The wing 2 region of the FOXC1 forkhead domain is necessary for normal DNA–binding and transactivation functions.

Abstract

Abstract: : Purpose: FOXC1 is a member of the forkhead/winged–helix family of transcription factors. Mutations in FOXC1 cause Axenfeld–Rieger (AR) malformations and increase the risk of early onset glaucoma. Two novel missense mutations, G165R and R169P and previously reported mutation, M161K, have been identified in wing 2 of the forkhead domain. The effects of these mutations on the ability of the FOXC1 protein to localize to the cell nucleus, bind DNA, and activate gene expression were investigated. Methods: AR patient DNA was amplified by PCR and sequenced manually using ³³P sequencing. Patient DNA and 100 controls were sequenced to confirm that the mutations were not present in the normal population. Site–directed mutagenesis was used to introduce the three mutations into the FOXC1 cDNA. Epitope–tagged FOXC1 constructs were expressed in COS–7 cells, producing products of the correct size by Western analysis. Nuclear localization was examined by immunofluoresence. The effect of each missense mutation on FOXC1–DNA interactions was tested by electrophoretic mobility shift assays (EMSAs) while the effect on the transactivation ability of FOXC1 was tested using a dual luciferase reporter assay. Results: G165R, M161K and R169P reside in the C–terminal nuclear localization signal (NLS) of FOXC1. The three wing 2 mutations are not sufficient to perturb the nuclear localization of FOXC1. EMSAs reveal that G165R is able to maintain wild type levels of DNA binding but, M161K and R169P result in reduced DNA binding for the FOXC1 binding site. G165R, M161K, and R169P all reduced FOXC1 transactivation ability from a luciferase reporter by 20 –36%. Conclusions: Based on the crystal structure of FOXA3/ DNA, wing 2 has been predicted to play a role in DNA binding through a sole side chain–base contact in the minor groove. Mutation of R169P and M161K demonstrate that wing 2 plays a role in DNA binding by reducing the DNA binding ability of FOXC1. The severe reduction in transactivation ability of all three FOXC1 wing 2 mutations indicates an important role for wing 2 in transactivation, independent of its role in DNA binding. These experiments help define our understanding of how disease–causing missense mutations perturb FOXC1 function and elaborate on the functional significance of wing 2, a poorly characterized region. Funded by the CIHR, the University of Alberta and GAP.

Keywords: mutations • transcription factors • protein structure/function