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

• 批准号: 10397700
• 负责人: Dalton Lee Greenwood
• 金额: $ 2.89万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10397700
• 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; gene regulatory network; 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

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.

总结 造血干细胞（HSC）具有不同的代谢程序，调节自我更新或再生的决定。 区分。代谢途径现在被认为是调节表观遗传标记，通过可及性， 代谢中间体作为底物，包括α-酮戊二酸（α KG）和乙酰辅酶A，用于翻译后 脱甲基化或乙酰化。最近的研究表明，谷氨酰胺和 醋酸盐代谢可能通过改变表观遗传介导的染色质引起谱系特异性分化 可接近性和基因表达负责谱系决定。事实上，来自拉斯梅尔实验室的数据 表明谷氨酰胺酶（GLS）的破坏，谷氨酰胺进入代谢池的入口点， 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， 抑制Th17 CD4+效应T细胞分化，改变细胞因子Ifng和I117基因座的可及性。GLS 处理谷氨酰胺以补充三羧酸（TCA）循环的碳库，促进TCA循环 中间体也调节表观遗传修饰反应。具体来说，α KG、琥珀酸盐和富马酸盐 作为组蛋白和DNA去甲基化酶的调节剂和底物。类似地，ATP柠檬酸裂解酶（ACLY） 将TCA循环通量与组蛋白乙酰化底物池连接， 检验报告。维持乙酰辅酶A和α KG水平对表观遗传稳态至关重要，因为表观遗传稳态降低， 酶底物和调节剂已经显示出广泛地限制表观遗传修饰反应速率。 初步数据表明，抑制ACLY可促进培养的小鼠造血细胞的髓样分化。 干细胞（HSC）。在这里，我建议使用条件敲除动物先前分析的T细胞 本发明的目的是为了使HSC和LSC中的GLS和ACLY分化破坏，并测试这些酶在髓样细胞中的作用。 分化我假设，破坏ACLY和GLS将抑制干细胞自我更新，同时促进干细胞自我更新。 骨髓单核细胞分化我将通过两个目标来处理这个中心假设。目标1：确定如何 GLS或ACLY缺陷足以调节HSC自我更新和分化。第一个目标 代表了体外和体内HSC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 基因修饰、基因调控网络和代谢活性随着GLS和ACLY缺乏而改变。 HSC。第二个目标集中在干细胞自我更新和分化变化背后的机制 在目标1中检查。我们将评估染色质可及性、组蛋白修饰、转录组 谱和代谢物浓度，以确定GLS和ACLY缺陷如何影响干细胞调节 网络.该项目有可能揭示表观遗传学和代谢之间的新的相互作用， 正常造血和急性髓性白血病，具有确定新的治疗靶点的前景， 血液恶性肿瘤