Figure 6. The repression activity of FOG-2 is hindered by SUMOylation in HeLa cells and in rat cardiomyocytes.

HeLa cells were co-transfected with a reporter construct containing the luciferase gene under the control of the BNP promoter and the indicated vectors. Dual luciferase assays were performed 24 h post-transfection. (A) The BNP promoter was activated by GATA-4 and, as expected, wt FOG-2 repressed this activation. Expression of the SUMOylation mutant molecule (FOG-2-4KR) resulted in significantly stronger repression activity. The immunoblots represent total cell lysates from the transfected cells probed with the antibodies indicated in the Figure. (B) The BNP promoter was activated by GATA-4 and this activation was strongly repressed by FOG-2-4KR. The constitutively SUMOylated fusion protein SUMO-1-FOG-2-4KR failed to repress GATA-4-mediated activation. The immunoblots represent total cell lysates from the transfected cells probed with the antibodies indicated in the Figure. Data represent the mean ± SD from 3 independent experiments (C) Neonatal rat cardiomyocytes were nucleofected with the vectors indicated in the Figure. In agreement with the findings in A and B, FOG-2-4KR showed stronger repression activity than the wt, while the SUMO-1-FOG-2-4KR fusion demonstrated significantly reduced repression capacity. Of note, co-expression of GATA-4 in cardiomyocytes did not activate the BNP promoter significantly most likely due to quenching by the presence of endogenous GATA-4. In addition, the repression exerted by FOG-2 and FOG-2-4KR reduced luciferase activity to levels lower than the reporter itself, again suggesting that the promoter alone was activated by endogenous GATA-4 and that FOG-2 repressed this activity. Data represent the arithmetic mean from 2 independent experiments. IB, immunoblot; ns, not significant; nr, no reporter; S-1, SUMO-1; F2m, FOG-2-4KR; S-1F2m, SUMO-1-FOG-2-4KR.