Fig. 4.

SUMO-1 modification is required for transcriptional repression by NZFP. (A) Derepression of gene expression by desumoylation of NZFP. HEK 293T cells were transfected with pcDNA4/TO/LacZ, pG5TK-luc reporter plasmids, and effectors as indicated on the X-axis. The effectors were fused to Gal4DBD-VP16 as described in the text. Cell extracts were prepared at 24 h after transfection and used for luciferase assays. Relative luciferase activities were obtained after normalization with β-galactosidase activity. Fold repression was calculated by dividing the relative luciferase activity of VP16 with that of each effector molecule. Experiments were performed in triplicate and error bars denote the standard deviation from the mean of three independent experiments. EGFP-C1 designates backbone vector used for SENP1 or SENP2 (desumoylating protein) cloning and was used as control. (B) The effect of mutations in SUMO acceptor sites on transcriptional repression activity. Experimental conditions are the same as that used in (A).