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

项目摘要 细胞命运的改变最终通过获得特定细胞类型的基因表达而发生 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产物，而几种酶可以去除这些 马克使用代谢物作为其酶反应的一部分。因此，细胞代谢活动可以塑造 基因表达程序通过对染色质组织的代谢物依赖效应来实现。一个健壮的 基因调控网络和允许的染色质景观是天真多能性的标志 胚胎干细胞(ESCs)的状态，但细胞内代谢途径如何对 这种独特的染色质景观的建立仍然不清楚。我们之前的工作证明了 幼稚的小鼠胚胎干细胞处于多能性的基础状态，改变了它们的代谢流量，以支持更大的 代谢产物α-酮戊二酸(αKG)的胞内池与其更承诺的 对口单位。用外源性、细胞透性的αKG补充更可靠的ESCs 足以增加自我更新。然而，幼稚的胚胎干细胞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，仍然是悬而未决的问题。 这项研究建议的目的是确定支持αKG积累和 确定αKG促进自我更新的机制。PI3K/Akt信号轴是一个很好的- 已知的细胞新陈代谢调节器，并已被证明支持ESC自我更新。是否 该信号轴在胚胎干细胞代谢中的作用，特别是对αKG的调节尚不清楚。 使用质谱分析结合药理学和遗传学方法，我们将 验证Akt信号介导的葡萄糖氧化增加是 在幼稚的ESCs中观察到αKG积聚。鉴于αKG作为预留共基质用于 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的积累导致在Nanog位点的抑制性染色质标记的丢失， 一种核心多能转录因子，从而推动Nanog表达增强和稳定性 与多能性相关的基因调控网络。我们将使用遗传学和药理学 确定αKG蓄积是否通过增强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胚胎干细胞是如何 将代谢途径与多能性基因调控网络和意志相结合 提供有关代谢调节如何影响细胞特性变化的重要见解。

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.