Analysis of multi-way chromatin interactions at sub-nucleosome resolution identifies synergistic nano-scale interactions within super-enhancers

In-depth understanding of the 3D organization of the genome and its role in the regulation of gene expression requires techniques to measure complex chromosome conformations at single alleles at high resolution. Here, we present multi-way Micro-Capture-C (mwMCC), a method to detect concurrent interactions between multiple chromatin regions at single alleles at sub-nucleosome resolution. mwMCC resolves the internal 3D structure of super-enhancers and identifies synergistic multi-way interactions between their constituent elements and gene promoters, providing a structural basis for cooperation within super-enhancers in nano-scale chromatin hubs. The dynamic formation and dissolution of these hubs during cellular differentiation correlates with up- and down-regulation of gene expression. Although large-scale chromatin hubs are dependent on the architectural protein CTCF, nano-scale hubs are robust to CTCF depletion. Instead, integration of footprinting analysis and protein occupancy data indicates a role for transcription factors and the co-activator Mediator in the formation of synergistic enhancer-promoter interactions within nano-scale chromatin hubs.