Single amino acid probing of the function and molecular topology of the forkhead/winged–helix transcription factor FOXC1

Abstract

Abstract: : Purpose: The forkhead domain–containing transcription factor FOXC1 is mutated in patients presenting with Axenfeld–Rieger malformations. Positive, negative or neutral charged amino acids were substituted into positions within the FOXC1 forkhead domain, predicted to be critical on the basis of bioinformatic analyses, to better understand essential structural and functional determinants within the forkhead domain. Methods: Molecular models for the forkhead domain of FOXC1 were generated using MODELLER based on the NMR structure of FOXC2 (pdb|1D5V). Site directed mutagenesis was used to introduce mutations into the FOXC1 expression plasmid FOXC1 pcDNA His/Max4. Epitope–tagged FOXC1 constructs were expressed in COS–7 cells. The effect of each substitution on FOXC1–DNA interactions was tested by electrophoretic mobility shift assays (EMSAs) while the effect of each alteration on the transactivation ability of FOXC1 was tested using a dual luciferase reporter assay. Results: FOXC1 is intolerant of mutations at I87. Additionally, alterations of amino acids within alpha–helix 1 of the FOXC1 forkhead domain affected both nuclear localization and transactivation. Amino acids within alpha–helix 3 were also found to be necessary for transactivation and can have roles in correct localization. Interestingly, changing amino acids within alpha–helix 3, particularly R127, resulted in altered DNA binding specificity and granted FOXC1 the ability to bind to a novel DNA sequence. Conclusions: These studies demonstrate the functional importance of not only individual amino acids within the FHD of FOXC1, but also demonstrate the how alpha–helix 1 and alpha–helix 3 secondary structures in the FHD are involved in FOXC1 function. This merging of bioinformatic analyses with experimental cell biology has generated a model of FOXC1 protein structure with predictive value. Given the limited topological variation of forkhead domains, due to the high conservation of residues, we anticipate that models of forkhead domain function derived from these data will be relevant to other members of the FOX family of transcription factors.

Keywords: transcription factors • anterior chamber • protein structure/function