Dissecting the role of the major cleft gene, IRF6, in primary palatal epithelia
Parada Sanchez, Monica Tatiana
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Mutations and common polymorphisms in Interferon Regulatory Factor 6 (IRF6) are associated with cleft lip/palate (CLP). IRF6 is expressed in the epithelium that mediates the outgrowth and contact of the embryonic tissue masses that fuse to form the upper lip and primary palate as well as in the epithelium that mediates fusion of the palatal shelves to form the secondary palate. Unfortunately, mice deficient in Irf6 display only secondary palatal clefts and abnormal intraoral adhesions but not classic CLP. To provide insights into the role of IRF6 in the pathogenesis of CLP, yeast two hybrid screens were performed to identify IRF6 protein interactors. Using this approach, NME2 was identified and confirmed as a bona fide IRF6 partner protein and this interaction is regulated by serine phosphorylation at the C-terminus of IRF6. In vivo studies revealed that NME2 co-localizes with IRF6 in the cytoplasm of primary palatal epithelial cells. Furthermore, NME1, the member of the NME family most closely related to NME2, heteromultimerizes with NME2 to form part of the same complex. Notably, NME proteins are NDP kinases that have been implicated in regulation of epithelial cell adhesion and E-cadherin endocytosis. The lack of a suitable IRF6 CLP model prompted the development of an in ovo electroporation protocol to facilitate use of the chick as an alternative model system. The application of a dominant negative form of IRF6 and shRNA constructs using this technique reproduced the phenotype seen in humans, demonstrating that IRF6 is required in epithelial cells to promote outgrowth and fusion of the facial prominences, with secondary effects on the differentiation of the underlying mesenchyme. It is proposed that localization and interactions of NME proteins with other factors may provide clues as to the cellular pathways in which IRF6 is involved, and therefore an explanation of the cellular etiology of the CLP. The data generated during this study have opened new directions not only for future studies on IRF6 itself, but in particular on the possible role of NME proteins in governing the behavior of facial epithelia during fusion of the primary palate and hence susceptibility to CLP.
- Dentistry