Fig. 4.
PIASy enhances GATA-1 sumoylation and represses GATA-1 transcriptional activity. (A) PIASy increases GATA-1 sumoylation. WT GATA-1 or the K137R mutant were transfected in 293T cells together with GFP-SUMO-1 and a vector expressing T7-PIASy as indicated. Lysates were separated by SDS/PAGE, and GATA-1 was detected by immunoblotting. (B) PIASy inhibits GATA-1-dependent transactivation. A GATA-1-responsive luciferase reporter plasmid was transfected in U2OS cells together with vectors expressing GATA-1, GFP-SUMO-1, and T7-PIASy as indicated. A plasmid constitutively expressing Renilla luciferase (pRL-CMV) was included as a control for transfection efficiency. GATA-1 transcriptional activity was measured by a dual luciferase assay (Upper), and GATA-1 protein was analyzed by immunoblotting (Lower). The band indicated by an arrowhead in lane 3 likely corresponds to GATA-1 conjugated to endogenous SUMO. (C) Sumoylation is not required for PIASy inhibition of GATA-1 transcriptional activity. Reporter experiments were performed in U2OS cells by using either WT GATA-1 or the nonsumoylatable K137R mutant as described above. Dual luciferase assays were done 36 h after transfection. Lower shows the expression levels of GATA-1 and T7-PIASy in one representative experiment as detected by immunoblotting. (D) PIASy represses GATA-1 transcriptional activity in erythroid cells. Reporter experiments were performed in the indicated cell lines transfecting WT GATA-1 with or without T7-PIASy as indicated. Dual luciferase assays were done 36 h after transfection. Relative luciferase activity is expressed as percent of the activity of the GATA reporter in the absence of PIASy.