Induced expression of EEN by AML1-ETO. (A) AML1-ETO targets the promoter region, as revealed by chromatin immunoprecipitation (ChIP) assay. The primers for the promoter region and the last exon (as control) are indicated by arrows. The numbers above the bar indicate the exon numbers. pSG5M, pSG5M-AML1b, pSG5M-AML1-ETO, and pSG5M-AML1-ETO (L148D) (10 μg each) were electroporated into the HL60 cells and immunoprecipitated by anti–FLAG M2 antibody. Input indicates input chromatin; ChIP, immunoprecipited chromatin. (B) AML1-ETO activates the wild-type (wt) promoter of the EEN gene. The EEN promoter–luciferase reporter gene construct (pGB-EENP7) was cotransfected with the expression vectors of AML1b, AML1-ETO, or mutant AML1-ETO (L148D). AML1b slightly increased the promoter activity, but the mutant AML1-ETO (L148D) did not. Mutant EEN promoter without the AML1 binding site (ΔAML1) was not activated by AML1-ETO or AML1b. The average firefly luciferase / Renilla luciferase activity on pGL3-EENP7 of the vector control was assigned a value of 1. Results are expressed as the means of 3 independent experiments plus or minus standard deviations. (C) AML1-ETO binds the wild-type (wt) promoter of the EEN gene. Flag-tagged recombinant proteins were prepared by in vitro translation. AML1b, AML1-ETO, and AML1-ETO (L148D) (lanes 2-4) were incubated with γ-³²P–labeled double-strand probe and run on a 4.5% native polyacrylamide gel. A retarded band was seen in AML1-ETO and AML1b, but not in AML1-ETO (L148D). Lane 1 was a negative control without template in in vitro translation. For the EMSA supershift assay the antibody against ETO was added to nuclear extracts from Kasumi-1 cells before incubation with radiolabeled probe. A supershift band is indicated by the asterisk (lane 6). Lane 7 was obtained with 100-fold molar excess of unlabeled competing oligonucleotides.