Detection of newly synthesized RNA reveals transcriptional reprogramming during ZGA and a role of Obox3 in totipotency acquisition

Zygotic genome activation (ZGA) after fertilization is a conserved transcriptional activation process that enables the maternal-to-zygotic transition. However, the global view of ZGA, particularly at the initiation, is not yet completely understood. Here, we develop a method to capture and sequence newly synthesized RNA in the early mouse embryos, which provides a view of transcriptional reprogramming events during ZGA. Our data demonstrate that gene activation associated with major ZGA, previously thought to occur exclusively during the mid-to-late 2-cell stage, is already initiated in zygotes. Furthermore, we identify a set of genes activated during minor ZGA and observe an enrichment of the homeobox-containing Obox factor motif at the promoters of minor ZGA genes. Among several Obox factors, Obox3 overexpression in mouse embryonic stem cells activates ZGA genes. Notably, the expression of Obox3 is severely impaired in somatic cell nuclear transfer (SCNT) embryos, and restoring its expression corrects the ZGA profile and greatly improves SCNT embryo development. Hence, our study reveals the dynamic transcriptional reprogramming during ZGA and underscores the crucial role of Obox3 in facilitating totipotency acquisition. RNA-seq for newly synthesized RNAs, total RNA-seq and CUT&RUN were performed using mouse early embryos or mESCs.