Multiple allelic configurations govern long-range Shh enhancer-promoter communication in the embryonic forebrain.

Developmental gene regulation requires input from enhancers spread over large genomic distances. Our understanding of long-range enhancer-promoter (E-P) communication, characterized as loops, remains incomplete without addressing the role of intervening chromatin. Here, we examine the topology of the entire Sonic hedgehog (Shh) regulatory domain in individual alleles from the mouse embryonic forebrain. Through sequential Oligopaint labeling and super-resolution microscopy, we find that the Shh locus maintains a compact structure that adopts several diverse configurations independent of Shh expression. The most frequent configuration contained distal E-P contacts at the expense of those more proximal to Shh, consistent with an interconnected loop. Genetic perturbations demonstrate that this long-range E-P communication operates by Shh-expression-independent and dependent mechanisms, involving CTCF binding sites and active enhancers, respectively. We propose a model whereby gene regulatory elements secure long-range E-P interactions amid an inherent architectural framework to coordinate spatiotemporal patterns of gene expression.