Figure 4. SUMOylation of E2F1 at K226 controls E2F1 binding to the EZH2 promoter.

(A) E2F1 expression was not affected by SAE2 knockdown. Western blot of E2F1 and SUMO1 in HCT116 cells with (+Dox) or without (−Dox) SAE2 knockdown and HT29 cells with (shSAE2) or without (shCtrl) SAE2 knockdown. E2F1 SUMO1 modification was reduced by SAE2 knockdown. (B) E2F1 is SUMOylated at lysine 266. HCT116 cells were transfected with HA-tagged E2F1 WT or K226R mutant expression plasmid together with a UBC9-expression plasmid. The expression of WT and K226 mutant E2F1 was detected by western blots with an anti-HA-tag antibody in input (left panel). Cell lysates were immunoprecipitated with an anti-HA-tag antibody followed by western blot with an anti-HA-tag antibody (middle panel) or an anti-SUMO1 antibody (right panel). (C) K226R mutation significantly decreased E2F1 transcriptional activation of the EZH2 promoter and the transcriptional activity of E2F1-K226R mutant was not affected by SAE2 knockdown. HCT116 cells containing Dox-inducible SAE2 shRNA were pretreated with (+Dox) or without (−Dox) Dox for 24 h. Cells were transfected with E2F1 WT or K226R mutant, together with EZH2 promoter luciferase reporter and Renilla plasmids. After transfection for 6 h, cells were treated with or without Dox for another 48 h and dual-luciferase assay was performed. (D) SUMO1 increased transcriptional activity on EZH2 promoter when co-expressed with WT E2F1 but showed no effect when co-expressed with E2F1 K266R mutant. HCT116 cells were transfected with E2F1 WT or K226R mutant along with a SUMO1-expressing plasmid (SUMO1) or empty vector (EV). All wells were transfected with the same amount of EZH2 promoter luciferase reporter and Renilla plasmids. Dual-luciferase assay was performed after 48 h. (E) Schematic of the genomic region surrounding the transcriptional start site (TSS) of the human EZH2 gene and the E2F1 binding region on the promoter. The annealing positions of primers used for ChIP experiments (labeled as P1, P2, P3, and P4) are indicated at the bottom of the figure. (F) SAE2 knockdown decreased the occupancy of E2F1 on the EZH2 promoter as measured by ChIP assay. ChIP was performed using an anti-E2F1 antibody in HCT116 cells without (−Dox) or with (+Dox) SAE2 knockdown. The occupancy was normalized to DNA input and calculated relative to IgG control. (G) E2F1 SUMOylation site mutation significantly decreased the occupancy of E2F1 on the EZH2 promoter. HCT116 cells were transfected with HA-tagged E2F1 WT or K226R expression plasmids, followed by ChIP assay using an anti-HA antibody. (H) GSEA analysis shows that “REN_BOUND_BY_E2F” gene set was enriched in HT29 control group (shCtrl) compared to the SAE2 knockdown group (shSAE2). Estimated variation is indicated as SD, p values were derived using a two-tailed Student’s t-test or ANOVA. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.