Functional evolution of polyQ repeats in the N-terminal domain of FOXP2

Homopolymeric amino acid repeats are frequently found in eukaryotic proteins where they mediate protein-protein interactions and regulate protein localization and function. In particular, the human FOXP2 protein, whose genetic mutations have been linked to vocalization and language disorders, contains an N-terminal glutamine-rich region with multiple polyQ repeats, which is phylogenetically ancient and has been recently proposed to play functional and evolutionary roles. Notably, the length of these polyQ repeats varies extensively throughout phylogenesis, suggesting functional implications. However, the relationship between the polyQ evolutionary dynamics and FOXP2 function is still poorly understood. Thus, we investigate here whether polyQ evolution in FOXP2 may have functional implications, through a combination of bioinformatics, biophysical, cell biological, and confocal imaging approaches in living cells. Specifically, our experiments searched for functional differences between human FOXP2 and a mutant form of this protein in which we replaced part of the polyQ repeats with those found in the ortholog of Rhinolophus ferrumequinum, a bat emitting constant-frequency ultrasonic vocalizations, which displays a very distinct polyQ repeat configuration. We compared the supramolecular organization, subcellular distribution, mobility, and functional activity of the human wild type and bat-like FOXP2 forms by means of atomic force microscopy, confocal fluorescence microscopy, fluorescence recovery after photobleaching, and transcriptional luciferase assays. We find significant differences between the two polyQ variants in all the morphological and functional aspects that were examined. Taken together, these results support the notion that polyQ repeats in FOXP2 are functionally relevant and that their phylogenetic variation may play important evolutionary roles.