Figure 6. Pc2-mediated E2F1 SUMOylation Is Required for Growth Control Gene Activation.

(A) Serum-induced E2F1 SUMOylation. HeLa cells were serum-starved followed by restimulation and the cell lysates were immunoprecipitated with E2F1 antibodies under denaturing conditions followed by immunoblotting with antibodies indicated. (B) Pc2 promotes E2F1 SUMOylation in vivo. HeLa cells were transfected with indicated siRNAs/plasmids and the cell lysates were immunoprecipitated with E2F1 antibodies under denaturing conditions followed by immunoblotting with E2F1 (left panel) or Myc tag (right panel) antibodies. (C) Pc2 promotes E2F1 SUMOylation in vitro. Bacterially-expressed His-Pc2, His-E2F1 were incubated with recombinant E1, E2 and SUMO1 or SUMO1 mutant. The reactions were separated by SDS-PAGE followed by Coomassie blue staining (left panel), immunoblotting with E2F1 (middle panel) or SUMO1 (right panel) antibodies. (D and E) E2F1 is SUMOylated at lysine 266. HeLa cells were transfected with wt or single K mutated E2F1 (D) or K266R mutant (E) as indicated and the cell lysates were immunoprecipitated with FLAG antibodies under denaturing conditions followed by immunoblotting with E2F1 (upper panel) or Myc tag (bottom panel) antibodies (D) and E2F1 (left panel) or SUMO1 (right panel) antibodies (E). (F) E2F1 SUMOylation on growth control gene promoters. HeLa cells were transfected with indicated plasmids followed by two-step ChIP analyses using FLAG and SUMO1 antibodies on indicated regions. (G) K191 methylation inhibits Pc2-mediated E2F1 SUMOylation in vivo. HeLa cells were transfected with indicated plasmids and the cell lysates were immunoprecipitated with E2F1 antibodies under denaturing conditions followed by immunoblotting with E2F1 (left panel), Myc tag (middle panel) or SUMO1 (right panel) antibodies. (H) Pc2-mediated E2F1 SUMOylation is RNA dependent. Immunoprecipitates of Pc2K191me² or Pc2K191 from HeLa cells (right panel) were incubated with His-tagged E2F1, recombinant E1, E2 and SUMO1 or SUMO1 mutant with or without RNase I treatment. The reaction products were subjected to immunoblotting using antibodies targeting E2F1 (left panel). (I) TUG1 and NEAT2 regulate Pc2-mediated E2F1 SUMOylation. Bacterially-expressed His-E2F1 were incubated with recombinant E1, E2, SUMO1 (wt or mutant), and His- Pc2 (unmethylated or methylated) in the presence of synthesized sense or antisense TUG1 or NEAT2 RNA oligos. The reactions were subjected to immunoblotting using antibodies against E2F1.

See also Figure S6.