Cooperation between the transcription factors p63 and IRF6 is essential to prevent cleft palate in mice
Helen A. Thomason, Huiqing Zhou, Evelyn N. Kouwenhoven, Gian-Paolo Dotto, Gaia Restivo, Bach-Cuc Nguyen, Hayley Little, Michael J. Dixon, Hans van Bokhoven, Jill Dixon
Helen A. Thomason, Huiqing Zhou, Evelyn N. Kouwenhoven, Gian-Paolo Dotto, Gaia Restivo, Bach-Cuc Nguyen, Hayley Little, Michael J. Dixon, Hans van Bokhoven, Jill Dixon
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Research Article

Cooperation between the transcription factors p63 and IRF6 is essential to prevent cleft palate in mice

Abstract

Cleft palate is a common congenital disorder that affects up to 1 in 2,500 live human births and results in considerable morbidity to affected individuals and their families. The etiology of cleft palate is complex, with both genetic and environmental factors implicated. Mutations in the transcription factor–encoding genes p63 and interferon regulatory factor 6 (IRF6) have individually been identified as causes of cleft palate; however, a relationship between the key transcription factors p63 and IRF6 has not been determined. Here, we used both mouse models and human primary keratinocytes from patients with cleft palate to demonstrate that IRF6 and p63 interact epistatically during development of the secondary palate. Mice simultaneously carrying a heterozygous deletion of p63 and the Irf6 knockin mutation R84C, which causes cleft palate in humans, displayed ectodermal abnormalities that led to cleft palate. Furthermore, we showed that p63 transactivated IRF6 by binding to an upstream enhancer element; genetic variation within this enhancer element is associated with increased susceptibility to cleft lip. Our findings therefore identify p63 as a key regulatory molecule during palate development and provide a mechanism for the cooperative role of p63 and IRF6 in orofacial development in mice and humans.

Authors

Helen A. Thomason, Huiqing Zhou, Evelyn N. Kouwenhoven, Gian-Paolo Dotto, Gaia Restivo, Bach-Cuc Nguyen, Hayley Little, Michael J. Dixon, Hans van Bokhoven, Jill Dixon

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Figure 4

Irf6 in p63-deficient cells.

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Irf6 in p63-deficient cells. (A) qPCR analysis of palatal shelves d...
(A) qPCR analysis of palatal shelves dissected from E13.5 wild-type, p63+/–, and p63–/– embryos indicated that Irf6 transcripts were reduced to approximately 90% and 63%, respectively, of normal levels. **P = 0.01 versus p63+/– and wild-type (Mann-Whitney U test). (B) siRNA knockdown of p63 in mouse primary keratinocytes reduced p63 levels 5-fold. *P = 0.05 versus control scrambled siRNA (Mann-Whitney U test). (C) Irf6 transcript levels after p63 siRNA knockdown were reduced more than 60%. *P = 0.05 versus control scrambled siRNA (Mann-Whitney U test). (D) Western analysis reveals reduced Irf6 protein levels in mouse primary keratinocytes after p63 siRNA knockdown. Quantitation, shown above the Western blot, shows Irf6 levels relative to tubulin. Samples were run on the same gel but were noncontiguous (white line). (E) qPCR analysis of IRF6 in human primary keratinocytes derived from normal control individuals and patients with EEC syndrome (R204W, R279H, and R304W) showed reduced IRF6 levels when the DNA-binding function of p63 was impaired. *P = 0.05, ***P = 0.001 versus control (Kruskal-Wallis 1-way ANOVA followed by post-hoc Dunn’s test). Data represent mean ± SEM.