Analyses of the effects that disease-causing missense mutations have on the structure and function of the winged-helix protein FOXC1

Five missense mutations of the winged-helix FOXC1 transcription factor, fou nd in patients with Axenfeld-Rieger (AR) malformations, were investigated f or their effects on FOXC1 structure and function. Molecular modeling of the FOXC1 forkhead domain predicted that the missense mutations did not alter FOXC1 structure. Biochemical analyses indicated that, whereas all mutant pr oteins correctly localize to the cell nucleus, the I87M mutation reduced FO XC1- protein levels. DNA-binding experiments revealed that, although the S8 2T and S131L mutations decreased DNA binding, the F112S and I126M mutations did not. However, the F112S and I126M mutations decrease the transactivati on ability of FOXC1. All the FOXC1 mutations had the net effect of reducing FOXC1 transactivation ability. These results indicate that the FOXC1 forkh ead domain contains separable DNA-binding and transactivation functions. In addition, these findings demonstrate that reduced stability, DNA binding, or transactivation, all causing a decrease in the ability of FOXC1 to trans activate genes, can underlie AR malformations.