Abstract
Gene expression often requires interaction between promoters and distant enhancers, which occur within the context of highly organized topologically associating domains (TADs). Using a series of engineered chromosomal rearrangements at the Shh locus, we carried out an extensive fine-scale characterization of the factors that govern the long-range regulatory interactions controlling Shh expression. We show that Shh enhancers act pervasively, yet not uniformly, throughout the TAD. Importantly, changing intra-TAD distances had no impact on Shh expression. In contrast, inversions disrupting the TAD altered global folding of the region and prevented regulatory contacts in a distance-dependent manner. Our data indicate that the Shh TAD promotes distance-independent contacts between distant regions that would otherwise interact only sporadically, enabling functional communication between them. In large genomes where genomic distances per se can limit regulatory interactions, this function of TADs could be as essential for gene expression as the formation of insulated neighborhoods.