Enhancer activity sensitive to the orientation of the gene it regulates in the chordategenome

► Enhancers are flexible in terms of their location and orientation relative to the genes they regulate. ► Little is known about whether the flexibility can be applied in every combination of enhancers and genes. ► We identified enhancers sensitive to the orientation of the gene they regulate in the chordate genome. ► This suggests that the relative positions and orientations of genes and enhancers are not randomly determined. ► Such constraints may have determined the positions of enhancers and genes during the evolution of animal genomes.