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Fig. 5 | Journal of Neurodevelopmental Disorders

Fig. 5

From: Functional characterization of rare FOXP2 variants in neurodevelopmental disorder

Fig. 5

Characterization of FOXP2 variants with ancestral amino acid substitutions. a Schematic representation of FOXP2 variants with ancestral amino acid substitutions. The N303T, S325N and N303T/S325N constructs are synthetic variants of human FOXP2 carrying ancestral amino acid substitutions. The chimpanzee and mouse orthologues of FOXP2 are included for comparison. Amino acid differences relative to human FOXP2 are indicated by arrowheads: N303T (red), S325N (blue), D80E (green), glutamine insertion (+Q, black), and glutamine deletion (−Q, white). 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 minimal CTBP interaction region determined using the BRET assay is indicated by the grey-shaded box. b Fluorescence micrographs of HEK293 cells transfected with FOXP2 variants with ancestral amino acid substitutions. Nuclei were stained with Hoechst 33342. c Fluorescence-based measurement of the expression level of FOXP2 variants with ancestral amino acid substitutions. HEK293 cells were transfected with YFP-FOXP2, together with mCherry for normalization. Fluorescence intensity was measured 48 h post-transfection. Values are mean YFP/mCherry fluorescence ratios ± S.D. (n = 3), relative to the value for human FOXP2. d, e Luciferase reporter assays for transcriptional regulatory activity of FOXP2 variants with ancestral amino acid substitutions. HEK293 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 human 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, >0.9999 for the N303T, S325N, and N303T/S325N variants and 0.2730 for the mouse protein. Exact p values for e are <0.0001 for the control and >0.9999 for the N303T, S325N, N303T/S325N and mouse variants. fh BRET assays for protein-protein interactions of FOXP2 variants with ancestral amino acid substitutions. HEK293 cells were transfected with FOXP2 variants with ancestral amino acid substitutions fused to Renilla luciferase (donor), together with YFP (acceptor) fusions of the same FOXP2 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, >0.9999 for the N303T, S325N, and N303T/S325N variants and 0.434 for the mouse protein. Exact p values for g are <0.0001 for the control and >0.9999 for the N303T, S325N, N303T/S325N and mouse variants. Exact p values for h are <0.0001 for the control, >0.9999 for the N303T, S325N, and mouse variants and 0.10 for the N303T/S325N variant

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