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

细胞自噬（Autophagy）是一类保守的细胞机制，可通过降解多余的细胞质蛋白与细胞器以循环利用其组分，现已被证实对胚胎干细胞（ESC）的自我更新及体细胞重编程至关重要。然而，细胞自噬在胚胎发育中的作用仍尚不明确，且目前尚无关于其在原始态至始发态多能性转变过程中功能的相关报道。本研究借助胚胎干细胞向其上胚层样细胞（EpiLCs）的体外转化模型，揭示依赖Atg7的细胞自噬的动态变化，既是原始态向始发态多能性转变所必需的过程，同时对生殖系特化亦不可或缺。通过RNA测序（RNA-seq）与转座酶可及性测序（ATAC-seq）分析显示，Nanog可作为屏障阻碍多能性转变；而依赖细胞自噬的Nanog降解，可通过失活与原始态多能性相关的活性增强子，对终止原始态多能性的表达程序发挥关键作用。机制层面的研究发现，在多能性转变期间，自噬衔接蛋白Sqstm1（p62）定位于细胞核，且可优先结合泛素化修饰的Nanog以介导其选择性降解。体内实验结果表明，敲低Atg7以消除细胞自噬会破坏胚胎着床后发育，同时观察到Nanog的染色质结合水平升高，该变化可通过激活一类神经外胚层发育相关增强子，进而影响神经元分化。综上，本研究阐明了原始态至始发态多能性转变的调控机制，并揭示依赖细胞自噬的Nanog调控是退出原始态多能性所必需的，同时在谱系特化过程中界定了独特的细胞命运分配模式。