The orofacial cleft risk gene IRF6 is a target gene of SOX9 in cranial neural crest cells

Abstract Orofacial clefts represent the most prevalent congenital anomalies affecting the craniofacial region. They can be evoked by a disturbed development of either oral epithelium or cranial neural crest-derived mesenchyme, or by disruptions of their interplay. IRF6 is a well-known risk gene associated with orofacial clefts. Its expression in the oral epithelium depends on the transcription factor AP-2α, encoded by TFAP2A. We here show by immunofluorescence on mouse embryonic sections and by mining of single cell RNA-seq data that IRF6 is also expressed in cranial neural crest-derived tissue in mice and humans, together with TFAP2A and SOX9. The IRF6 enhancer MCS-9.7 can be activated by the transcription factor SOX9, mutations of which cause Pierre Robin sequence, a craniofacial anomaly that includes cleft palate. This SOX9-dependent activation is influenced by the single nucleotide variant rs76145088 that is associated with orofacial clefting. Inactivation of Sox9 in a murine neural crest cell line by CRISPR/Cas9 results in loss of Irf6 expression. We conclude that dysregulation of the SOX9–IRF6 axis in cranial neural crest cells could be relevant for the pathogenesis of orofacial clefting.