Figure 9.

SUMO-dependent recruitment to chromatin. (A) ChIPs were performed in a HEK 293 reporter cell line (33) harbouring an integrated transgene as illustrated [drawing based on Fig.2 in ref. (33)]. The reporter cell line was transfected with Gal-fusion derivatives of c-Myb as illustrated. Occupancy of factors and presence of chromatin marks were assessed on the luciferase promoter 5×Gal4 responsive elements (5×GRE) and on an intronic element in the neighbouring NCOA5 gene. Arrows indicate the regions amplified during analysis of ChIP samples by quantitative real-time PCR (ChIP–qPCR). (B) HEK 293 cells were transfected with plasmids expressing Gal4p-DBD (GBD) fused to c-Myb (amino acid residues 233–640) wild-type or 2KR. The output from the integrated luciferase was normalized to the effect of GBD alone, which was set to 100. The results are presented as RLU ± SEM. Immunoblotting was performed to control protein expression. Cells were lysed directly in SDS–PAGE loading buffer to maintain the modifications, and analysed using anti-HA antibody. GBD-Myb 1S and 2S: GBD-c-Myb modified with one or two SUMO proteins, respectively. (C) HEK 293 cells were transfected with plasmids expressing Gal4p-DBD fused to c-Myb wild-type, Sp3 wild-type or the kee SUMO-negative mutant of Sp3. The luciferase output was treated and presented as in (B). Occupancies of (D) Gal-Myb, (E) Mi-2α, (F) p300, (G) acetylated histone H3 and (H) acetylated histone H4 on the 5×GRE promoter and on the NCOA5 intron were analysed using ChIP–qPCR with cells transfected with GBD-Myb wild-type, 2KR or empty vector. In (E) Gal-Myb was co-transfected with FLAG-tagged Mi-2α.