Gene expression patterns during development are orchestrated in part by thousands of distant-acting transcriptional enhancers. Genetic perturbation of individual enhancers in some cases results in profound molecular and developmental phenotypes, and in mild or no phenotypes in others. Topological maps of long-range regulatory interactions may provide the means to identify enhancers essential for regulation of their target genes. Here, we leveraged chromatin topology to identify and disrupt the major long-range promoter-enhancer interaction for Pitx1, which is essential for hindlimb development. The Pitx1 promoter forms a robust, hindlimb-specific interaction with a distal 13-kb enhancer. We used CRISPR genome editing to delete this element in the mouse. Surprisingly, although the deletion completely disrupted the predominant topological interaction in the Pitx1 locus, this resulted in only minor reductions in gene acetylation and Pitx1 expression, and did not recapitulate any of the characteristic morphologies of thePitx1-/- mutant. These results suggest that Pitx1 exhibits regulatory robustness, insensitive to the loss of an associated enhancer, while chromatin topology is not sufficient to predict enhancers of large effect size.
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