High-throughput functional characterization of enhancers in totipotent-like cells.

Zygotic genome activation (ZGA) marks the initial transcription event in embryogenesis, yet the cis-regulatory mechanism remains unclear. Here, utilizing massively parallel reporter assays, we functionally dissect enhancers across mouse genome in DUX-induced 2C-like cells (2CLCs). Through integrated analysis with epigenomic and transcriptomic data from 2CLCs and 2C embryos, active enhancers in totipotent cells are depicted. Among them, a notable proportion of promoters exhibit enhancer activities, showing elevated active chromatin features and correlating with enhanced gene expression during ZGA. Transcription factors preferentially enriched at enhancer-promoter regions play critical roles in regulating ZGA and early embryonic development. Furthermore, only half of the MT2_Mm exhibit enhancer activities in 2CLCs. Notably, 2CLC enhancers augment transcription in 2C embryos. Finally, deleting enhancer regions in both 2CLCs and 2C embryos, together with dCas9-KRAB-MeCP2-mediated CRISPRi in vitro, collectively underscores their crucial role in facilitating transcription of ZGA genes. These findings advance our comprehension of the cis-regulatory mechanism governing ZGA process.