GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE283nnn/GSE283389/primaryOK200GenomicsMus musculusGenome binding/occupancy profiling by high throughput sequencing Expression profiling by high throughput sequencing Otherhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE283389GEOGSEfalseHigh-throughput functional characterization of enhancers in totipotent-like cellsZygotic 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.2026/07/19GSE283389GSM8660598GSM8660597GSM8660596GSM8660595GSM8660613GSM8660612GSM8660611GSM8660610GSM8660599GSM8660594GSM8660593GSM8660592GSM9797981GSM9797980GSM8660606GSM8660605GSM8660604GSM8660626GSM8660603GSM8660625GSM8660609GSM8660608GSM8660607GSM8660620GSM8660624GSM8660602GSM8660601GSM8660623GSM8660600GSM8660622GSM8660621GSM8660617GSM8660616GSM8660615GSM8660614GSM8660619GSM8660618170212127324247283389Mus musculus