Connecting TCA cycle flux and epigenetic regulation of hematopoiesis

摘要 造血干细胞(HSCs)具有独特的新陈代谢程序，调节自我更新或 差异化。新陈代谢途径现在被认为通过可及性调节表观遗传标记 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 分别去甲基化或乙酰化。最近的研究表明，谷氨酰胺和谷氨酰胺的扰动 醋酸盐代谢可能通过改变表观遗传介导的染色质而引发谱系特异性分化 可获得性和基因表达决定了血统。事实上，来自Rathmell实验室的数据 证明破坏谷氨酰胺酶(GLS)是谷氨酰胺进入代谢池的切入点 负责将谷氨酰胺催化为谷氨酸，改变组蛋白甲基化模式以促进Th1和Th1 抑制Th17 CD4+效应器T细胞分化，改变细胞因子IFNG和IL17基因的可及性。GLS 处理谷氨酰胺以补充三羧酸(TCA)循环的碳库，有助于TCA循环 也调节表观遗传修饰反应的中间体。具体地说，αKG、琥珀酸和富马酸 作为组蛋白和DNA去甲基化酶的调节剂和底物。同样，三磷酸腺苷柠檬酸裂解酶(ACLY) 通过催化胞液柠檬酸转化为乙酰基，将TCA循环通量与组蛋白乙酰化底物池连接起来. 可口可乐。维持乙酰辅酶A和αKG水平对于表观遗传动态平衡至关重要，因为表观遗传减少了 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度。 初步数据表明，抑制ACLY可促进培养的小鼠造血细胞的髓系分化 干细胞(HSCs)。在这里，我建议使用先前分析过的T细胞的条件性基因敲除动物 分化干扰HSCs和LSCs中的GLS和ACLY并检测这些酶在髓系中的作用 差异化。我推测，干扰ACLY和GLS将抑制干细胞的自我更新，同时促进 骨髓单核细胞分化。我将通过两个目标来解决这一核心假设。目标1：确定如何 GLS或ACLY缺乏足以调节HSC的自我更新和分化。这是第一个目标 代表了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、CRISPR筛查实验。目标2：确定表观遗传学 GLS和ACLY缺乏症患者的基因修饰、基因调控网络和代谢活性改变 造血干细胞。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。 我们将评估染色质可及性、组蛋白修饰、转录组的变化 和代谢物浓度来确定GLS和ACLY缺陷如何影响干细胞调节 网络。这个项目有可能发现表观遗传学和新陈代谢之间的新的相互作用 正常造血和急性髓系白血病寻找新的治疗靶点的前景 恶性血液病。

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.