siRNA knock-down of DNMT1 produces decreased γ-globin promoter methylation but no increase in γ-globin mRNA or fetal hemoglobin. siRNA for the DNMT1 gene was transiently transfected into cells on day +11 of in vitro erythroid differentiation. Controls for this experiment were cells from the same initial culture that were treated with a nonspecific siRNA (siCTRL) or underwent mock transfection without siRNA (CTRL). (A) The effect of siRNA treatment on DNMT1 mRNA levels during differentiation as determined by quantitative RT-PCR. (B) The effect of siRNA treatment on DNMT3a mRNA levels. (C) The effect of siRNA treatment on DNMT1 protein levels as determined by Western blotting. β-actin and DNMT3a serve as nonspecific controls. (D) The effect of siRNA treatment on global DNA methylation as determined by bisulfite conversion analysis of LINE elements. Note that for the day 15 control sample no error bars are shown. This is because all 5 sequences, which included a total of 42 individual CpGs, were 100% methylated so the standard deviation for this data point was 0. (E) The effect of siRNA treatment on γ-globin promoter DNA methylation. (F) The effect of siRNA treatment on γ-globin mRNA during differentiation. (G) The effect of siRNA treatment on hemoglobin production as assessed by HPLC analysis of cell lysates at the end of in vitro differentiation. CD34⁺ cells from a single normal donor were used for this experiment. Error bars represent 1 SD. P values were determined by t test.