| Summary: | Forkhead (Fox) proteins are transcription factors that function in many processes including development, metabolism and cell cycle regulation. This gene family is divided into subfamilies that appear to originate from a common ancestor. I have identified the evolutionary selection pressures acting on individual amino acid positions in the FoxA, FoxC, FoxD, FoxI, FoxO and FoxP subfamilies. The patterns of selection observed allowed for the prediction of residue function and identification of residues that differentiate orthologs and paralogs. The subfamily structure and negative selection found within the subfamilies indicates that after gene duplication, differentiation of subfamilies through amino acid changes and subsequent negative selection on these changes has occurred. Meanwhile, the observed neutral changes and positive selection allow for further protein differentiation. Within the FoxC subfamily, positive selection was identified at one amino acid site in the inhibitory domain. Mutation of this site in FOXC1 alters transactivation activity and the effects of mutants on transactivation activity are different on different reporters. The mutant effects were consistent with those of known disease causing mutations, supporting the predicted positive selection. The inhibitory domain is known to function in reducing FOXC1 transactivation activity and influences protein stability. Here I additionally show that loss of the inhibitory domain and mutation of the positively selected site can reduce FOXC1 DNA binding. Co-transfection of FOXC1 and TLE4, a repressor protein that can potentially bind to the inhibitory domain, was shown to increase FOXC1 transactivation activity. The effects of a novel disease causing FOXC1 inhibitory domain mutation on FOXC1 function were also assessed. The mutation reduced FOXC1 transactivation activity and increased protein half-life both of which may lead to disease. Regulation of FOXC1 activity is critical for normal function and this work has furthered our knowledge of how the inhibitory domain influences FOXC1 activity. I have provided biological evidence for the theory that positive selection acts at the amino acid level to optimize protein function. I have also shown that both changes in transcription factor proteins and the cis-regulatory region of target genes have the potential to contribute to evolutionary adaptation.
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