RUNX1/ETO dimers lack DNA-binding capacity. (A) ABCD assay for the tetrameric, dimeric, and monomeric forms of REtr. Constructs were tested for binding to biotinylated oligonucleotides containing the RUNX1-binding sites of either RUNX3 (R3) or PU.1 (PU1) promoter sequences. Protein binding was detected by immunoblotting with an anti-HA antibody. (B) REtr and mutants were analyzed for PU.1-oligo binding as in panel A. (C) DNA binding of WT RUNX1 protein to the RUNX3-oligo. The RUNX1-R3-oligo interaction was detected with an anti-Flag antibody. (D) Comparison of the binding strength defined as the signal intensity ratio of R3-pulldown/R3-input to the R3-oligo. Values were obtained from the marked bands in panels A and C. DNA-binding activity of REtr was set to 100%. (E) Binding capacity of full-length RUNX1/ETO (RE) and RE-m5 to the R3 oligonucleotide (upper panel). The binding specificity was confirmed using cell extracts containing PML/RAR-α. (F) Analysis of DNA-binding capacity of RE, RE-m5, RE-cm5, RE-dNHR2, and RE-L148 mutants to the R3 and PU.1 oligonucleotides. (G) DNA-binding capacity of RE and RE-m5 to a radioactively labeled RUNX1 high affinity oligos by EMSA. (Bottom) Western blotting of lysates indicates equal loading for RE and RE-m5. (H) Target gene analysis of REtr and REtr-m5 transduced U937 cells. FACS-sorted (eGFP-positive) cell populations were analyzed for the expression of Trk-A, c-Jun, and p21. Expression of the REtr forms was verified by immunoblotting with an anti-Flag antibody. Actin serves as loading control. I indicates input; R3, RUNX3-oligo; PU1, PU.1-oligo; b, magnetic beads only; and C, RUNX1/ETO-DNA-oligo complex.