Figure 1.

A SUMO-mediated SC is operating on c-Myb. (A) Schematic picture of the different EMSA probes used to identify the minimal phasing between the MREs, and the luciferase reporters used in this study. The reporter plasmids contained increasing numbers of MREs (1× to 5×, all with 10 bp phasing) upstream of a core promoter from human MYC driving the luciferase reporter gene. (B) Recombinant c-Myb DBD (R123: 0–25 fmol) was bound to 2×MRE(GG) oligonucleotides (20 fmol each) with different phasing (6, 10, 15 or 20 bp) and complexes were separated on EMSA gels. F, free probe; */**, one or two R123 molecule(s) in complex with the probe, respectively. (C) CV-1 cells were transfected with reporter plasmids containing increasing numbers of MREs (1× to 5×) as indicated and a plasmid encoding c-Myb wild-type (inverted filled triangle) or a SUMO-negative c-Myb 2KR (filled diamond). Reporter activation is presented as relative luciferase units (RLU) ± SEM. (D) The definition of the SF. (E) CV-1 cells were transfected with reporters containing one or four MREs [(1×MRE(GG)-MYC or 4×MRE(GG)-MYC] and plasmids encoding c-Myb wild-type, c-Myb with both SUMO-conjugation sites mutated from lysine to arginine (2KR) or single SUMO-conjugation sites mutated (K503R/K527R, upper panel). CV-1 cells were co-transfected with plasmids encoding the different c-Myb SUMO-mutants and PIASy (ratio 4:1) to visualize the sumoylation pattern. The cells were lysed directly in SDS–PAGE loading buffer to maintain the modifications (lower panel). c-Myb-S and c-Myb-2S: c-Myb modified with one or two SUMO moieties, respectively. (F) SFs were measured using wild-type c-Myb and c-Myb 2KR expression plasmids together with two variants of a luciferase reporter activated by four MREs, differing only in their helical phasing. In the cis reporter, pGL4-4×MRE(GG)-MYC, the MRE phasing is 10 bp, while in the trans reporter, pGL4-4×MRE(GG)-MYC abab, the MRE phasing is 15 bp. The results are presented as SF ± SEM.