基化在发育中的调节作用”。这一要求在 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月获得了早期研究者R35/MIRA补助金。我 目前，我正处于融资期的第一年。因此，我们尚没有未支配资金需要报告; 然而，根据我们目前的支出率，我们预计我们将有大约20,000 - 40000美元 这些未支配资金将进入第二年，其中大部分是由于与COVID相关的实验室限制。我们计划 用这些资金来测序我们在过去几个月里收集的大规模基因组样本 正在等待分析。 家长奖励汇总 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， 部分是由表观遗传机制驱动的，如组蛋白修饰，有助于建立和 随后保持小区身份。然而，证明单个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，因为多个拷贝的典型组蛋白基因被 存在于哺乳动物基因组中。此外，许多组蛋白标记受几个冗余的 酶，这是很难同时和在生理环境中扰动。我们的长期目标是 研究的目的是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 系统.我们的方法是创新的，因为它克服了该领域目前的局限性， 组蛋白H3上的赖氨酸到甲硫氨酸（K-to-M）突变，其作为显性负性抑制剂， 甲基化在各自的位置。这项资助的目的是描述甲基化在 残基H3K9和H3K36在分化和发育过程中发生显著变化。我们的中央 假设H3K9和H3K36甲基化在发育转变中具有不同且关键的作用。到 为了验证这一假设，我们将在早期胚胎中表达突变组蛋白H3K9M和H3K36M， 干细胞具体地说，我们将跟踪母系到合子的过渡和早期谱系决定， 胚胎中H3K9和H3K36甲基化的抑制。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。一个关键 我们的方法的特点是突变组蛋白的表达是强力霉素诱导的， 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。使用这个工具，我们会问 细胞是否能够重建组蛋白修饰以挽救突变后的适当分化 组蛋白撤退。总的来说，这项工作是重要的，因为它将提供宝贵的见解， 组蛋白修饰在细胞命运改变中的功能作用。了解监管机制， 控制干细胞功能是实现其巨大潜力的关键一步。因此，我们预计， 这项工作将对基础科学和医学产生重要影响。 简要介绍迄今取得的进展（目前状况）。我们的父母补助金的主要目的是澄清如何特别

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