AML1-ETO binds to PU.1 and down-regulates transactivation capacity of PU.1.

(A) AML1-ETO binds to PU.1 in vivo. (i) Kasumi-1 cell nuclear extracts (200 μg) were immunoprecipitated with rabbit IgG (lane 1), anti-AML1 antibody (lane 2), goat IgG (lane 3), or anti-ETO antibody (lane 4). The immunoprecipitates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) along with in vitro translated PU.1 (lane 6) and nuclear extracts (NE; lane 7) and further subjected to immunoblotting with PU.1 antibody. (ii) Kasumi-1 nuclear extracts were immunoprecipitated with anti-PU.1 (lane 2) or IgG (lane 3) and subjected to SDS-PAGE along with nuclear extracts of Kasumi-1 cells (lane 4) and blotted with anti-ETO antibody. (B) AML1-ETO inhibits transactivation capacity of PU.1. (i) 293T cells were transiently transfected with human monocyte-specific M-CSF receptor promoter or promoterless vector pXP2 or pSRE (serum response element) and with expression plasmids of PU.1 (100 ng), c-Jun (50 ng), AML1-ETO (20 ng), and activated Ras (50 ng). Promoter activities (fold) were determined 24 hours after transfection and normalized to the activities of the internal control plasmid pRL0. Data represent mean values of 3 independent experiments. Error bars represent +SEM. (ii) AML1-ETO does not change the expression of cotransfected PU.1. The 293T cells were transfected as shown in Figure 1Bi, and whole cell lysates were subjected to SDS-PAGE followed by immunoblot assay with PU.1-specific antibody. (C) AML1 does not affect transactivation capacity of PU.1. The 293T cells transfected with p(PU.1)4TK-luc and expression plasmids of PU.1 (100 ng), c-Jun (50 ng), AML1 (50 ng), or CBFβ (50 ng), PU.1, c-Jun, AML1, and CBFβ had no effects on negative control p(mut.PU.1)4TK (data not shown).