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