TITLE: “Enhancer Loss and Gene Dosage Sensitivity Drives a Human Craniofacial Disorder”
ABSTRACT: In recent years, the importance of understanding the role of non-coding regulatory mutations in human disease has become increasingly apparent. This is illustrated by a hotspot of non-coding mutations at the far end of a large gene desert surrounding the SOX9 gene that result in a human craniofacial disorder called Pierre Robin sequence (PRS). To test whether this locus harbours developmental regulatory elements that are perturbed in PRS patients, we utilized a well-characterized in vitro differentiation model of facial development. Within the PRS-associated region we identified two clusters of enhancers that regulate SOX9 expression during a restricted window of facial progenitor development, at distances of up to 1.45 Mb. Enhancers within these clusters exhibit highly synergistic activity that is dependent on the Coordinator motif and TWIST1 binding and are conserved in activity down to Coelacanth fish. We observe dramatic changes in 3D conformation of the SOX9 locus during facial progenitor development and propose a mechanism for extreme long-range gene regulation. Using mouse models, we demonstrate that specificity of PRS manifestations arises from the convergence of two developmental features: confinement of Sox9 dosage perturbation to the developing facial structures due to context-specific enhancer activity, and a heightened sensitivity of the lower jaw to Sox9 level reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations described to date, provide molecular insights into disease aetiology to directly classify PRS as an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation.