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F.B. Berry, M.A. Lines, J.M. Oas, T. Footz, D.A. Underhill, P.J. Gage, M.A. Walter; Functional Interactions Between FOXC1 and PITX2 Underlie the Sensitivity to FOXC1 Gene Dose in Axenfeld–Rieger Syndrome and Anterior Segment Dysgenesis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5915.
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© ARVO (1962-2015); The Authors (2016-present)
Axenfeld–Rieger ocular dysgenesis is associated with mutations of the human PITX2 and FOXC1 genes, which encode transcription factors of the homeodomain and forkhead classes, respectively. We sought to identify a functional link between FOXC1 and PITX2 through protein–protein interactions.
Expression patterns for FOXC1 and PITX2 proteins were assessed in the developing mouse eye primordia by immunofluorescence microcopy. Physical interactions between FOXC1 and PITX2 were determined by immunoprecipitation experiments. Dual–luciferase assays were performed to monitor activation of transcription by FOXC1 and PITX2.
We demonstrate that both PITX2 and FOXC1 exhibit a dynamic coexpression pattern during ocular development. Furthermore we present the first biochemical evidence of a common functional role for PITX2 and FOXC1. Coimmunoprecipitation reveals that recombinant PITX2A and FOXC1 associate in a physical complex and this interaction requires crucial functional domains on both proteins: the C–terminal activation domain of FOXC1 and the homeodomain of PITX2. Immunofluorescence further shows PITX2A and FOXC1 to be co–localized within a common nuclear subcompartment. Furthermore, PITX2A can function as a negative regulator of FOXC1 transactivity.
This work ties both proteins into a common pathway and offers an explanation of why increased FOXC1 gene dosage produces a phenotype resembling that of PITX2 deletions and mutations. Ocular phenotypes arise though the deregulated expression of FOXC1–target genes through mutations in either FOXC1 or PITX2. Ultimately, in our model, PITX2 loss of function mutations have a compound effect: the reduced expression of PITX2–target genes coupled with the extensive activation of FOXC1–regulated targets. Our findings indicate that the functional interaction between FOXC1 and PITX2A underlies the sensitivity to FOXC1 gene dosage in Axenfeld–Rieger syndrome and related anterior segment dysgeneses.
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