Connecting TCA cycle flux and epigenetic regulation of hematopoiesis

连接 TCA 循环通量和造血的表观遗传调控

基本信息

批准号：
10383136
负责人：
Dalton Lee Greenwood
金额：
$ 3.07万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10383136
关键词：
ATP Citrate (pro-S)-Lyase Acetates Acetyl Coenzyme A Acetylation Acetylesterase Acute Myelocytic Leukemia Affect Binding Sites Biological Assay Blood Bone Marrow CRISPR screen Carbon Cell Culture Techniques Cell Lineage Cells Characteristics Chromatin Citrates Citric Acid Cycle Consensus DNA Data Dioxygenases Enzymes Epigenetic Process Equilibrium Erythroid Cells Flow Cytometry Fumarates Gene Expression Gene Expression Profile Generations Genes Genetic Transcription Glucose Glutamates Glutaminase Glutamine Hematologic Neoplasms Hematological Disease Hematopoiesis Hematopoietic Hematopoietic stem cells Histone Acetylation Histones Homeostasis ITGAM gene In Vitro Link Lymphoid Cell Malignant Neoplasms Measures Mediating Metabolic Metabolic Pathway Metabolism Methylation Methylcellulose Modification Mus Mutation Myelogenous Myeloid Cells Myeloid Progenitor Cells NMR Spectroscopy Oxaloacetates Oxidative Phosphorylation Play Population Process Reaction Regulation Regulator Genes Research Role Stimulus Succinates T cell differentiation Testing alpha ketoglutarate cellular development chemokine computerized tools conditional knockout cytokine demethylation effector T cell enzyme substrate epigenetic regulation experimental study extracellular hematopoietic differentiation hematopoietic stem cell differentiation hematopoietic stem cell fate hematopoietic stem cell self-renewal histone acetyltransferase histone methylation histone modification in vivo knockout animal methylation pattern mouse model multiple omics new therapeutic target novel therapeutics prevent programs reaction rate self-renewal single-cell RNA sequencing stem cell function stem cell self renewal stem cells stemness therapeutic target transcription factor transcriptome

项目摘要

Summary Hematopoietic Stem Cells (HSCs) possess distinct metabolic programs that regulate decisions to self-renew or differentiate. Metabolic pathways are now recognized to modulate epigenetic marks through accessibility of metabolic intermediates as substrates, including α-ketoglutarate (αKG) and acetyl-CoA for post-translational demethylation or acetylation, respectively. Recent research suggests that perturbations of glutamine and acetate metabolism may provoke lineage-specific differentiation by altering epigenetic-mediated chromatin accessibility and gene expression responsible for lineage determination. Indeed, data from the Rathmell lab demonstrates that disruption of Glutaminase (GLS), the entry point of glutamine into the metabolic pool responsible for catalyzing glutamine to glutamate, changes histone methylation patterns to promote Th1 and inhibit Th17 CD4+ effector T-cell differentiation, altering accessibility of the loci of cytokines Ifng and Il17. GLS processes glutamine to replenish the carbon pool of the tricarboxylic acid (TCA) cycle, contributing to TCA cycle intermediates that also regulate epigenetic modifying reactions. Specifically, αKG, succinate, and fumarate serve as regulators and substrates of histone and DNA demethylation enzymes. Similarly, ATP citrate lyase (ACLY) connects TCA cycle flux with the histone acetylation substrate pool by catalyzing cytosolic citrate into acetyl- CoA. Maintaining both acetyl-CoA and αKG levels is crucial to epigenetic homeostasis, as reduced epigenetic enzyme substrates and regulators have been shown to broadly limit epigenetic modifying reaction rates. Preliminary data suggest that inhibiting ACLY promotes myeloid differentiation in cultured murine hematopoietic stem cells (HSCs). Here, I propose to use conditional knockout animals previously analyzed for T cell differentiation to disrupt GLS and ACLY in HSCs and LSCs and test the role of these enzymes in myeloid differentiation. I hypothesize that disrupting ACLY and GLS will inhibit stem cell self-renewal while promoting myelomonocytic differentiation. I will tackle this central hypothesis through two aims. Aim 1: Determine how GLS or ACLY deficiency is sufficient to modulate HSC self-renewal and differentiation. This first aim represents a functional characterization of HSC self-renewal and differentiation both in vitro and in vivo, utilizing stem cell culture, flow cytometry, and CRISPR screen experiments. Aim 2: Establish how epigenetic modification, gene regulator networks, and metabolic activity alter with GLS and ACLY deficiency in HSCs. The second aim focuses on the mechanism behind changes in stem cell self-renewal and differentiation examined in Aim 1. We will assess changes in chromatin accessibility, histone modifications, transcriptome profiles, and metabolite concentrations to determine how GLS and ACLY deficiencies impact stem cell regulatory networks. This project has the potential to uncover new interactions between epigenetics and metabolism in normal hematopoiesis and acute myeloid leukemia with the prospect of identifying novel therapeutic targets for hematologic malignancies.

