<i>Dux</i> cluster duplication ensures full activation of totipotent genes

Zygotic genome activation (ZGA) confers to the mouse two-cell (2C) embryo a unique transcriptional profile characterized by transient up-regulation of many totipotency-related genes and <i>MERVL</i> retrotransposons. Intriguingly, those genes are duplicated and clustered in the genome during evolution, including <i>Dux</i> cluster, <i>Obox,</i> and <i>Zscan4</i> family members in mice. However, the contribution and biological significance of the totipotency-related gene duplication events in early embryo development remain poorly understood. Here, we focus on <i>Dux</i> cluster, the master regulator of ZGA that is necessary and sufficient for the induction of 2C-like cells and activation of totipotency-related genes in mouse embryonic stem cells (mESCs). By reducing <i>Dux</i> gene copies from 31 to 0 or 1 through CRISPR-Cas9 technology, we generate <i>Dux</i>-KO and <i>Dux</i> (n = 1) mESC lines, respectively. We uncover that the totipotency-related gene transcriptional profile is awakened to a much lesser extent in <i>Dux</i> (n = 1) mESCs compared to wild type mESCs following global DNA demethylation reprogramming or induction of DNA damage, mimicking the intrinsic events in preimplantation development. Together, <i>Dux</i> cluster duplication is critically required for full activation of ZGA transcripts.