Msl3-mediated H4K16ac suppresses 2C-like reprogramming by sustaining LINE1 expression in mouse embryonic stem cells

Two-cell-like cells (2CLCs), a rare totipotent-like population within mouse embryonic stem cells (mESCs) cultures, serve as a valuable model for studying early embryonic events like zygotic genome activation. However, the molecular mechanisms restraining their spontaneous emergence remain elusive. Here, we identify Msl3, an essential component of the MSL complex that is required for H4K16ac deposition, as a key suppressor of 2C-like reprogramming. Msl3 mediated H4K16ac promotes the expression of LINE1 retrotransposons, which act as upstream repressors of the 2C-lineage master regulator Dux. Consequently, Msl3 depletion reduces LINE1 expression, de-represses Dux, and robustly activates 2C-specific genes. Furthermore, LINE1 inhibition leads to heterochromatin dispersal and fragmented nucleoli. Our findings unveil a novel MSL3-H4K16ac-LINE1-Dux axis that actively safeguards mESCs pluripotency by suppressing a latent 2C-like program.