Dux cluster duplication ensures fully activation of totipotent genes. Dux cluster duplication ensures fully activation of totipotent genes

Mammalian zygotic genome activation (ZGA) following fertilization refers to the process that results in transcriptional awakening of the embryonic genome at two-cell (2C) stage in mice or 4-8 cell stage in human. ZGA confers to the mouse 2C embryo a unique transcriptional profile characterized by transient up-regulation of many totipotency-related genes and MERVL transposons. Intriguingly, many ZGA-related totipotent genes are duplicated and clustered in the genome during evolution, including Dux cluster, OBOX and Zscan4 family members in mice. Yet, the contribution and biological significance of the totipotency-related gene duplication events during evolution in totipotency acquisition remain poorly understood. Here, we focus on Dux cluster, the master regulator of ZGA that is necessary and sufficient for the emergence of 2C-like cells (2CLCs) and activation of target totipotent genes in mouse embryonic stem cells (ESCs). By reducing Dux gene copy number from 31 to 0 or 1 with CRISPR-Cas9 technology, we generated Dux-KO and Dux (n=1) ES cell lines, respectively. We found that the totipotent gene transcriptional profile could not be fully activated in Dux (n=1) mESCs compared to wild type (WT) mESCs after treatment with DNMT1 protein degrader, mimicking the DNA demethylation process during early embryo development. These data demonstrate that Dux cluster duplication is essential for fully activation of totipotency-related genes, ensuring ZGA and totipotency acquisition. 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 fully activation of ZGA transcripts during early embryo development associated with 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 1 uM GSK-3484862 were conducted.