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 non-syndromic orofacial clefts and causative for Van der Woude syndrome, an autosomal dominant syndrome characterized by orofacial clefting and lower lip pits. Its expression in the oral epithelium depends on the transcription factor 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 is bound and 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 cranial 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. Key Messages Irf6 is co-expressed with Sox9 – the major gene mutated in Pierre Robin Sequence (a craniofacial anomaly that includes cleft palate) – in a subset of cranial neural crest cells. The IRF6 enhancer MCS-9.7 is bound and activated by SOX9 and different alleles of the orofacial cleft risk SNV rs76145088 in MCS-9.7 confer differential activation of this enhancer by SOX9. Inactivation of Sox9 via CRISPR/Cas9 in a cranial neural crest cell line prevents Irf6 expression.