Figure 6.

hUHRF1 cooperates with G9a to enhance the transcriptional repression of p21 promoter. (A) hUHRF1-mediated transcriptional repression of the p21 promoter-luciferase reporter. COS-7 cells were cotransfected with the reporter (2 μg) and increasing amounts of EGFP-hUHRF1 as indicated at the bottom of the panel. (B) Enhanced transcriptional repression by G9a in the presence of exogenous hUHRF1. Luciferase activities were measured from COS-7 cells cotransfected with the same reporter described earlier and increasing amounts of EGFP-G9a (0.1–1 μg) with or without a constant amount (0.4 μg) of EGFP-hUHRF1. The luciferase assays were performed as described in Figure 4, and the data represent the means ± SD of duplicate determinations from three separate experiments. Western blot analyses of hUHRF1 and G9a expression by anti-GFP antibody are shown for each cotransfection group. (C) Loss of interaction between UHRF1 and G9a abolishes the UHRF1/G9a-mediated repression of p21 promoter. Reporter assays were performed as described in (B), using the wild-type G9a plasmid (EGFP-G9a) and its deletion mutant lacking the N-terminal UHRF1-interacting region (EGFP-NΔG9a). Expression of hUHRF1 and G9a/NΔG9a is shown by western blot analyses with anti-GFP antibody.