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

Abstract

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. Zygotic genome activation marks the first wave of gene transcription in embryogenesis, yet its regulatory mechanisms remain unclear. Here, Yang et.al mapped and functionally validated active enhancers driving this process in totipotent cells.