Dux 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 MERVL retrotransposons. Intriguingly, those genes are duplicated and clustered in the genome during evolution, including Dux cluster, Obox and Zscan4 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 Dux cluster, the master regulator of ZGA that is necessary and sufficient for the induction of 2C-like cells (2CLCs) and activation of totipotency-related genes in mouse embryonic stem cells (mESCs). By reducing Dux gene copies from 31 to 0 or 1 through CRISPR-Cas9 technology, we generate Dux-KO and Dux (n=1)mESC lines, respectively. We uncover that the totipotency-related gene transcriptional profile is awakened to a much lesser extent in Dux (n=1) mESCs compared to wild-type (WT) mESCs following global DNA demethylation reprogramming or induction of DNA damage, mimicking the intrinsic events in preimplantation development. Together, Dux cluster duplication is critically required for the full activation of ZGA transcripts. Overall design: To investigate the effects of Dux cluster duplication on the activation of two-cell specific transcripts during zygotic genome activation (ZGA), we generated Dux-KO and Dux (n=1) mESCs by CRISPR-Cas9 technology. We performed gene expression profiling analysis using data obtained from RNA-seq of WT, Dux-KO and Dux (n=1) mESCs. To determine whether Dux cluster duplication is critical for efficient and full activation of ZGA transcripts during early embryo development associated with the global DNA demethylation process, we treated WT, Dux-KO and Dux (n=1) mESCs with DNMT1 protein degrader GSK3484862 to mimic the DNA demethylation reprogramming events. Comparative gene expression profiling analyses of RNA-seq data from WT, Dux-KO and Dux (n=1) mESCs treated with DMSO and 2 uM GSK-3484862 for 3 days were conducted.