Fig. 4

Mapping of the CTBP binding site in FOXP2. a Schematic representation of synthetic truncated forms of FOXP2. The R328* and Q390Vfs*7 variants identified in patients are included for comparison. Known domains are labelled: glutamine-rich (Q-rich) region (hatched shading) including polyglutamine tracts (solid shading), zinc finger (ZF), leucine zipper (LZ), and forkhead domain (FOX). The PLNLV motif is indicated with a green bar. Nuclear localization signals are indicated with red bars. A synthetic nine-residue nuclear targeting sequence (hatched red bars) was appended to the C-terminus of variants which lack one or both of the endogenous nuclear localization signals. b Fluorescence micrographs of HEK293 cells transfected with synthetic truncated FOXP2 variants. Nuclei were stained with Hoechst 33342. c, d BRET assay for interaction of synthetic truncated FOXP2 variants with full-length FOXP2 and CTBP2. HEK293 cells were transfected with truncated FOXP2 variants fused to Renilla luciferase (donor) and FOXP2 (c) or CTBP2 (d) fused to YFP (acceptor). The control donor protein is a nuclear-targeted luciferase. Values are mean corrected BRET ratios ± S.D. (n = 3). Asterisks indicate significant differences compared to control (p < 0.05, one-way ANOVA followed by Bonferroni post hoc correction). NS not significant. Exact p values for c are 0.002 for FOXP2.330*, 0.025 for FOXP2.259* and <0.0001 for all other variants. Exact p values for d are 0.001 for FOXP2.423*, 0.012 for FOXP2.330*, 0.97 for FOXP2.259*, and <0.0001 for all other variants