The wing 2 region of the FOXC1 forkhead domain is necessary for normal DNA-binding and transactivation functions.
OBJECTIVE
To determine the biochemical defects that underlie Axenfeld-Rieger malformations, to determine a functional role for wing 2 in FOXC1, and to understand how mutations in this region disrupt FOXC1 function.


METHODS
Sequencing DNA from patients with Axenfeld-Rieger malformation resulted in the identification of two novel missense mutations (G165R and R169P) in wing 2 of FOXC1. Site-directed mutagenesis was used to introduce these mutations, as well as previously reported mutation (M161K), into the FOXC1 cDNA. These FOXC1 mutants were evaluated to determine their ability to localize to the nucleus, bind DNA and activate gene expression.


RESULTS
Two novel missense mutations were identified in unrelated patients, in wing 2 of the FOXC1 forkhead domain. Because there had been no previous biochemical analysis, the mutation M161K was also investigated. All three mutant proteins localized correctly to the nucleus. The G165R mutation maintained wild-type levels of DNA binding; however, both the M161K and R169P mutations displayed reduced DNA binding ability. Biochemical analysis showed that all three mutations disrupt FOXC1's transactivation ability.


CONCLUSIONS
Biochemical analysis of mutations G165R and R169P and of a previously reported mutation, M161K, demonstrate the functional significance of wing 2. M161K and R169P disrupt DNA binding of FOXC1, consistent with the hypothesis that wing 2 is necessary for DNA binding. The results also suggest that wing 2 plays a role in gene activation. These results provide the first insights into how mutations in wing 2 disrupt FOXC1 function.
Murphy
Tara C
TC
Department of Ophthalmology, University of Alberta, Edmonton, Canada.
Saleem
Ramsey A
RA
Footz
Tim
T
Ritch
Robert
R
McGillivray
Barbara
B
Walter
Michael A
MA
D000595:Amino Acid Sequence; D000818:Animals; D000869:Anterior Eye Segment / Q000002:abnormalities*; D019556:COS Cells; D002675:Child, Preschool; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D005124:Eye Abnormalities / Q000235:genetics*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D051858:Forkhead Transcription Factors; D005901:Glaucoma / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007498:Iris / Q000002:abnormalities*; D008875:Middle Aged; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010957:Plasmids; D011487:Protein Conformation; D014157:Transcription Factors / Q000737:chemistry / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology*
D004268:DNA-Binding Proteins; C112743:FOXC1 protein, human; D051858:Forkhead Transcription Factors; D014157:Transcription Factors