The Hbo1-Brd1 complex is responsible for the bulk of H3K14 acetylation. (A) Levels of acetylation at histone H3 in wild-type, Brd1^−/−, and Moz^−/− CD71⁺Ter119⁻ erythroblasts. Histones purified from purified CD71⁺Ter119⁻ erythroblasts were analyzed by Western blotting by use of the indicated antibodies. Levels of acetylated H3K9 and H3K14 were normalized to the amount of H3 and are indicated relative to wild-type control values. (B) Levels of H3K14 acetylation at the promoters of erythroid regulator genes. A ChIP analysis was performed with CD71⁺ erythroblasts from wild-type (W) and Brd1^−/− (K) 12.5 dpc fetal livers with an anti-acetylated H3K14 antibody. The relative amount of immunoprecipitated DNA is depicted as a percentage of input DNA. The data are shown as the mean ± SE for triplicate PCRs. The Alb promoter served as a negative control. (C) Hbo1 knockdown in fetal liver progenitor cells. c-Kit⁺ CD71⁻ cells were sorted from fetal livers at 12.5 dpc and cultured in the presence of SCF and IL3. Twenty-four hours later, cells were infected with lentiviruses against Hbo1 (#2 and #3) and the culture medium was changed to that containing EPO to induce erythroid differentiation (top left). After a 3-day induction, cells were stained with the indicated antibodies and analyzed by flow cytometry. The knockdown cells were monitored for expression of GFP, a marker antigen for infection. The flow cytometric profiles of GFP⁺ cells are indicated (bottom left) and their differentiation defined by the expression of CD71 and Ter119 is shown as the mean ± SE for triplicate cultures (right). * P < .05, *** P < .0005. (D) Levels of acetylation of histones H3 and H4 in Hbo1-knockdown erythroblasts. Histones were prepared from CD71⁺ erythroblasts purified from the Hbo1-knockdown culture in (C) and analyzed by Western blotting by the use of the indicated antibodies (left). Levels of acetylation of H3 and H4 at each residue were normalized to the amount of H3 and H4, respectively. The acetylation levels relative to the sh-Luc controls are indicated (right).