Myc and Dnmt1 impede the pluripotent to totipotent state transition in embryonic stem cell [RNA-Seq]

Totipotency is defined as the ability of a cell to generate all the cell types of an organism, including those of the extraembryonic tissues. Unlike pluripotency, the molecular features and the establishment of totipotency are poorly understood. In mouse embryonic stem cell (ESC) culture, a small percentage of cells transits into a totipotent state by expressing a group of genes that are only expressed in 2-cell-stage embryos. To understand how this transition takes place, we performed single cell RNA-seq analysis which revealed a two-step transcriptional reprogramming process characterized by downregulation of pluripotent genes in the first step and upregulation of the 2-cell embryo-specific genes in the second step. To identify factors controlling the transition process, we performed a CRISPR/Cas9-mediated genetic screen which revealed Myc and Dnmt1 as two factors preventing the transition. Mechanistic studies demonstrate that Myc prevents down-regulation of the genes in the first step, while Dnmt1 impedes gene activation in the second step. Collectively, our study reveals insights into the mechanism underlying establishment and regulation of totipotent state in ESCs.

全能性（totipotency）被定义为单个细胞能够生成生物体所有细胞类型的能力，包括胚外组织细胞。与多能性（pluripotency）不同，目前对全能性的分子特征及其建立机制尚缺乏深入认知。在小鼠胚胎干细胞（ESC）培养体系中，仅小部分细胞会通过表达仅在2细胞期胚胎中特异性表达的基因簇，转变为全能性状态。为阐明该转变的发生机制，我们开展了单细胞RNA测序（single cell RNA-seq）分析，结果揭示了以两步转录重编程为特征的过程：第一步为多能性基因的下调，第二步为2细胞胚胎特异性基因的上调。为筛选调控该转变过程的因子，我们实施了CRISPR/Cas9介导的遗传筛选，鉴定出Myc与Dnmt1为两个抑制该转变的因子。机制研究表明，Myc可阻止第一步中多能性基因的下调，而Dnmt1则会阻碍第二步中2细胞胚胎特异性基因的激活。综上，本研究为解析胚胎干细胞中全能性状态的建立与调控机制提供了新的见解。