Histone acetylation status of ex vivo–generated CD34⁺ cells and promoters of erythroid-specific genes. (A) Analyses of histone acetylation. Representative flow cytometric analysis of the histone H3K9 acetylation status of primary CD34⁺cells, VPA-treated CD34⁺ cells, and CD34⁺ cells cultured in the presence of cytokines alone after 7 days of culture. Cells were stained with H3K9 antibody to assess the acetylation level of lysine 9 residue of histone H3 (n = 3). One of 3 representative experiments is shown. (B) Analyses of acetylated histone H3K9/14 and H3K27 on the promoters of erythroid lineage-specific genes, a stem cell gene (c-Kit), and a nonhematopoietic gene (MyoD) of cytokines alone and VPA-treated CD34⁺ cells as determined by ChIP assay. A reduction of H3K9/14 and H3K27 acetylation was observed with all of the promoters on the CD34⁺ cells isolated from cell culture with cytokines alone compared with cells cultured cytokines + VPA. ChIP efficiency, in terms of the percentage of input DNA recovered by immunoprecipitation, was determined by Q-PCR (primers were designed within −1-kb promoters of the erythroid lineage-related genes). A sample with no antibody (No Ab) was used as a background control. The histogram represents mean percentage of fold change relative to input chromatin and SE (n = 3). (C) Percent acetylation of H3K9/14 and H3K27 relative to histone H3 in CD34⁺ cells from cultures containing cytokines alone and cytokines + VPA. The percentage of acetylation of H3K9/14 and H3K27 relative to total histone H3 was determined on the erythroid lineage-specific promoters, a stem cell gene (c-Kit), and a non hematopoietic gene (MyoD) of CD34⁺ cells isolated from cultures performed in the presence of cytokines alone and cytokines + VPA cultures after 7 days of incubation. Histogram represents mean ± SE of ChIP Q- PCR (n = 3).