5-Azacytidine decreases methylation of the γ-globin promoter. (A) The sodium bisulfite modification method was used to determine the methylation status of the 6 CpGs between −162 and +50 of the γ-globin promoter during in vitro differentiation. Each bar represents the proportion of methylated CpGs at each site for different time points during differentiation. (B) Methylation of individual γ-globin CpGs on day +11 of differentiation with () and without (□) 300 nM 5-azacytidine treatment. For comparison, the methylation of the CpGs at day +5 without 5-azacytidine is shown (). (C) Methylation of individual γ-globin CpGs on day +14. (D) Methylation of individual γ-globin CpGs on day +18. (D) Comparison of total γ-globin promoter methylation during differentiation with and without 300 nM 5-azacytidine. (E) Comparison of total γ-globin promoter methylation during differentiation with and without 300 nM 5-azacytidine. (F) As a measure of genome-wide DNA methylation, the effect of 300 nM 5-azacytidine on LINE methylation was determined. CD34⁺ cells from a single normal donor were used for this experiment. Error bars represent 1 SD. P values were determined by t test.