FIGURE 4.

Protein complex containing EVI1, P/CAF, and CtBP on the EVI1-binding site in the GATA2 promoter during transcriptional activation of GATA2. A, ternary protein complex containing EVI1, P/CAF, and CtBP was detected by co-immunoprecipitation (IP) Various combinations of FLAG-EVI1, P/CAF, and/or FLAG-CtBP2 expression vectors were transfected into COS7 cells, and protein complexes containing P/CAF were precipitated by anti-P/CAF antibodies. Precipitated proteins were detected by anti-EVI1, P/CAF, or CtBP antibodies. Asterisks indicate nonspecific bands. B, after transfection with four kinds of expression vectors (EVI1, P/CAF, CtBP2, or HDAC1), formalin-fixed DNA fragments were immunoprecipitated with anti-EVI1, anti-P/CAF, anti-CtBP2, or anti-HDAC1 antibody. After immunoprecipitation, purified DNA was amplified by specific pairs of primers in the GATA2 promoter region. Each species-specific IgG was used for immunoprecipitation as an isotype control. C, loss of GATA2 transcriptional activation by transfection with an EVI1-K564A mutant. EVI1 or EVI1-K564A mutant expression vectors were transfected into COS7 cells either with CtBP2 alone or CtBP2 with P/CAF expression vectors for luciferase assays of GATA2 promoter activity. D, chromatin immunoprecipitation analysis of the EVI1-binding site in the GATA2 promoter region after transfection with the lysine-mutated EVI1 expression vector. After transfection with FLAG-tagged EVI1 (WT), -K561A, or -K564A mutants with GATA2 promoter constructs, formalin-fixed DNA fragments were immunoprecipitated with anti-FLAG antibody or control mouse IgG. The precipitated DNA was then amplified using specific primers for detecting the EVI1-binding sites. IB, immunoblot. E, P/CAF enhanced the binding of EVI1 to the EVI1-binding site in the GATA2 promoter region. After co-transfection of the EVI1 expression vector with CtBP2 or P/CAF, formalin-fixed DNA fragments were precipitated with the anti-FLAG antibody or control mouse IgG. F, gel mobility shift assay for EVI1 using the EVI1-binding site (D1/b or D2/i) of the GATA2 promoter region. After transfection of EVI1 or EVI1-K564A mutant expression vectors into COS7 cells, EVI1 protein purified by anti-FLAG M2 antibody was used for gel mobility shift assay with EVI1-binding oligonucleotides specific for D1/b or D2/i. Purified EVI1 protein was detected with anti-FLAG-M2 antibody, and a 100-fold excess of cold binding oligonucleotides (D1/b or D2/i) was added to the gel mobility shift assay reaction. G, levels of EVI1, P/CAF, and GATA2 were determined in UCSD/AML1 cells during shP/CAF expression by semi-quantitative RT-PCR. UCSD/AML1 cells with an shLuc expression vector were used as a control. H, levels of P/CAF and EVI1 acetylation at Lys⁵⁶⁴, and EVI1 were determined by immunoblot using each specific antibody with β-actin as a control in both UCSD/AML1 cells with shLuc and with shP/CAF. I, chromatin immunoprecipitation analysis of the EVI1-binding site in the GATA2 promoter region after transfection of shP/CAF or shLuc expression vectors into UCSD/AML1 cells. DNA was precipitated with an anti-EVI1 antibody or control mouse IgG.