Preformed Chromatin Topology Assists Transcriptional Robustness ofShhduring Limb Development

Long-range gene regulation involves physical proximity between enhancers and promoters to generate precise patterns of gene expression in space and time. However, in some cases proximity coincides with gene activation, whereas in others preformed topologies already exist before activation. In this study, we investigate the preformed configuration underlying the regulation of theShhgene by its unique limb enhancer, theZRS, in vivoduring mouse development. Abrogating the constitutive transcription covering theZRSregion led to a shift within theShh-ZRScontacts and a moderate reduction inShhtranscription. Deletion of the CTCF binding sites around theZRSresulted in a loss of theShh-ZRSpreformed interaction and a 50% decrease inShhexpression but no phenotype, suggesting an additional, CTCF-independent mechanism of promoter-enhancer communication. This residual activity, however, was diminished by combining the loss of CTCF binding with a hypomorphic ZRS allele resulting in severeShhloss-of-function and digit agenesis. Our results indicate that the preformed chromatin structure of theShhlocus is sustained by multiple components and acts to reinforce enhancer-promoter communication for robust transcription.