SUMOylation of the Forkhead Transcription Factor FOXL2 Promotes Its Stabilization/Activation through Transient Recruitment to PML Bodies
Figure 4
FOXL2 recruitment to PML Bodies enhances its transactivation ability.
A and B) FOXL2 colocalization with PML, SUMO1 and SP100 in nuclear bodies. COS-7 cells overexpressing SUMO-FOXL2-GFP, FOXL2-WT-GFP, FOXL2-KFULL-GFP or NLS-GFP (as indicated, green color) were stained with an anti-PML antibody (panel A, red) or an anti-SUMO1 anti-body (panel B, first three lines, red) In the fourth line of panel B, COS-7 cells overexpressing FOXL2-WT-GFP and SP100A-HSV were stained with an anti-HSV antibody. Representative cells with FOXL2 enriched in nuclear bodies are shown. DNA was stained using Hoechst 33342 (in blue). Scale bar: 5 µm. Scale bar is valid for all micrographs. (C) Interaction between FOXL2 and SP100. SP100-HSV was expressed alone (lane 1) or co-expressed with V5-tagged FOXL2-WT (lane 2) or KFULL (lane 3). FOXL2 was precipitated using anti-V5-conjugated affinity resin for western blot analysis. Upper panel shows a 50 kDa band detected with anti-FOXL2 antibody. Second panel shows a 70 kDa band detected with an anti-HSV antibody. Third and fourth panels display a western blot analysis of the lysates, with anti-FOXL2 and anti-HSV antibodies, respectively. (D) Quantification of cells displaying subnuclear structures in COS-7 expressing SUMO-FOXL2-GFP or FOXL2-WT-GFP with or without overexpression of SUMO1, PML-IV-RFP, PML-V-RFP or SP100A-HSV. At least 300 GFP-positive cells were scanned per condition (in groups of 50 to estimate the standard deviations). Error bar: SEM. Letters a, b, c, d refer to statistical categories in a Student's t-test. Conditions with different letters are statistically different with p<0.05. (E) PML localisation in COS-7 or KGN cells overexpressing PML-V-RFP. Green: anti-PML directed against all isoforms of PML. Red: PML-V-RFP. Blue: DNA stained with Hoechst 33342. In non-transfected cells, PML is mostly nucleoplasmic with a few dots (1–5 in COS-7 cells, 5–20 in KGN cells) indicative of PML NBs. With PML-V-RFP expressed, PML NBs are much enlarged and the proportion of nucleoplasmic PML is decreased. Scale bar: 10 µm. Scale bar is valid for all micrographs. F and G) Luciferase assays in KGN cells transfected with Per2-luc and pSODluc-3340 (40 ng/well), respectively, with or without FOXL2 and PML-V-RFP overexpression (each 20 ng/well). Each value is representative of six biological replicates. Error bars represent SEM. Statistical significance in Student t-tests: n.s.: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.001. H) Western blot of whole cell extracts of KGN cells transfected with or without FOXL2-V5 and PML-V-RFP (2 µg/well in a 6-well plate), using pcDNA3.1 as a mock vector. Lic- (expressing a fusion protein His-Tag∶S-Tag∶HSV-Tag∶His-Tag) was used as a transfection control (500 ng). First panel: FOXL2. Second panel: Lic- (anti-HSV). Third panel: PML-V (anti-HA). Below second panel is indicated the relative intensity of the FOXL2 band compared to the Lic- band. All experiments were repeated at least three times with consistent results.