Enhancer-promoter interactions form independently of genomic distance and are functional across TAD boundaries

Developmental enhancers are essential regulatory elements that drive precise spatio-temporal gene expression patterns. They do so by interacting with the promoter of their target genes, often across large genomic distances, in a highly specific manner. However, it is unclear how such specificity can be achieved. While several studies have suggested that Topologically Associating Domains (TADs)1-3 facilitate and constrain enhancer-promoter interactions, the role of TAD boundaries in effectively restricting enhancer-promoter interactions is heavily debated. Here we show that enhancers can establish long-range interactions across TAD boundaries and even between different chromosomes. Moreover, some of these interactions are functional in vivo, illustrating their functional importance. Using the twist locus in Drosophila embryos, we systematically relocated one of its enhancers to different regulatory contexts and distances from the twist promoter. We found that the twist promoter can engage in functional enhancer-promoter interactions across a TAD boundary and that distal interactions are sometimes favored over proximal ones. Our results demonstrate that TAD boundaries are not sufficient to constrain enhancer-promoter interactions and that the formation of long-range interactions is not solely driven by distance. These observations suggest that other general mechanisms must exist to establish and maintain specific enhancer-promoter interactions across large distances.