Molecular Mechanisms and Effects of Mutant Irf6 Gene on Epithelial Cells

Cleft lip/palate is one of the most common human craniofacial congenital diseases, occurring in one of 600 live births. However, its pathogenesis and molecular mechanism remain unknown. Van der Woude syndrome is a unique group among patients with cleft lip/palate (CLP) caused by an autosomal dominant gene, and interferon regulatory factor 6 (IRF6) gene mutation is the causative factor. Recently, IRF6 gene mutation has been found to cause other non-syndromic CLP in 12% of the patients. Its underlying molecular mechanism is unclear. Study of the effect and molecular mechanism of IRF6 mutant gene on embryonic palatal shelves fusion should help to understand its disease pathogenesis, and therefore clinical translation to disease treatment and prevention. In the past study of IRF6 gene silencing on the palatal shelves fusion, we have successfully used virus as a vector to transfer small interference RNA (RNAi) into in vitro embryonic palatal shelves culture. It effectively shut down the expression of IRF6 gene. We have found that the IRF6 gene silencing does not prevent the fusion of palatal shelves among in vitro culture system, which is contradictory to the findings among in vivo system. This may imply that IRF6 gene does not directly involve in the process of palatal shelves fusion. Therefore, we speculate that the IRF6 gene may exert other functions onto the epithelial cells of palatal shelves, as the gene has been found to appear in these cells during the fusion process. Recent reports have shown that IRF6 gene may inhibit cellular growth, but its regulatory mechanism is not clear. Our study aims to evaluate the regulatory mechanism of IRF6 gene onto the cellular growth. We have constructed wild type IRF6, IRF6-R84C, IRF6-R250Q, IRF6-V274I and IRF6-R400W expression plasmids. We find that IRF6-R250Q and IRF6-R400W protein have better stability and longer half life than wild type IRF6. IRF6-R400W protein has shorter half life than that of normal IRF6 protein. We will further investigate that whether different IRF6 mutations have different influences on cells. To further delineate the IRF6 functions and the influences from IRF6 gene mutations, we would investigate if IRF6 involves in cell cycle and cell apoptosis. It is known that deltaNp63α can work with IRF6, and has negative regulatory function. DeltaNp63α is reported to regulate cell cycle and apoptosis. We would like to investigate if IRF6 regulate cell growth through interaction with deltaNp63α. In addition, we will use yeast two hybrid method to find out proteins that are connected to IRF6 protein, which may reveal the regulatory mechanism and signal pathway. Microarray will be applied to discover the downstream genes regulated by IRF6 protein. The effect of IRF6 gene mutation onto expression of IRF6 protein, other proteins, and its downstream genes will be searched. Knowledge of the effect of the IRF6 protein and mutation on the signal pathway will help to understand its role in palate development and pathogenesis of cleft palate. The molecular mechanism will be used in the future study to evaluate embryonic palatal development in mouse model, and IRF6 mutation will be used to study cleft palate formation.

Project ID:PC10107-0356
External Project ID:NSC101-2314-B182-026