Integration of 3D genome architecture and local chromatin features uncovers enhancers underlying craniofacial-specific cartilage defects

Aberrations in tissue-specific enhancers underlie many developmental defects. Disrupting a distal noncoding region of the human SOX9 gene causes the Pierre Robin sequence (PRS) characterized by the undersized lower jaw. Such a craniofacial-specific defect has been previously linked to enhancers transiently active in cranial neural crest cells (CNCCs). We demonstrate the PRS region also strongly regulates Sox9 in CNCC-derived Meckel's cartilage (MC), but not in limb cartilages. The MC-specific regulatory function of the PRS region cannot be attributed to the CNCC enhancers that have been inactivated in cartilages, but correlates with its MC-specific interactions with the Sox9 gene. By integrating the enhancer signatures and chromatin topology, we uncovered ~20,000 enhancers that function differentially between MC and limb cartilages and demonstrated their association with human diseases. Our findings highlight the importance of lineage-dependent chromatin topology in instructing enhancer usage and provide critical insights for interpreting the genetic basis of craniofacial pathologies. In this study, we systematically identified the enhancer atlas in mouse mandibular Meckel's cartilage (MC) and forelimb cartilage with multiple high-throughput sequencing techiniques including ATAC-seq, H3K27Ac CUT & Tag and Hi-C. By integrating local chromatin features and 3D genome architecture, our analyses uncovered several distal enhancers that specifically regulate Sox9 expression in MC, which could possibly contribute to the incidence of Pierre Robin sequence when disrupted.