Disrupting the three-dimensional regulatory topology of the locus results in overtly normal development.

Developmental gene expression patterns are orchestrated by thousands of distant-acting transcriptional enhancers. However, identifying enhancers essential for the expression of their target genes has proven challenging. Maps of long-range regulatory interactions may provide the means to identify enhancers crucial for developmental gene expression. To investigate this hypothesis, we used circular chromosome conformation capture coupled with interaction maps in the mouse limb to characterize the regulatory topology of , which is essential for hindlimb development. We identified a robust hindlimb-specific interaction between and a putative hindlimb-specific enhancer. To interrogate the role of this interaction in regulation, we used genome editing to delete this enhancer in mouse. Although deletion of the enhancer completely disrupts the interaction, expression in the hindlimb is only mildly affected, without any detectable compensatory interactions between the promoter and potentially redundant enhancers. enhancer null mice did not exhibit any of the characteristic morphological defects of the mutant. Our results suggest that robust, tissue-specific physical interactions at essential developmental genes have limited predictive value for identifying enhancer mutations with strong loss-of-function phenotypes.