Coupling metabolic pathways with pluripotent gene regulation in mouse embryonic stem cells

小鼠胚胎干细胞中代谢途径与多能基因调控的耦合

• 批准号: 9760563
• 负责人: Paige Arnold
• 金额: $ 4.5万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-24 至 2022-06-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9760563
• 关键词: AKT Signaling Pathway; Address; Automobile Driving; Biochemical Reaction; Cells; Cellular Metabolic Process; Chemicals; Chromatin; Coupling; DNA; DNA Methylation; Deposition; Development; Enzymes; Epiblast; Excision; Formulation; Gene Expression; Gene Expression Regulation; Genetic; Glucose; Histone H3; Histones; Inner Cell Mass; Lysine; Mediating; Metabolic; Metabolic Pathway; Metabolism; Modification; Mus; Pathway interactions; Permeability; Pharmacology; Phenotype; Play; Production; Regulation; Regulator Genes; Research; Research Proposals; Role; Shapes; Signal Pathway; Signal Transduction; Stimulus; Supplementation; Testing; Work; alpha ketoglutarate; cell type; chromatin modification; embryonic stem cell; enzyme substrate; experimental study; genetic approach; glucose metabolism; histone demethylase; improved; insight; oxidation; pluripotency; preimplantation; preservation; programs; self-renewal; transcription factor

Project Summary Changes in cell fate ultimately occur through the acquisition of cell type-specific gene expression programs that are enabled by cooperation between the chromatin landscape and transcription factor availability. The deposition and removal of the chemical modifications that decorate chromatin require metabolites that are intermediates of metabolic pathways, while several enzymes that remove these marks use metabolites as part of their enzymatic reaction. Thus, cellular metabolic activity can shape gene expression programs through metabolite-dependent effects on chromatin organization. A robust gene regulatory network and permissive chromatin landscape are hallmarks of the naïve pluripotent state in embryonic stem cells (ESCs), yet how intracellular metabolic pathways contribute to the establishment of this distinct chromatin landscape remains unclear. Our previous work demonstrated that naïve mouse ESCs in the ground state of pluripotency alter their metabolic flux to support larger intracellular pools of the metabolite alpha-ketoglutarate (αKG) compared to their more committed counterparts. Supplementation of more committed ESCs with exogenous, cell-permeable αKG is sufficient to increase self-renewal. However, how naïve ESCs rewire metabolic pathways to promote αKG accumulation, and how αKG enhances self-renewal, remain open questions. The aim of this research proposal is to identify the pathways that support αKG accumulation and determine the mechanism by which αKG promotes self-renewal. The PI3K/Akt signaling axis is a well- known regulator of cellular metabolism and has been shown to support ESC self-renewal. Whether this signaling axis plays a role in ESC metabolism, particularly αKG regulation, remains unexplored. Using mass spectrometric analysis combined with pharmacologic and genetic approaches, we will test the hypothesis that increased glucose oxidation mediated by Akt signaling is a major driver of the αKG accumulation observed in naïve ESCs. Given that αKG serves as an obligate co-substrate for multiple enzymes that catalyze the removal of DNA methylation and repressive histone marks, we hypothesize that αKG accumulation drives loss of repressive chromatin marks at the locus of Nanog, a core pluripotency transcription factor, thereby driving increased Nanog expression and stabilization of the pluripotency-associated gene regulatory network. We will use genetic and pharmacologic approaches to determine whether αKG accumulation stimulates self-renewal by enhancing Nanog expression through a chromatin-mediated mechanism. These studies will address how mouse ESCs couple metabolic pathways with regulation of the pluripotency gene regulatory network and will provide critical insight into how metabolic regulation contributes to changes in cell identity.

项目概要 细胞命运的改变最终通过获得细胞类型特异性基因表达而发生 通过染色质景观和转录因子之间的合作实现的程序 可用性。装饰染色质所需的化学修饰的沉积和去除 代谢物是代谢途径的中间体，而一些酶可以去除这些代谢物 标记使用代谢物作为酶反应的一部分。因此，细胞代谢活动可以塑造 通过对染色质组织的代谢依赖性影响来进行基因表达程序。坚固耐用 基因调控网络和允许的染色质景观是幼稚多能性的标志 胚胎干细胞 (ESC) 的状态，但细胞内代谢途径如何促进 这种独特的染色质景观的建立仍不清楚。我们之前的工作证明了 处于多能性基态的幼稚小鼠 ESC 会改变其代谢通量以支持更大的 与更稳定的代谢物 α-酮戊二酸 (αKG) 的细胞内池相比 同行。向更稳定的 ESC 补充外源性、细胞可渗透的 αKG 是 足以增强自我更新能力。然而，幼稚的 ESC 如何重新连接代谢途径以促进 αKG 的积累，以及 αKG 如何增强自我更新，仍然是一个悬而未决的问题。 本研究计划的目的是确定支持 αKG 积累和 确定αKG促进自我更新的机制。 PI3K/Akt 信号轴是一个良好的 已知的细胞代谢调节剂，并已被证明支持 ESC 自我更新。无论 该信号轴在 ESC 代谢中发挥作用，特别是 αKG 调节，目前尚未被探索。 使用质谱分析结合药理学和遗传学方法，我们将 检验这样的假设：Akt 信号传导介导的葡萄糖氧化增加是 在幼稚 ESC 中观察到 αKG 积累。鉴于 αKG 作为专性共底物 多种酶催化 DNA 甲基化和抑制性组蛋白标记的去除，我们 假设 αKG 积累导致 Nanog 基因座上抑制性染色质标记的丢失， 核心多能性转录因子，从而驱动 Nanog 表达增加和稳定性 多能性相关基因调控网络。我们将利用遗传和药理学 确定 αKG 积累是否通过增强 Nanog 刺激自我更新的方法 通过染色质介导的机制表达。这些研究将解决小鼠 ESC 如何 将代谢途径与多能性基因调控网络的调控结合起来，并将 提供关于代谢调节如何影响细胞特性变化的重要见解。