Preformed chromatin topology assists transcriptional robustness of <i>Shh</i> during limb development

Abstract

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 the <i>Shh</i> gene by its unique limb enhancer, the <i>ZRS</i>, in vivo during mouse development. Abrogating the constitutive transcription covering the <i>ZRS</i> region led to a shift within the <i>Shh-ZRS</i> contacts and a moderate reduction in <i>Shh</i> transcription. Deletion of the CTCF binding sites around the <i>ZRS</i> resulted in the loss of the <i>Shh-ZRS</i> preformed interaction and a 50% decrease in <i>Shh</i> expression 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 <i>ZRS</i> allele, resulting in severe <i>Shh</i> loss of function and digit agenesis. Our results indicate that the preformed chromatin structure of the <i>Shh</i> locus is sustained by multiple components and acts to reinforce enhancer-promoter communication for robust transcription.