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.

远端增强子的转录激活对于细胞命运的规范和细胞特性的维持是必不可少的。如何在物理上实现远程基因调控，特别是在非二元增强子-启动子配置的复杂调控环境中，仍然难以捉摸。最近的纳米技术进展将启动子动力学和~100 到 200 nm 大小的增强子相关调节因子 (RF) 簇在重要的发育基因上定量联系起来。在这里，我们进一步剖析了小鼠胚胎干细胞中 RF 聚集和转录激活的机制。RF 募集到簇中涉及对同源 DNA 和染色质结合位点的特异性分子识别，表明潜在的顺式-元素聚类。引人注目的是，在活细胞中以≤1 kb 和约 20 纳米的精度对标记的基因组位点进行成像，揭示了扩展位点上的远端增强子簇，它们经常靠近靶基因——在 RF 聚类距离内。这些高交互频率增强子簇“超簇”创造了纳米环境，其中聚集的 RF 激活靶基因，为相关基因组组织、焦点 RF 积累和转录激活提供结构框架。