Domain stacking enables a limb enhancer to act across multiple TAD boundaries

Abstract

Many developmentally important genes have their promoter and enhancers within different TADs. Although there are hypotheses about molecular mechanisms enabling such cross‐TAD interactions between these cis‐elements, but they 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 cis‐elements adjacent to TAD boundaries are collected in transient hub‐like structures when neighboring boundaries are stacked with each other as a result of loop‐extrusion. Through molecular dynamics simulations, we further propose that increasing boundary strengths (eg. by increasing CTCF occupancy) facilitates the formation of the stacked boundary conformation. 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.