概括造血干细胞 (HSC) 具有独特的代谢程序，可调节自我更新或自我更新的决定现在认识到代谢途径可以通过可及性来调节表观遗传标记。代谢中间体作为底物，包括用于翻译后的 α-酮戊二酸 (αKG) 和乙酰辅酶 A 最近的研究分别表明谷氨酰胺和乙酰化的干扰。醋酸盐代谢可能通过改变表观遗传介导的染色质来引发谱系特异性分化事实上，数据来自 Rathmell 实验室。表明谷氨酰胺酶 (GLS) 被破坏，谷氨酰胺进入代谢库的入口点负责催化谷氨酰胺转化为谷氨酸，改变组蛋白甲基化模式以促进 Th1 和抑制 Th17 CD4+ 效应 T 细胞分化，改变细胞因子 Ifng 和 Il17 位点的可及性。处理谷氨酰胺以补充三羧酸（TCA）循环的碳库，为TCA循环做出贡献还调节表观遗传修饰反应的中间体，具体来说，αKG、琥珀酸和富马酸。类似地，ATP 柠檬酸裂解酶 (ACLY) 作为组蛋白和 DNA 去甲基化酶的调节剂和底物。通过将胞质柠檬酸盐催化成乙酰基，将 TCA 循环通量与组蛋白乙酰化底物库连接起来维持乙酰辅酶 A 和 αKG 水平对于表观遗传稳态至关重要，因为表观遗传水平降低。酶底物和调节剂已被证明可以广泛限制表观遗传修饰反应速率。初步数据表明，抑制 ACLY 可促进培养小鼠造血细胞的骨髓分化在这里，我建议使用之前分析过的 T 细胞的条件敲除动物。分化破坏 HSC 和 LSC 中的 GLS 和 ACLY，并测试这些酶在骨髓中的作用我认为破坏 ACLY 和 GLS 会抑制干细胞的自我更新，同时促进干细胞的自我更新。我将通过两个目标来解决这一中心假设：确定如何分化。 GLS 或 ACLY 缺乏足以调节 HSC 自我更新和分化，这是第一个目标。代表 HSC 在体外和体内自我更新和分化的功能特征，利用干细胞培养、流式细胞术和 CRISPR 筛选实验目标 2：确定表观遗传的机制。修饰、基因调节网络和代谢活性随着 GLS 和 ACLY 缺乏而改变 HSCs的第二个目标集中于干细胞自我更新和分化变化背后的机制。在目标 1 中进行检查。我们将评估染色质可及性、组蛋白修饰、转录组的变化概况和代谢物浓度，以确定 GLS 和 ACLY 缺陷如何影响干细胞调节该项目有可能揭示表观遗传学和新陈代谢之间的新相互作用。正常造血和急性髓系白血病，有望确定新的治疗靶点血液系统恶性肿瘤。