Myc and Dnmt1 impede the pluripotent to totipotent state transition in embryonic stem cells

Totipotency refers to the ability of a cell to generate all of the cell types of an organism. Unlike pluripotency, the establishment of totipotency is poorly understood. In mouse embryonic stem cells, Dux drives a small percentage of cells into a totipotent state by expressing 2-cell-embryo-specific transcripts. To understand how this transition takes place, we performed single-cell RNA-seq, 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 elements in the second step. To identify factors controlling the transition, we performed a CRISPR-Cas9-mediated screen, which revealed Myc and Dnmt1 as two factors preventing the transition. Mechanistic studies demonstrate that Myc prevents downregulation of pluripotent genes in the first step, while Dnmt1 impedes 2-cell-embryo-specific gene activation in the second step. Collectively, the findings of our study reveal insights into the establishment and regulation of the totipotent state in mouse embryonic stem cells.