FIGURE 2.

EVI1 was acetylated by the histone acetyltransferase P/CAF during transcriptional activation of GATA2. A, EVI1 co-precipitated with P/CAF, but not with p300 or CBP. After transfection of EVI1 and P/CAF into COS7 cells, EVI1 and P/CAF protein complexes were immunoprecipitated (IP) with anti-EVI1 or anti-P/CAF antibody, respectively, and the precipitated proteins were immunoblotted with anti-P/CAF or anti-EVI1 antibody. B, transcriptional activation of the GATA2 promoter is dependent on P/CAF. EVI1 and/or various amounts of P/CAF expression vector were transfected into COS7 cells, and GATA2 transcriptional activity was measured by a Dual-Luciferase assay system. C, activation of GATA2 transcription by transfection of EVI1 with P/CAF, p300, or CBP was determined by using a luciferase assay system with the GATA2 promoter. D, acetylation of EVI1 was detected in COS7 cells transfected with EVI1 expression vectors using an anti-acetylated antibody. Co-transfection of P/CAF with an EVI1 expression vector and treatment with trichostatin A (TSA) as an HDAC inhibitor enhanced the acetylation of EVI1 in COS7 cells. E, endogenous EVI1 was acetylated and co-precipitated with endogenous P/CAF in UCSD/AML1 cells. After immunoprecipitation with an anti-acetylated antibody, the precipitated proteins were immunoblotted with an anti-EVI1 antibody. After immunoprecipitation with an anti-P/CAF antibody, the precipitated proteins were immunoblotted with an anti-EVI1 or anti-P/CAF antibody. F, EVI1 co-localized with P/CAF in the nuclei of UCSD/AML1 cells, and most of the EVI1 in the nucleus was acetylated, as revealed by immunohistochemical staining under confocal microscopy. Nuclear DNA was stained with TOTO-3. Anti-human EVI1 antibody was stained with Alexa Fluor 488-labeled secondary antibody (green), anti-acetylated lysine was stained with Alexa Fluor 546-labeled secondary antibody (red) and anti-P/CAF antibody was stained with Alexa Fluor 555-labeled antibody (red).