Fig. 6

Characterization of synthetic FOXP2 variants with reduced polyglutamine tracts. a Schematic representation of synthetic FOXP2 variants with reduced polyglutamine tracts. Known domains are labelled: glutamine-rich (Q-rich) region (hatched shading) including the long polyglutamine tract (Q _L ) and short polyglutamine tract (Q _S ), zinc finger (ZF), leucine zipper (LZ), and forkhead domain (FOX). The ΔS variant has a short polyglutamine tract reduced from 10 to 3 residues. The ΔL variant has a long polyglutamine tract reduced from 40 to 3 residues. The ΔS + L variant has shortened versions of both tracts. b Fluorescence micrographs of HEK293 cells transfected with FOXP2 variants with reduced polyglutamine tracts. Nuclei were stained with Hoechst 33342. c Fluorescence-based measurement of expression level for FOXP2 variants with reduced polyglutamine tracts. HEK293 cells were transfected with YFP-FOXP2 variants together with mCherry for normalization. Fluorescence intensity was measured 48 h post-transfection. Values are mean YFP/mCherry fluorescence ratios ± S.D. (n = 3), expressed relative to the value for full-length FOXP2. d, e Luciferase reporter assays for transcriptional regulatory activity of FOXP2 variants with reduced polyglutamine tracts. Cells were transfected with a luciferase reporter vector containing the SV40 promoter (d) or the human SRPX2 promoter (e), together with a Renilla luciferase normalization plasmid, and YFP-FOXP2 or YFP alone (control). Values are mean relative luciferase activity ± S.D. (n = 3), expressed relative to the control. Asterisks indicate significant differences compared to full-length FOXP2 (p < 0.05, one-way ANOVA followed by Bonferroni post hoc correction). NS not significant. Exact p values for d are <0.0001 for the control, 0.4892 for ΔS, 0.0003 for ΔL, and 0.0010 for ΔS + L. Exact p values for e are <0.0001 for the control, 0.0768 for ΔS, 0.0633 for ΔL, and 0.1506 for ΔS + L. f–h BRET assays for protein-protein interactions of FOXP2 variants with reduced polyglutamine tracts. HEK293 cells were transfected with FOXP2 variants with reduced polyglutamine tracts fused to Renilla luciferase (donor) and YFP (acceptor) fusions of the same variants (f), CTBP1 (g) or CTBP2 (h). The control donor protein is a nuclear-targeted luciferase and the control acceptor protein is a nuclear-targeted YFP. Values are mean corrected BRET ratios ± S.D. (n = 3). Asterisks indicate significant differences compared to wild-type (WT) FOXP2 (p < 0.05, one-way ANOVA followed by Bonferroni post hoc correction). NS not significant. Exact p values for f are <0.0001 for the control, ΔL and ΔS + L, and >0.9999 for ΔS. Exact p values for g are <0.0001 for the control, >0.9999 for ΔS, 0.038 for ΔL, and 0.024 for ΔS + L. Exact p values for h are <0.0001 for the control, 0.4 for ΔS, and >0.9999 for ΔL and ΔS + L