YTHDF2 suppresses the 2C-like state in mouse embryonic stem cells via the DUX-ZSCAN4 molecular circuit

Mouse embryonic stem cells (ESCs) consist of a rare population of heterogeneous 2-cell-like cells (2CLCs). These cells transiently recapitulate the transcriptional and epigenetic features of the 2-cell embryos, serving as a unique model for studying totipotency acquisition and embryonic development. Accumulating evidence has demonstrated that transcription factors and epigenetic modifications exert crucial functions in the transition of ESCs to 2CLCs. However, the roles of RNA modification in the regulation of the 2C-like state remain elusive. Using a DUX-induced 2CLCs system, we examine N⁶-methyladenosine (m⁶A) modification landscape transcriptome-wide and observe dynamic regulation of m⁶A during DUX-driven 2C-like reprogramming. Notably, many core 2C transcripts like Dux and Zscan4 are highly methylated. We identify the m⁶A reader protein YTHDF2 as a critical regulator of 2C-like state. Depletion of YTHDF2 facilitates robust expression of 2C-signature genes and ESCs-to-2CLCs transition. Intriguingly, YTHDF2 binds to a subset of m⁶A-modified 2C transcripts and promotes their decay. We further demonstrate that YTHDF2 suppresses the 2C-like program in a manner that is dependent on both m⁶A and the DUX-ZSCAN4 molecular circuit. Mechanistically, YTHDF2 interacts with CNOT1, a key component of the RNA deadenylase complex. Consistently, silencing of CNOT1 upregulates the 2C program and promotes ESCs-to-2CLCs transition. Collectively, our findings reveal novel insights into the epitranscriptomic regulation of the 2C-like state in mouse ESCs.