Nanoscale dynamics of enhancer–promoter interactions during exit from pluripotency

While compelling genetic evidence supports the role of enhancers in regulating promoter activity even over large genomic distances, it remains unclear to what extent physical proximity to promoters is required. To address this, we combined fluorescence in situ hybridization (FISH) with super-resolution microscopy and Tri-C to examine enhancer-promoter (E-P) distances and regulatory element clustering at regulated loci (Nanog, Dppa3, Dnmt3a, Sox2, Prdm14) during the transition from naive to primed pluripotency in mouse embryonic stem cells. Despite transcriptional changes of several orders of magnitude, most genes show no major alterations in median E-P distances or in the probability of multiway contacts across states. However, Tri-C reveals a weak enrichment of multiway contacts at Nanog in naive cells, where it is highly expressed. Because transcription often occurs in transient bursts within a subset of cells, we combined RNA and DNA FISH to identify active alleles. For Nanog and Dppa3, reduced E-P distances correlate with transcriptional activity. Together, these findings support models in which transcription is associated with transient E-P proximity and suggest that multiway contact formation among regulatory elements may contribute to gene regulation.