Figure 1

FOXP2 interacts with members of the PIAS family of SUMO E3 ligases.

(a) Top: schematic representation of human PIAS proteins. Domains are shaded in dark blue: SAP domain (SAP); PINIT domain (PINIT); SP-RING domain (SP-RING); acidic domain (AD); serine/threonine-rich domain (S/T). The number of amino acid residues is shown to the right of the schematic. Bottom: identity matrix for PIAS proteins. (b) BRET assay for interaction between FOXP2 and PIAS proteins. HEK293 cells were transfected with luciferase-FOXP2 (donor) and YFP-PIAS (acceptor). The control donor protein is a nuclear-targeted luciferase. Values are mean corrected BRET ratios ± S.E.M. (n = 3). (c) Fluorescence micrographs of HEK293 cells transfected with mCherry-PIAS (red) and YFP-FOXP2 (green). Nuclei were stained with Hoechst 33342 (blue). (d) Left panel: Schematic representation of synthetic truncated forms of FOXP2. The number of amino acid residues is shown on the left; 1–715 represents the full-length protein. Known domains are shown in dark blue: glutamine-rich region (Q-rich); zinc finger (ZF); leucine zipper (LZ); forkhead domain (FOX). A nuclear-targeting signal (shown in black) was appended to the C-terminus of variants 1–487, 1–329 and 1–258 because these variants lack endogenous nuclear targeting signals. Centre panel: BRET assay for interaction between synthetic FOXP2 truncations and wild-type FOXP2. HEK293 cells were transfected with luciferase-FOXP2 truncations (donor) and YFP-FOXP2 (acceptor) Right panel: BRET assay for the interaction between synthetic FOXP2 truncations and PIAS1. HEK293 cells were transfected with luciferase-FOXP2 truncations (donor) and YFP-PIAS1 (acceptor). The control donor protein is a nuclear-targeted luciferase. Values are mean corrected BRET ratios ± S.E.M. (n = 3).