Interferon Regulatory Factors transcriptionally regulate development and differentiation of the innate and adaptive immune systems. Within this family, IRF6 is unique because it regulates cutaneous and orofacial development in humans and mice. Common variants in IRF6 are associated with 12% of all orofacial clefting risk. Critically, a DNA variant in the IRF6 enhancer MCS9.7, rs642961, is found in 30% of the world's population. Biochemically, we know that rs642961 abrogates one of four TFAP2a binding sites, suggesting regulatory function. Mutations in TFAP2a can lead to Branio-oculo-facial Syndrome, a dominantly inherited orofacial clefting syndrome that includes upper lip pits. However, functional studies have not shown if Tfap2a regulates MCS9.7 activity or endogenous Irf6 expression in the mouse. In addition, rare mutations in IRF6, located within 1q32-q41, lead to Van der Woude and Popliteal Pterygium Syndromes, dominantly inherited orofacial clefting disorders. Currently, 70% of VWS families have mutations in IRF6. While the remaining 30% have unknown etiology, prior linkage analysis suggest locus heterogeneity.We use a mouse models to determine how common variants in IRF6 may be associated with orofacial clefting and to investigate locus heterogeneity in VWS. We find that knocking out Tfap2a leads to loss of MCS9.7 enhancer activity and Irf6 expression in vivo. On the other hand, Irf6 also appears to stabilize Tfap2a protein in epidermis. The necessity of Tfap2a for Irf6 expression contributes to our understanding of the association between rs642961 and isolated orofacial clefting. Significantly, we also find that Irf6 transcriptionally activities Grhl3 in epithelium. Consistent with prior work showing locus heterogeneity, we find that mutations in GRHL3 can also led to Van der Woude Syndrome. These results suggest that TFAP2a, IRF6, and GRHL3 share a conserved genetic pathway that is required for proper development of the lip and palate in humans and mice. In the mouse, loss of Grhl3 and Tfap2a leads to skin, limb, craniofacial and neural tube defects. Because Irf6 is an intermediate node between Tfap2a and Grhl3 in oral epithelium, we predict and find that changes in Irf6 expression can lead to neural tube defects. Over-expressing Irf6 leads to rostral neural tube defects, including loss of the cranial vault, i.e. acrania, and a split face. In addition, both reducing and over-expressing Irf6 leads to caudal neural tube defects, a curled and kinked tail, respectively. Consistent with orofacial genetic regulation, we find that Irf6 represses Tfap2a in rostral neural tube development. In the caudal neural tube, we find that Irf6 activates both Tfap2a and Grhl3 expression and that Tfap2a and Grhl3 interact in caudal neurulation. Consistently, human sequencing reveals a rare IRF6 mutation in an individual with spina bifida. Finally, we show that Irf6 expression in skin development rescues perinatal lethality but not limb, tail and palatal development. These results suggest that Tfap2a-Irf6-Grhl3 regulate the development of multiple ectodermal lineages. We conclude that cross-fertilization in orofacial and neural tube development provides candidate genes and potential therapeutic strategies for two congenital diseases with significant morbidity and mortality.
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