Boundary stacking interactions enable cross-TAD enhancer-promoter communication during limb development

Abstract While long-range enhancers and their target promoters are frequently contained within a TAD, many developmentally important genes have their promoter and enhancers within different TADs. Hypotheses about molecular mechanisms enabling such cross-TAD interactions remain to be assessed. To test these hypotheses, we use Optical Reconstruction of Chromatin Architecture (ORCA) to characterize the conformations of the Pitx1 locus on thousands of single chromosomes in developing mouse limbs. Our data supports a model in which neighboring boundaries are stacked with each other as a result of loop-extrusion, bringing boundary-proximal cis -elements into contact. This stacking interaction also explains the appearance of architectural stripes in the population average maps (e.g. Hi-C data). Through molecular dynamics simulations, we further propose that increasing boundary strengths facilitates the formation of the stacked boundary conformation, counter-intuitively facilitating border bypass. This work provides a revised view of the TAD borders’ function, both facilitating as well as preventing cis -regulatory interactions, and introduces a framework to distinguish border-crossing from border-respecting enhancer-promoter pairs.