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 空房的修饰染色质的化学修饰的沉积和去除需要 代谢物是代谢途径的中间体，而几种酶，消除这些 标记使用代谢物作为其酶促反应的一部分。因此，细胞代谢活动可以塑造 通过对染色质组织的代谢物依赖性作用的基因表达程序。一个强大 基因调控网络和允许的染色质景观是幼稚多能 胚胎干细胞（ESCs）中的状态，但细胞内代谢途径如何有助于 这种独特的染色质景观的建立仍然不清楚。我们之前的工作表明 处于多能性基态的幼稚小鼠胚胎干细胞改变了它们的代谢通量， 代谢产物α-酮戊二酸（αKG）的细胞内池与其更致力于 同行用外源性细胞渗透性αKG补充更多定型的ESC， 足以增强自我更新能力。然而，幼稚的胚胎干细胞如何重新连接代谢途径， 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。 本研究提案的目的是确定支持αKG积累的途径， 确定αKG促进自我更新的机制。PI 3 K/Akt信号轴是一个良好的- 已知是细胞代谢的调节剂，并已显示支持ESC自我更新。是否 该信号轴在ESC代谢中起作用，特别是αKG调节，仍然未被探索。 使用质谱分析结合药理学和遗传学方法，我们将 测试Akt信号介导的葡萄糖氧化增加是葡萄糖代谢的主要驱动力这一假设。 在未处理ESC中观察到αKG蓄积。考虑到αKG作为一种专性共底物， 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积累驱动Nanog位点抑制性染色质标记的丢失， 核心多能性转录因子，从而驱动增加Nanog表达和稳定 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胚胎干细胞 将代谢途径与多能性基因调控网络的调控相结合， 提供关键的洞察如何代谢调节有助于细胞身份的变化。

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.