Disrupting Pitx1 regulatory topology results in overtly normal limb development

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Abstract Gene expression patterns during development are orchestrated in part by thousands of distant-acting transcriptional enhancers. However, identifying enhancers that are essential for expression of their target genes has proven challenging. Genetic perturbation of individual enhancers in some cases results in profound molecular and developmental phenotypes, but in mild or no phenotypes in others. Topological maps of long-range regulatory interactions may provide the means to identify enhancers critical for developmental gene expression. Here, we leveraged chromatin topology to characterize and disrupt the major promoter-enhancer interaction for Pitx1 , which is essential for hindlimb development. We found that Pitx1 primarily interacts with a single distal enhancer in the hindlimb. Using genome editing, we deleted this enhancer in the mouse. Although loss of the enhancer completely disrupts the predominant topological interaction in the Pitx1 locus, Pitx1 expression in the hindlimb is only reduced by ~14%, with no apparent changes in spatial distribution or evidence of regulatory compensation. Pitx1 enhancer null mice did not exhibit any of the characteristic morphological defects of the Pitx1 −/− mutant. Our results indicate that Pitx1 expression is robust to the loss of its primary enhancer interaction, suggesting disruptions of regulatory topology at essential developmental genes may have mild phenotypic effects.

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