IRF6 loss-of-function causes disturbances in epithelial polarity, enamel formation, and root patterning

Abstract

Interferon regulatory factor 6 (IRF6) variants are associated with common isolated forms of cleft lip with or without clefting of the palate (CLP). Based on high sequence and structural homology with other IRFs, IRF6 has been predicted to act as a transcription factor, but its function and role in CLP is largely unknown. Studies in the Cox lab have found that IRF6 binds, in a phosphorylation-enhanced manner, to the NME (non-metastatic) complex, which has been reported to interact with factors that activate the PAR (PARD3-PARD6) polarity complex. This provides a novel mechanism in which IRF6 may contribute to proper epithelial function. NME regulated PARD3 activation stimulates establishment of epithelial apical and basolateral domains (i.e. polarity), ultimately impacting epithelial cell adhesion, shape, and behavior. Thus, via protein-protein interactions, IRF6 is hypothesized to contribute to the regulation of epithelial polarity, which when disrupted, leads to epithelial defects observed in CLP. Specific aims were developed to investigate the contribution of IRF6 to primary palate and tooth development. Using site-directed mutagenesis, reported CLP associated VWS patient mutations were introduced into yeast and mammalian expression constructs and subsequently compared with wild type IRF6 in their ability to interact with NME. Out of the twelve mutations tested, nine mutations disrupted the IRF6:NME interaction. Additionally, mutating three conserved serines to alanines resulted in loss of IRF6:NME binding and lowered nuclear levels of IRF6. Potential effects of the IRF6:NME interaction on epithelial polarity were assessed by measuring activity of the GTPases Rac1 and RhoA, in HEK293T cells ectopically expressing either a wild type or mutant IRF6. Disruption of the IRF6:NME complex also caused active Rac1 and RhoA up-regulation. In human patients, CLP is often associated with tooth abnormalities, and in mice, Irf6 was detected in ameloblasts and has roles in tooth epithelial invagination. A murine model with Irf6 conditionally ablated in ameloblasts (driven by the Pitx2-Cre promoter) was developed. Irf6 conditional knockouts-(Irf6 cKO) exhibited a variety of crown morphological alterations, including loss of cusp patterning. Hypodontia was also observed; 3rd molars were occasionally missing. Additionally, Irf6-cKO molars displayed rapid enamel attrition with wear reaching dentin 7 days post-eruption. Irf6-cKO enamel was hypomineralized compared to controls (p<0.05), and a delay in maturation was observed. Histological sections revealed persistence of an immature enamel matrix in Irf6-cKO samples (not present in controls) in addition to disruptions in ameloblast polarity. Immunohistochemistry revealed upregulation of amelogenin and downregulation of kallikrein-like 4 in Irf6-cKO P14 molars. Root patterning defects were also observed in Irf6-cKO mice, including severely taurodontic mandibular second molars. In P4 Irf6-ckO molars, mRNA expression of enamel matrix proteins was unchanged, yet Wnt10b and Osterix were downregulated (p<0.05). By P13, Wnt10b and Osterix expression in Irf6-cKO mice were comparable to controls. Overall, our findings contributed toward the understanding of the role of Irf6 in CLP, as well as tooth abnormalities associated with epithelial disorders. Disruptions of IRF6:NME complex and altered Rac1/RhoA expression by IRF6/NME mutations suggest IRF6 can impact processes such as epithelial polarity. Tooth-targeted Irf6 deletion in mice demonstrated enamel and root patterning defects consistent with CLP patients featuring IRF6 mutations. These results identified a critical and non-redundant role for IRF6 in crown and root formation, suggesting diversity of IRF6 function in epithelial-derived tissues.