Effects of different concentrations of butyrate on the expression and the epigenetic modifications of γ-globin genes. (A) Mononuclear cells from a patient with SCD were cultured at 50 and 150 μM concentrations of butyrate. BFU-E–derived colonies were counted and expressed as number of colonies per 10⁶ nucleated cells (colonies/10⁶ NC). Error bars are SD. (B) γ-Globin chain synthesis was assessed by polyacrylamide gel electrophoresis and fluorography and expressed as percentage of γ-globin chain synthesis (ie, % γ/[γ + β]). (C,D) mRNA levels were measured by quantitative real-time RT-PCR and expressed as fold change relative to the control (0 μM) and as percent γ/(β + γ). (E) Acetylation of histones H3 and H4 at the γ-globin promoters was measured by quantitative real-time PCR-based ChIP assay and is expressed in relative units and is presented as the sum of acH3 and acH4 (acH3 + acH4). (F) DNA methylation at 5 CpG sites in the γ-globin promoters was determined by pyrosequencing and is expressed as the mean of all the methylation measurements at all 5 sites. Medium and large symbols indicate that the changes relative to the control and to the 50 μM butyrate were statistically significant, respectively. This experiment is representative of 3 independent experiments.