Functional analysis of AML1-TRPS1. (A-B) AML1-TRPS1 and their mutants were subjected to EMSA in the presence (+) or absence (−) of PEBP2β/CBFβ. RD and AT indicate the Runt domain and AML1-TRPS1, respectively. RD and AML1 served as positive controls. The position of the indicated factor or complex with DNA is shown. Mutant constructs of AML1-TRPS1 were generated by PCR-based mutagenesis, using the following primers: AT/R174Q, 5′-gtggatgggccccaagaacctcgaagaca-3′; and AT/GATAm, 5′-ggatatgtaggcaacgcgggtggcctctacc -3′. (C) AML1-TRPS1 seems to form a homodimer through the Ikaros-like ZF domain. The deletion of Ikaros-like ZF (AT/ΔIKAROS) resulted in a decrease in the upper band (arrowhead), which might indicate the homodimer of AML1-TRPS1, and an increase in the lower band (AT, AML1-TRPS1 monomer). The AT/ΔIKAROS mutant was made by PCR-based mutagenesis using 5′-gaagtactcaagatgaactttcaacataatgtgrgcactgtggc-3′ as a primer. (D) A fixed amount of GATA-1 (1 U) and increasing relative amounts (0.15, 0.3 and 0.6 U) of AML1-TRPS1 were subjected to EMSA. Relative amounts of expressed proteins were evaluated by Western blotting. (E-F) Wild-type AML1, GATA-1, AML1-TRPS1, or its mutants were cotransfected with pBXH2-LTR-luc or pRBGP3-MαP reporter at varying relative doses, as indicated, into NIH3T3 cells by the nonliposomal transfection reagent FuGENE6 (Roche Applied Science, Basel, Switzerland). Luciferase activities are expressed as fold changes relative to the control transfected with the backbone expression vector alone. The total input of plasmid DNAs was kept constant (0.6 μg) by supplementing appropriate amounts of the backbone pEF-Bos plasmid so as to avoid potential artifacts due to uneven overall DNA dosages. Error bars indicate standard deviations for 3 independent experiments. (G) Expression levels of AML1-TRPS1 and its mutants were comparable with each other. COS7 cells were transfected with expression plasmids for the indicated genes, and whole-cell extracts were prepared 48 hours after transfection and subjected to Western blotting using rabbit polyclonal anti-AML1 antibody (Active Motif, Carlsbad, CA). (H-I) The Runt domain of AML1 physically interacted with GATA-1 (H), while GATA ZF of TRPS1 interacted with AML1 (I) in immunoprecipitation (IP) assays. COS7 cells were cotransfected with pEF-Bos expression vectors for FLAG-tagged AML1 (FL-AML1), FLAG-tagged AML1 mutant R174Q (FL-AML1/R174Q), and GATA-1, or FLAG-tagged TRPS1 (FL-TRPS1), FLAG-tagged TRPS1 GATA ZF mutant (FL-TRPS1/GATAm), and AML1, as indicated. Cell lysates were immunoprecipitated using anti-FLAG antibody (M2 monoclonal antibody; Sigma). Immunoprecipitates were detected by immunoblotting (IB) using rat monoclonal anti-GATA1 antibody (N6; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-AML1 antibody (Active Motif), or anti-FLAG antibody. (J) AML1-TRPS1 inhibits GATA-2–mediated transcription. GATA-2 and AML1-TRPS1 were cotransfected with MαP reporter at the indicated doses, into HL-60 cells. Luciferase activity is expressed as fold changes relative to the control. (K) HDAC inhibitor TSA did not greatly affect the repression mediated by intact AML1-TRPS1, but dramatically relieved that of AT/Δ Ikaros. GATA-1, AML1-TRPS1, or AT/ΔIKAROS were cotransfected into NIH3T3 cells with the MαP reporter at the indicated doses. Cells were treated with TSA (500 nM) for 36 hours from 12 hours after transfection. Luciferase activity is expressed as fold changes relative to the control. The results represent 3 independent experiments.