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L. Huang, Y. Tamimi, M. A. Walter; Regulation of the Fibroblast Growth Factor Gene FGF19 by Both FOXC1 and FOXC2 Provides Evidence for Non-Allelic Non-Complementation of FOXC1 and FOXC2 in the Eye. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2975.
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FOXC1 and FOXC2 are related members of the Forkhead Box transcription factor family. Mutations in FOXC1cause Axenfeld-Rieger malformations often leading to glaucoma. Mutations in FOXC2 cause hereditary lymphedema with distichiasis. Foxc1 and Foxc2 have overlapping expression patterns, and Foxc2+/- mice and Foxc1+/- mice have similar eye phenotypes. Foxc1+/- and Foxc2+/- double heterozygous mice present with more severe ocular defects than in either single heterozygote, which strongly suggests overlapping function of FOXC1 and FOXC2 in the developing eye. We therefore hypothesize that FOXC1 and FOXC2 co-regulate some downstream target genes. Recent work from our laboratory revealed that FOXC1 directly regulates FGF19 expression. Here we tested if FOXC2 also regulates the FGF19 expression.
A 1.3Kb upstream element (FGF19-5’-UPE) and a 354bp amplicon (FGF19-RE) of the FGF19-5’-UPE containing FOXC binding site were cloned into the pGL3-TK-luciferase vector. Luciferase activities were monitored after co-transfecting HeLa cells with pcDNA4-FOXC2 and FGF19-5’-UPE-pGL3-TK or FGF19-RE-pGL3-TK luciferase reporter vector. A reporter containing 6 FOXC-binding sites (6XBS) was used as a positive control. HeLa cells were also co-transfected with FOXC1 and FOXC2 plus the FGF19-RE-pGL3-TK luciferase reporter to study the regulation of FGF19 expression by both FOXC1 and FOXC2.
FOXC2 significantly activated FGF19-5’-UPE and FGF19-RE luciferase reporters 200% more than the control. The FGF19-5'UPE or FGF19-RE cloned in the reverse orientation resulted in significantly reduced reporter activation. In addition, mutation of the putative FOXC binding site in the FGF19-RE completely abolished the FOXC2 responsiveness of the FGF19 construct. Co-transfection with both FOXC1 and FOXC2 resulted in FGF19 transcriptional activation levels similar to those when either transcription factor was used alone.
FGF19 is a shared downstream target gene of both FOXC1 and FOXC2, strongly supporting the hypothesis that both factors are key regulators of transcription in overlapping ocular genetic pathways. Our finding that FOXC1 and FOXC2 independently regulate FGF19 provides an explanation for the similar phenotypes of double heterozygous Foxc2+/- /Foxc1+/- mice to those of mice with homozygous null alleles of either FOXC1 or FOXC2, and direct evidence for non-allelic non-complementation of FOXC1 and FOXC2 in the eye.
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