Abstract
Transcription activation by distal enhancers is essential for cell-fate specification and maintenance of cellular identities. How long-range gene regulation is physically achieved, especially within complex regulatory landscapes of non-binary enhancer-promoter configurations, remains elusive. Recent nanoscopy advances have quantitatively linked promoter kinetics and ~100- to 200-nm-sized clusters of enhancer-associated regulatory factors (RFs) at important developmental genes. Here, we further dissect mechanisms of RF clustering and transcription activation in mouse embryonic stem cells. RF recruitment into clusters involves specific molecular recognition of cognate DNA and chromatin-binding sites, suggesting underlying cis-element clustering. Strikingly, imaging of tagged genomic loci, with ≤1 kilobase and ~20-nanometer precision, in live cells, reveals distal enhancer clusters over the extended locus in frequent close proximity to target genes-within RF-clustering distances. These high-interaction-frequency enhancer-cluster 'superclusters' create nano-environments wherein clustered RFs activate target genes, providing a structural framework for relating genome organization, focal RF accumulation and transcription activation.