SETDB1 enables development beyond cleavage stages by extinguishing the MERVL-driven two-cell totipotency transcriptional program in the mouse embryo.

Loss of maternal SETDB1, a histone H3K9 methyltransferase, leads to developmental arrest prior to implantation, with very few mouse embryos advancing beyond the eight-cell stage, which is currently unexplained. We genetically investigate SETDB1's role in the epigenetic control of the transition from totipotency to pluripotency-a process demanding precise timing and forward directionality. Through single-embryo total RNA sequencing of two-cell and eight-cell embryos, we find that Setdb1mat-/+ embryos fail to extinguish one-cell and two-cell transient genes-alongside persistent expression of MERVL retroelements and MERVL-driven chimeric transcripts that define the totipotent state in mouse two-cell embryos. Comparative bioinformatics reveals that SETDB1 acts at MT2 LTRs and MERVL-driven chimeric transcripts, which normally acquire H3K9me3 during early development. The dysregulated targets substantially overlap with DUXBL-responsive genes, indicating a shared regulatory pathway for silencing the two-cell transcriptional program. We establish maternal SETDB1 as a critical chromatin regulator required to extinguish retroelement-driven totipotency networks and ensure successful preimplantation development.