Poising and connectivity of emergent human developmental enhancers in the transition from naive to primed pluripotency.

Poised enhancers (PEs), co-marked by H3K4me1 and Polycomb-associated H3K27me3, are common in primed human pluripotent stem cells (hPSCs) resembling post-implantation epiblast but scarce in naive hPSCs modeling pre-implantation epiblast. PEs form abundant chromosomal contacts with developmental genes, but the timing of their emergence, their relationship to enhancer poising, and their functional significance remain unclear. We devised high-resolution, PE-targeted Capture Hi-C to map PE contacts during the transition from naive to primed pluripotency. We find that enhancer poising emerges early in the transition, while the contacts show diverse dynamics. PROTAC-induced degradation of Polycomb repressive complex 2 early in the transition, but not inhibition of its H3K27 methyltransferase activity, weakens PE connectivity. Finally, PE contacts persist after developmental activation or ectopic CRISPRa targeting and can mediate long-range gene induction. Together, these findings reveal the temporal and mechanistic principles of PE connectivity and highlight a potential role of PE contacts in establishing developmental gene expression patterns.