Multi-feature clustering of CTCF binding creates robustness for loop extrusion blocking and Topologically Associating Domain boundaries

Topologically Associating Domains (TADs) separate vertebrate genomes into insulated regulatory neighborhoods that focus genome-associated processes. TADs are formed by Cohesin-mediated loop extrusion, with many TAD boundaries consisting of clustered binding sites of the CTCF insulator protein. Here we determined how this clustering of CTCF binding contributes to the blocking of loop extrusion and the insulation between TADs. We identified enrichment of three features of CTCF binding, whereby strongly bound and closely spaced binding sites are enriched around strong TAD boundaries. Using multi-contact Nano-C analysis in cells with normal and perturbed CTCF binding, we established that individual CTCF binding sites contribute to the blocking of loop extrusion, but in an incomplete manner. When clustered individual CTCF binding sites thus create a stepwise insulation between neighboring TADs. Based on these results, we propose a model whereby multiple instances of temporal loop extrusion blocking create strong isolation between TADs.