siDNMT1 increases γ-globin expression in chemical inducer of dimerization (CID)-dependent mouse βYAC bone marrow cells and in baboon erythroid progenitor cell cultures

Objective These studies were performed to test the hypothesis that DNMT1 is required for maintenance of DNA methylation and repression of the gamma-globin gene in adult stage erythroid cells Materials and Methods DNMT1 levels were reduced by nucleofection of small interfering RNA targeting DNMT1 in chemical Inducer of dimerization-dependent multipotential mouse bone marrow cells containing the human 13 globin gene locus in the context of a yeast artificial chromosome and in primary cultures of erythroid progenitor cells derived from CD34(+) baboon bone marrow cells The effect of reduced DNMT1 levels on globin gene expression was measured by real-time polymerase chain reaction and the effect on globin chain synthesis in primary erythroid progenitor cell cultures was determined by biosynthetic radio labeling of globin chains followed by high-performance liquid chromatography analysis The effect on DNA methylation was determined by bisulfite sequence analysis Results Reduced DNMT1 levels in cells treated with siDNMT1 were associated with Increased expression of gamma-globin messenger RNA, an increased gamma/gamma+beta chain ratio in cultured erythroid progenitors, and decreased DNA methylation of the gamma globin promoter Similar effects were observed in cells treated with decitabine, a pharmacological inhibitor of DNA methyltransferase Inhibitor Conclusions DNMT1 is required to maintain DNA methylation of the gamma-globin gene promoter and repress gamma-globin gene expression in adult stage erythroid cells (C) 2011 ISEH Society for Hematology and Stem Cells Published by Elsevier Inc