Single-cell Micro-C profiles 3D genome structures at high resolution and characterizes multi-enhancer hubs.

In animal genomes, regulatory DNA elements called enhancers govern precise spatiotemporal gene expression patterns in specific cell types. However, the spatial organization of enhancers within the nucleus to regulate target genes remains poorly understood. Here we report single-cell Micro-C (scMicro-C), a micrococcal nuclease-based three-dimensional (3D) genome mapping technique with an improved spatial resolution of 5 kb, and identified a specialized 3D enhancer structure termed 'promoter-enhancer stripes (PESs)', connecting a gene's promoter to multiple enhancers. PES are formed by cohesin-mediated loop extrusion, which potentially brings multiple enhancers to the promoter. Further, we observed the prevalence of multi-enhancer hubs on genes with PES within single-cell 3D genome structures, wherein multiple enhancers form a spatial cluster in association with the gene promoter. Through its improved resolution, scMicro-C elucidates how enhancers are spatially coordinated to control genes.