FIG. 5.

Pax5 and Ets1 collaborate to activate the Ebf1β but not Ebf1α promoter. (A) Ba/F3 cells were transiently transfected with 1 μg cytomegalovirus β-galactosidase reporter and 2 μg of Ebf1α or Ebf1β luciferase reporter construct, together with 3 μg of Pax5 and 3 μg of Ets1 expression plasmids alone or in pairwise combinations as indicated. (B) Overview of the Ebf1β promoter luciferase reporter constructs. Indicated are the Pax5-binding sites at positions −318, −25, +222, and +257 and the Ets1-binding sites at −75 and +303. (C) Electrophoretic mobility shift assays show binding of Pax5 to binding sites at −318, +222, and +257 in the Ebf1β promoter. Recombinant Pax5 protein (0, 50, or 200 ng) was incubated with labeled wild-type or mutant oligonucleotide probes. (D) Mutation of all four Pax5-binding sites in the Ebf1β promoter leads to a twofold reduction of transactivation by Pax5. Ba/F3 cells were transiently transfected with 1 μg cytomegalovirus β-galactosidase reporter and 2 μg of Ebf1β(0.5)-luc or Ebf1β(Pax5mut)-luc reporter construct, together with increasing amounts of Pax5 expression plasmids (0.3, 1, and 3 μg). (E) Electrophoretic mobility shift assays show binding of Ets1 to binding sites at −75 and +303 in the Ebf1β promoter. The indicated amount of recombinant Ets1 protein (0, 100, or 300 ng) was incubated with labeled oligonucleotide probes and assayed in the presence of a 200-fold excess of specific (mb1) or unspecific competitor. (F) Mutation of the Ets1-binding sites at positions −75 and +303 reduces fourfold the potential transactivation by Ets1 and Pax5 on the Ebf1β promoter. Ba/F3 cells were transiently transfected with 1 μg cytomegalovirus β-galactosidase reporter and 2 μg of Ebf1β(0.2)-luc or Ebf1β(Ets1mut)-luc reporter construct, together with 3 μg of Pax5 and 3 μg of Ets1 expression plasmids as indicated.