siRNA Targeting DNA Methyltransferase I Increases ε- and γ-Globin Expression.

Abstract 973

Pharmacological inhibitors of DNA methyltransferase (DNMT) increase fetal hemoglobin (HbF) levels in experimental primates and patients with sickle cell disease. Therefore we hypothesize that DNMT is directly involved in maintaining repression of the γ-globin gene in adult stage erythroid cells. To test this hypothesis, levels of DNMT1 in mouse chemical-of-dimerization (CID) bone marrow (BM) cells containing the human β-globin gene locus in the context of a yeast artificial chromosome (βYAC) and primary cultured erythroid progenitor cells (EPC) derived from baboon CD34+ BM cells were reduced by treatment with siRNA targeting DNMT1 (siDNMT1) and the effect on globin gene expression determined. Nucleofection conditions that achieved 80-90% transfection efficiency were used to introduce siRNA into CID-dependent mouse βYAC BM cells. Real time PCR analysis showed that expression of DNMT1 was decreased 70-80% in cells treated with siDNMT1 compared to cells transfected with nonsilencing control siRNA while DNMT3A and 3B were not decreased. Results of real time PCR analysis of six independent experiments showed that ε-globin expression was increased 65.3+/−37.8 fold, γ-globin 230.3+/−147.8 fold, and β-globin 6.0+/−3.3 fold in cells treated with siDNMT1 compared to untreated controls, while cells treated with nonsilencing siRNA showed minimal (<2 fold) changes. The difference in ε- and γ-globin expression between cells treated with siDNMT1 and nonsilencing RNA was significant (p<.01). Bisulfite sequence analysis showed that DNA methylation of the ε-globin promoter and γ-globin promoters were reduced to 25.7 and 53.3% dmC, respectively, in cells treated with siDNMT1 compared to 68.8 and 98.8% dmC, respectively, in cells treated with nonsilencing siRNA. Nucleofection of cultured baboon EPC with siDNMT1 or nonsilencing siRNA was performed on d7 and d8 of culture. Transfection efficiencies of 45-50 % were achieved. Expression of DNMT1 was decreased >80% in cells treated with siDNMT1 compared to those treated with nonsilencing siRNA. Real time PCR analysis of duplicate samples showed that γ-globin expression was increased 2.06 and 2.25 fold relative to untreated controls following treatment of cells with siDNMT1 while nonsilencing siRNA had no effect. Expression of ε-globin was increased 35.26 and 25.4 fold relative to untreated controls in cells treated with siDNMT1 while a lesser effect was observed in cells treated with nonsilencing siRNA (7.41 and 8.16 fold). HPLC analysis of biosynthetically radiolabelled globin chains showed that γ-globin chain synthesis (γ/γ+β ratio) was increased in cells treated with siDNMT1 (0.59 and 0.63) compared to cells treated with nonsilencing siRNA (0.41 and 0.37), untreated controls (0.39) and mock-transfected controls (0.43). DNA methylation of 3 CpG residues within the 5' ε-globin promoter region and 5 CpG residues within the 5' γ-globin promoter region was reduced to 52.8 and 45.0% dmC, respectively, in cells treated with siDNMT1, compared to 100 and 85.4% dmC, respectively, in cells treated with nonsilencing siRNA. Our results demonstrate that siDNMT1 reduces DNMT1, reduces levels of DNA methylation of the ε- and γ-globin gene promoters, and increases ε- and γ-globin gene expression and γ-globin chain synthesis in CID-dependent mouse BM cells and in primary baboon EPC cultures derived from CD34+ BM cells. We conclude that DNMT1 is critically involved in the mechanism responsible for repression of γ-globin expression in adult-stage erythroid cells and therefore inhibition of DNMT1 activity by pharmacological inhibitors of DNA methyltransferase likely plays a fundamental role in the ability of these drugs to increase HbF in vivo.