Loss of extreme long-range enhancers in human neural crest drives a craniofacial disorder

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). In this study we utilise epigenomic profiling of histone modifications and coactivator binding using ChIP-seq and chromatin accessibility using ATAC-seq to map cis-regulatory enhancer elements within the PRS locus during differentiation of human embryonic stem cells to cranial neural crest and chondrocytes. Strikingly, these enhancers are located up to 1.45 Mb away from the SOX9 gene promoter. We further perform RNA-sequencing to determine gene expression changes during the differentiation and identify potential transcription factors that regulate enhancer activity during CNCC development, including TWIST1. We utilise Capture-C from the SOX9 promoter to profile long-range interactions with the distal PRS-associated enhancer elements, and we confirm these interactions using reciprocal Capture-C from the identified candidate enhancer elements. Finally, we phase the H9 human embryonic stem cell line using 10X genomics linked-read sequencing in order link genetically edited alleles to gene expresssion changes in cis. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and identify one factor that may contribute to enhancer function in a developmentally-regulated manner.