Metabolic control of exit from naïve pluripotency

项目摘要 细胞代谢途径在发育和发育过程中的不同细胞类型之间表现出显着的可塑性， 疾病除了伴随细胞状态的变化，代谢重新布线已被证明可以驱动细胞命运 通过改变染色质景观来决定程序。装饰性化学修饰的沉积 染色质需要代谢途径的中间产物，而去除这些标记的几种酶使用 代谢物作为其酶促反应的一部分。因此，代谢物水平的波动有能力 形成染色质以影响细胞命运特异性基因表达，但驱动染色质的代谢变化 在早期发育过程中，重组和介导细胞命运代谢控制的酶仍然存在， 大部分未知。我们以前已经确定了特定的代谢物，控制自我更新的小鼠胚胎 干细胞（ESCs）。当胚胎干细胞退出自我更新的多能状态时，代谢是否改变， 这些代谢变化是多谱系分化所必需的，仍然是一个悬而未决的问题。 这项研究计划的目标是描述在退出过程中发生的代谢重新布线， 幼稚的多能性，并确定这种重新连接控制小鼠ESC的机制 分化我们的初步数据表明，退出幼稚多能性伴随着增加 在线粒体柠檬酸盐的输出中。在目标1中，我们将使用遗传学和药理学方法来靶向 线粒体柠檬酸转运蛋白SLC 25 A1或下游柠檬酸分解代谢酶ATP-柠檬酸裂解酶， 检验早期分化需要来源于尿道的柠檬酸盐的假设。我们将调查 这种代谢变化是否通过柠檬酸盐衍生的组蛋白乙酰化的沉积来调节细胞命运 标记.初步数据还显示，细胞氧化还原状态的变化，其标志是细胞质中 在早期分化过程中的NAD+/NADH比率。在目标2中，我们将确定这种代谢变化是否是 通过使用药理学或遗传学工具调节NAD+/NADH比率来退出幼稚多能性。进一步 实验将确定细胞氧化还原状态向染色质景观发出信号的机制， 决定细胞的命运这些研究将揭示退出幼稚多能性过程中的代谢控制机制 并将提供关键的洞察如何代谢调节有助于细胞身份的变化， 胚胎发育本建议书中概述的工作和培训计划将在实验室完成 Lydia Finley博士与纪念斯隆凯特琳癌症中心的Kristian Helin博士合作， 将理想地准备申请人进一步的临床培训和职业生涯作为一个独立的医生，科学家。

PROJECT SUMMARY Cellular metabolic pathways exhibit remarkable plasticity across different cell types in both development and disease. In addition to accompanying changes in cell state, metabolic rewiring has been shown to drive cell fate decisions programs by altering the chromatin landscape. The deposition of chemical modifications that decorate chromatin requires the intermediates of metabolic pathways, and several enzymes that remove these marks use metabolites as part of their enzymatic reaction. Therefore, fluctuations in metabolite levels have the capacity to shape chromatin to effect cell fate-specific gene expression, but the metabolic changes that drive chromatin reorganization and the enzymes that mediate metabolic control of cell fate during early development remain largely unknown. We have previously identified specific metabolites that control self-renewal of mouse embryonic stem cells (ESCs). Whether metabolism is altered as ESCs exit the self-renewing pluripotent state, and whether these metabolic changes are required for multi-lineage differentiation remains an open question. The goal of this research proposal is to characterize the metabolic rewiring that occurs during exit from naïve pluripotency and to determine the mechanisms by which this rewiring controls mouse ESC differentiation. Our preliminary data indicate that exit from naïve pluripotency is accompanied by an increase in the mitochondrial export of citrate. In Aim 1, we will use genetic and pharmacologic approaches to target the mitochondrial citrate transporter SLC25A1 or the downstream citrate-catabolizing enzyme ATP-citrate lyase to test the hypothesis that mitochondrially-derived citrate is required for early differentiation. We will investigate whether this metabolic change regulates cell fate through the deposition of citrate-derived histone acetylation marks. Preliminary data also shows changes in cellular redox state marked by an increase in the cytosolic NAD+/NADH ratio during early differentiation. In Aim 2, we will determine if this metabolic change is required for exit from naïve pluripotency by modulating the NAD+/NADH ratio using pharmacological or genetic tools. Further experiments will identify the mechanism by which cellular redox state signals to the chromatin landscape to dictate cell fate. These studies will reveal the mechanisms of metabolic control during exit from naïve pluripotency and will provide critical insight into how metabolic regulation contributes to changes in cell identity during embryonic development. The work and training plan outlined in this proposal will be completed in the laboratory of Dr. Lydia Finley with the co-advisement of Dr. Kristian Helin at Memorial Sloan Kettering Cancer Center and will ideally prepare the applicant for further clinical training and a career as an independent physician-scientist.