Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder

Abstract

Summary Non-coding mutations at the far end of a large gene desert surrounding the SOX9 gene result in a human craniofacial disorder called Pierre Robin sequence (PRS). Leveraging a human stem cell differentiation model, we identify two clusters of enhancers within the PRS-associated region that regulate SOX9 expression during a restricted window of facial progenitor development at distances up to 1.45 Mb. Enhancers within the 1.45 Mb cluster exhibit highly synergistic activity that is dependent on the Coordinator motif. Using mouse models, we demonstrate that PRS phenotypic specificity arises from the convergence of two mechanisms: confinement of Sox9 dosage perturbation to developing facial structures through context-specific enhancer activity and heightened sensitivity of the lower jaw to Sox9 expression reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation.

A gene desert required for regulatory control of pleiotropicShox2expression and embryonic survival

“…Furthermore, our results demonstrate that craniofacial Shox2 expression and in particular Shox2 transcripts in the mandibular and nasal processes critically depend on the presence of the gene desert. A recent study uncovered that human (and mouse) extreme long-range enhancers located in a large gene desert upstream of Sox9 are acting across nearly 1.5 Mb to regulate Sox9 expression in craniofacial regions, such as the nasal, maxillary and mandibular processes 50 . Similarly, our study identifies gene desert enhancers with activities in nasal and maxillary-mandibular regions, the latter likely critical for the formation of the temporomandibular joint 18 .…”

Phenotypes, Developmental Basis, and Genetics of Pierre Robin Complex

“…It is unknown whether the etiology of PR anomalies varies according to diagnostic category. There are excellent reviews of mandible, tongue, and palate development (e.g., [ 35 , 36 , 37 ]) and limited studies of mouse models that show PR phenotypes [ 38 , 39 , 40 , 41 ], but most studies are descriptive, without a focus on how these anomalies might be mechanistically, molecularly, or developmentally related.…”

“…Heterozygous inactivation of Sox9 results in a shortened mandible, abnormal tongue, and CP [ 77 ]. Conditional, heterozygous deletion of Sox9 in the neural crest also results in a shortened mandible and CP [ 41 , 78 ]. One model involves deletion of a long-range enhancer element that regulates Sox9 expression in mice and is conserved in humans in the region affected by deletions and translocations in some PR-Plus cases [ 41 ]; however, it does not display the full PR triad, lacking tongue and palate defects.…”

“…Conditional, heterozygous deletion of Sox9 in the neural crest also results in a shortened mandible and CP [ 41 , 78 ]. One model involves deletion of a long-range enhancer element that regulates Sox9 expression in mice and is conserved in humans in the region affected by deletions and translocations in some PR-Plus cases [ 41 ]; however, it does not display the full PR triad, lacking tongue and palate defects. Loss-of-function mutations in collagen genes were found in syndromes associated with PR phenotypes and mice homozygous for chondrodysplasia ( Col11a1 cho/cho ), cartilage matrix deficiency ( Acan cmd/cmd ), and disproportionate micromelia ( Col2a1 Dmm/Dmm ) exhibited macroglossia and tongue obstruction during palatogenesis resulting in CP, thereby supporting the hypothesis for the PR sequence [ 79 , 80 ].…”

Retrieving high-resolution chromatin interactions and decoding enhancer regulatory potentialin silico

“…4,6 Within these domains, the directed interplay between enhancers and gene promoters fine-tunes gene expression, although such regulatory interactions have now been recorded over larger scales too. 7–9…”