Purchase this article with an account.
M.V. Carle, Y. Tamimi, M.A. Walter; Analysis of 2 Mutations of FOXC2 in Lymphedema–Distichiasis: Functional Comparison to Potentially Analogous Known FOXC1 Axenfeld–Rieger Mutations . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3140.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Forkhead box (FOX) transcription factors contain a conserved 110 amino acid DNA–binding domain, the forkhead domain, that we have shown is required for nuclear localization, DNA–binding, and transactivation activity of FOX proteins. The FOX gene family plays key roles in development, cell fate determination, and tissue–specific gene expression in both vertebrates and invertebrates. Recently a number of disease–causing mutations have been identified in human FOX genes. FOXC2 mutations are responsible for hereditary lymphedema–distichiasis syndrome, and FOXC2 has also been implicated in hypertriglyceridemia, obesity, and diet–induced insulin resistance. Mutations in FOXC1 underlie human Axenfeld–Rieger (AR) anterior segment malformations in the eye that result in a spectrum of glaucoma phenotypes, dental anomalies, and redundant periumbilical skin. The overall effect of missense mutations found in AR patients has been determined to be reduced transactivation function of FOXC1. Methods: A series of parallel experiments of wild–type FOXC1 and FOXC2 and the analogous mutated counterparts of FOXC1 and FOXC2 (S131L and S125L; R127H and R121H) were performed. FOXC2 was amplified from genomic DNA by PCR, and subcloned into a modified pcDNA–C mammalian expression plasmid in which the Apa restriction site had been abolished. The missense mutants R121H and S125L were subcloned into FOXC2 using internal restriction sites. The functional aspects of these mutations were analized by immunofluorescence to measure the ability of FOXC2 to localize to the cell nucleus, Electrophoretic Mobility Shift Assays (EMSA) to measure DNA binding; and Dual–Luciferase Assays of reporter plasmids to measure transactivation capacity. Results: The three parameters tested for the FOXC1 mutations have shown: R127H had a decreased nuclear localization and no detectable DNA binding, S131L showed normal nuclear localization, and a decreased DNA binding, while both had significantly decreased transactivation. The investigation of these same parameters on the FOXC2 mutations are underway. Conclusions: These mutations in FOXC1change the ability of this transcription factor to correctly localize to the nucleus, bind DNA, and activate transcription. While the results for FOXC2 are pending, this model allows a better understanding of the forkhead family and homeobox function. Molecular modeling of these two genes will result in improvements in the models of FOX family function and may enable better predictive value for similarly structed proteins.
This PDF is available to Subscribers Only