FIG. 6.

HBZ disrupts CREB binding at a cellular CRE. (A) HBZ represses CREB transactivation from a minimal promoter carrying only the cellular CREs. The pminLuc-cellular CRE reporter plasmid (400 ng) was transfected into CEM T cells that were subsequently treated with forskolin (20 μM for 4 h) as indicated or dimethyl sulfoxide (carrier). pcDNA-HBZ-MycHis (400 ng) and pcDNA-HBZΔATG (400 ng) were cotransfected with the reporter plasmid as indicated. Reported values are the average luminescence from two experiments performed in duplicate and are representative of four independent experiments. The standard error is indicated. (B) HBZ-bZIP displaces CREB-bZIP from a cellular CRE. Binding reactions in lanes 1 to 14 contained 6 fmol of ³²P-labeled cellular CRE probe with 0.12 pmol of CREB-bZIP (lanes 2 to 5 and lanes 7 to 14). Reactions in lanes 3 to 5 also contained increasing amounts of HBZ-bZIP (0.6, 1.2, and 1.8 pmol). Reactions in lane 8 also contained 1.2 pmol of HBZ-bZIP, and lanes 9 to 11 also contained increasing amounts of HBZ-ΔbZIP (1.2, 1.8, and 2.4 pmol). An antibody directed against the bZIP domain of CREB (500 ng, lane 13) or an antibody against histone H3 (500 ng, lane 14) were also added in the reactions. The positions of the free probe and relevant protein-DNA complexes are labeled.