Relative association of class I HDACs at the integrated β- and γ-globin gene promoters. Antibodies to HDAC1, HDAC2, and HDAC3 were used to immunoprecipitate chromatin isolated from GM979 cells cultured in either control conditions or 200 μM RB7. Precipitated DNA was amplified and quantitated by real-time PCR using primers flanking either the β-globin gene promoter (A) or the γ-globin gene promoter (B). For each HDAC antibody used, the presence of the HDAC protein bound at the promoter is represented relative to its control (arbitrarily set at 100). Data represent average of 3 independent experiments; error bars represent the standard error of the mean. (C) Agarose gel showing relative association of HDAC1, HDAC2, and HDAC3 with the beta- and gamma-globin promoters in GM979 cells cultured in either control conditions (C), or in the presence of 200 μM RB7 (R). Results are representative of 3 independent experiments. (Di) ChIP of NCoR at the γ-globin gene promoter. NCoR association with the human γ-globin gene promoter in control and RB7 (200 μM)–treated cells; values are reported relative to the control and are an average of 3 experiments. Error bar indicates standard error of the mean. (Dii) Dissociation of HDAC3/NCoR complex after RB7 treatment. Immunoprecipitation was carried out using HDAC3 antibodies. The immunoblot was performed using antibodies to HDAC3 and NCoR. Lane 1: control cells; lane 2: 200 μM RB7 treatment. (E) Acetylation status of β- and γ-globin gene promoters. Change in acetylation status of histones H3 and H4 at the β- and γ-globin gene promoter 24 hours after GM979 cells were treated with RB7. Values are represented as a percentage of the untreated control (C) and are an average of 3 independent experiments. Error bars represent the standard errors of the mean.