Fig. 4: Loss of TRIM28 SUMO-E3-ligase activity in granulosa cells phenocopies Trim28 conditional knock-out.
From: TRIM28-dependent SUMOylation protects the adult ovary from activation of the testicular pathway
a Schematic of the SUMO pathway with TRIM28 E3-SUMO ligase activity. After proteolytic maturation by sentrin-specific proteases (SENPs), SUMO C-terminus is activated by the heterodimeric SUMO-activating enzyme E1 (SAE1/SAE2), and then transferred to a cysteine of E2 (UBC9). Subsequently, the E3 ligases (TRIM28) transfer SUMO from E2 to a lysin residue(s) of target proteins. SUMO2 and 3 diverge by only one residue, making them indistinguishable by antibodies, thus they are currently referred to as SUMO2. b RT-qPCR analysis of ovarian- and testicular-specific genes in 8-week-old Trim28cKO, Trim28Phd/cKO, Trim28Phd/+, and control ovaries. Bars are the mean ± SEM, n = 5 animals (gonad pairs). P: < 0.0001 (****), 0.0002(***), 0.0021(**), 0.032(*) (Ordinary one-way ANOVA with Dunnett’s multiple comparisons test). Details of the statistical analysis are provided in the Source data file. Source data are provided as a Source Data file. c FOXL2 is expressed in control and Trim28Phd/+ ovaries, but not in Trim28Phd/cKO and Trim28cKO ovaries. Like in Trim28cKO ovaries, SOX9, SOX8 and DMRT1 are expressed in pseudo-tubules of Trim28Phd/cKO ovaries, but not in control and Trim28Phd/+ ovaries. Protein (green or red) is merged with DNA stains (blue). Scale bar: 50 µm. d Confocal microscopy shows strong SUMO1 and 2 nuclear staining in granulosa cells of control ovaries. The staining intensity is markedly decreased in Trim28cKO and Trim28Phd/cKO ovaries. SUMO1/2 staining is merged with DNA staining. Scale bar: 20 µm. Right panels: quantification of SUMO1 and SUMO2 signal intensity relative to DNA staining. For the three conditions (control and mutants) each column represents one experiment, n represents the number of cells analysed. P: < 0.0001 (****), 0.0002(***), 0.0021(**), 0.032(*)(two-way ANOVA with Dunnett’s multiple comparisons test). Details of the statistical analysis are provided in Source data file. Source data are provided as a Source Data file. For the immunofluorescences, at least three independent biological replicates were analysed, and the images presented are representative of all replicates.
