Functional dissection of enhancers in 2-Cell embryo with an in vitro model [STARR-seq]

Zygotic genome activation (ZGA) marks the initial transcription event in embryogenesis, yet the mechanisms cis-regulating gene activation remain unclear due to material constraints. Here, we functionally dissect the enhancers across the mouse genome in DUX-induced 2C-like cells. Integrated analysis with the epigenetic modifications and RNA transcriptiome in both 2C-like cells and 2C embryos, regions which show enhancer activities and their potential downstream target gens were depicted. Among them, a notable proportion of promoters exhibits enhancer activities in totipotent cells. Those promoter regions are characterized by elevated levels of open chromatin-associated epigenetic modifications, which correlate with enhanced gene expression during ZGA. Our data further reveal that class Ⅲ mouse endogenous retrovirus (MERV)-related enhancers are enriched in 2C-like cells and nearly half of MERVL-LTR (Mt2_mm) elements exhibit enhancer activities, which have been mapped and characterized within the genome. Intriguingly, enhancers identified in 2C-like cells augment the transcription in vivo when injected into the embryos. Furthermore, the deletion of enhancer regions in both 2C-like cells and embryos demonstrated their role in promoting the transcription of their adjacent downstream target genes, with one of the deletions impaired the early embryo development. This study enhances our understanding of the regulatory elements guiding ZGA.