The EKLF-GATA1 fusion protein occupied the GATA1-binding motif of the δ-globin promoter proximal region and competed with GATA1 for binding to the promoter. (A) CD34⁺ bone marrow cells were transduced with EKLF, GATA1, EKLF-GATA1 (EG), or vector only at day 4 of the expansion stage. On day 6, transduced CD34⁺ cells were reseeded into differentiation medium with 3 μg/mL of blasticidin for 7 days. Cells were then harvested and subjected to ChIP assay using antibody against V5 (top first panel) to immunoprecipitate chromatin-protein complexes. A parallel ChIP assay was performed using mouse IgG for the immunoprecipitation (IP) step as a ChIP assay control (top second panel). DNA was amplified and quantitated by PCR with specific primers flanking the δ-globin gene promoter from −152 to +2 (which contains the GATA1-binding motif) and a pair of control primers flanking the δ-globin gene promoter from −619 to −473 that does not contain the GATA1-binding motif (bottom second-to-last panel). PCR using input DNA as template served as an internal control (top third panel and bottom last panel). (B) Graphical representation of data in panel A. Results are expressed as relative proportions of immunoprecipitated DNA (ratios of immunoprecipitated versus input DNA) normalized to the ratio obtained for the δ-globin promoter in GATA1-transduced CD34⁺ cells (arbitrarily set at 100%). *P < .05 versus vector only–transduced cells. (C) CD34⁺ bone marrow cells were transduced with various MOIs of medium-form EKLF-GATA1 (EG) or vector only at day 4 of the expansion stage. On day 6, transduced CD34⁺ cells were reseeded into differentiation medium with 3 μg/mL of blasticidin for 5 days. Cells were then harvested and subjected to ChIP assay using antibody against V5 (top panel) or GATA1 (directed at the C-terminus of GATA1; middle panel) to immunoprecipitate chromatin-protein complexes. DNA was amplified and quantitated by PCR. A parallel ChIP assay was performed using mouse IgG for the immunoprecipitation (IP) step as a negative control. (D) Graphical representation of data in panel C. Relative level of immunoprecipitated DNA (ratios of immunoprecipitated vs input DNA) normalized to the ratio obtained for the δ-globin promoter in mock CD34⁺ cells (arbitrarily set at 1). Error bars indicate SD of the mean of 3 independent experiments.