Atg7-dependent autophagy regulates the naive to primed transition by selective Nanog degradation in mouse embryonic stem cells [ATAC-seq]. Atg7-dependent autophagy regulates the naive to primed transition by selective Nanog degradation in mouse embryonic stem cells [ATAC-seq]

Autophagy is a conserved cellular mechanism to degrade unwanted cytoplasmic proteins and organelles to recycle their components, and it is proved to be critical for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. However, the role of autophagy in embryonic development remains elusive, and no information exists regarding its functions during the transition from naive to primed pluripotency. Here by using an in vitro transition model of ESCs to epiblast-like cells (EpiLCs), we describe that the dynamic changes in Atg7-dependent autophagy is required for the naive to primed transition, and it is also necessary for germline specification. RNA-seq and ATAC-seq profiling reveal that Nanog acts as a barrier to prevent pluripotency transition, and autophagy-dependent Nanog degradation is important for dismantling the naive pluripotency expression program through decommissioning of naive-associated active enhancers. Mechanistically, we found that autophagy adaptor protein Sqstm1 (p62) is nucleus located during the pluripotency transition period and it is preferentially associated with ubiquitinated Nanog for selective protein degradation. In vivo, loss of autophagy by Atg7 depletion disrupts peri-implantation development and we observed increased chromatin association of Nanog, which affects neuronal differentiation through activation of a subset of neuroectodermal development-associated enhancers. Taken together, our findings illuminate regulatory mechanisms underlying the naive to primed transition and reveal that autophagy-dependent regulation of Nanog is essential for exit from the naive state and marks distinct cell fate allocation during lineage specification. Overall design: The ATAC-Seq experiments of the Control and Atg7 KO groups were taken at each time point from the 4-day ESC to EpiLC transition.

自噬（Autophagy）是一种保守的细胞降解机制，可通过降解多余的细胞质蛋白与细胞器来循环利用其组分，现已证实其对胚胎干细胞（Embryonic Stem Cell, ESC）的自我更新及体细胞重编程至关重要。然而，自噬在胚胎发育中的作用仍有待阐明，目前尚无关于其在初始态多能性向始发态多能性转换过程中发挥功能的相关研究报道。本研究借助ESC向上胚层样细胞（epiblast-like cells, EpiLCs）的体外转换模型，发现依赖Atg7的自噬动态变化对于初始态到始发态的转换以及生殖细胞特化均不可或缺。RNA测序（RNA-seq）与转座酶可及性测序（ATAC-seq）谱分析显示，Nanog作为屏障阻碍多能性转换，而依赖自噬的Nanog降解可通过使初始态相关的活性增强子失活，拆解初始态多能性的表达程序，该过程具有重要意义。机制层面研究显示，自噬衔接蛋白Sqstm1（p62）在多能性转换期间定位于细胞核，且优先与泛素化的Nanog结合以介导选择性蛋白降解。体内实验中，通过敲除Atg7丧失自噬功能会破坏围植入期发育，我们还观察到Nanog的染色质结合水平升高，这会通过激活一组神经外胚层发育相关增强子影响神经分化。综上，本研究阐明了初始态向始发态转换的调控机制，并揭示依赖自噬的Nanog调控对于退出初始态至关重要，同时标志着谱系特化过程中不同细胞命运的分配。整体实验设计：本研究在ESC向EpiLCs为期4天的转换过程中，于各时间点采集对照组与Atg7敲除（KO）组样本开展ATAC-seq实验。