Fig. 3.
Chromatin sumoylation is associated with transcriptional repression by GAL4-UBC9. (A) Transcriptional repression by GAL4-UBC9. HeLa cells were cotransfected with 2 μg of 4XGAL14D luciferase reporter and indicated amount of GAL4, GAL4-UBC9 (full-length, amino acid 1–158), GAL4-UBC9ΔN (35–158), or GAL4-UBC9ΔC(1–92). After transfection (48 h), the luciferase activities were determined. The data represent the average of two independent experiments. Comparable expression of GAL4, GAL4-UBC9, GAL4-UBC9ΔN, and GAL4-UBC9ΔC was confirmed by anti-GAL4 immunoblotting (data not shown). (B) Chromatin sumoylation by GAL4-UBC9. HeLa cells were cotransfected with 4XGAL14D luciferase reporter and GAL4 or GAL4-UBC9. Two days later, sumoylation of chromatin near the GAL4 site was examined by anti-SUMO-1 chromatin immunoprecipitation. Mock lanes represent the chromatin immunoprecipitation without the Ab. Chromatin sumoylation by GAL4-UBC9 was detected by chromatin immunoprecipitation under both nondenaturing (Left) and denaturing (Right) conditions. (C) SUMO-conjugating activities of UBC9 deletion mutants. The 293T cells were cotransfected with HA-SUMO-1 or HA-SUMO-3 together with a vector, UBC9, UBC9DN, or UBC9DC. Whole-cell lysate (30 μg) was analyzed by anti-HA immunoblotting for sumoylation of cellular proteins.