基于TurboID技术揭示LSD1通过激活氧化磷酸化调控多能性退出的具体机制

LSD1可以通过调控细胞能量代谢抑制多能性重建和促进多能性退出。因此，申请人提出：“LSD1通过影响能量代谢关键基因上的表观修饰调控多能干细胞由原始态向始发态的转变（NPT）”的假说。由于LSD1在胚胎干细胞中多组ChIP-seq数据之间重复性差，难以直接用于鉴定LSD1的调控位点及下游靶标基因。申请人计划利用TurboID技术（邻近蛋白生物素标记技术）将表观因子与组蛋白的短暂结合转变成组蛋白上稳定的生物素修饰，进而结合CUT&Tag技术揭示表观因子的调控位点和下游靶标基因。申请人将在NPT过程中的不同时间点利用上述新技术体系确定LSD1的调控位点和下游靶标基因，利用蛋白质组技术确定与LSD1直接结合的其他表观因子，以此为基础确定LSD1通过调控能量代谢关键基因上的表观修饰促进氧化磷酸化和NPT的具体机制。项目将通过揭示LSD1调控NPT过程的具体机制推动多能性退出过程中表观机制研究。

LSD1 inhibits pluripotency re-establishment and facilitates pluripotency exit by modulating energy metabolism in cells. Thus, the applicant suggests that LSD1 can regulate the transition of embryonic stem cells (mESCs) from naïve to primed state (NPT) by controlling the epigenetic modifications on the key genes related to energy metabolism. Since the consistency among five ChIP-seq datasets of LSD1 in mESCs is low, it is difficult to identify the targeting sites and genes of LSD1 in mESCs with these datasets. Therefore, the applicant tries to convert the transient interaction between epigenetic factors and histones to biotin-labels on histones by using TurboID system, which will be followed by a CUT&Tag system to identify the targeting sites and genes of epigenetic factors. At different time points during the NPT process, the applicant will identify the targeting sites and genes of LSD1 by using abovementioned method, identify the proteins which directly interact with LSD1 by using proteomic methods, and then explore how LSD1 facilitates oxidative phosphorylation and NPT by controlling the epigenetic modifications on the key genes related to energy metabolism. By understanding how LSD1 regulates NPT, new insights will be provided for epigenetic mechanisms during pluripotency exit.