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

Abstract

Objective: These studies were performed to test the hypothesis that DNMT1 is required for maintenance of DNA methylation and repression of the γ-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 β-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 radiolabeling 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 γ-globin messenger RNA, an increased γ/γ+β chain ratio in cultured erythroid progenitors, and decreased DNA methylation of the γ-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 γ-globin gene promoter and repress γ-globin gene expression in adult-stage erythroid cells.

FIGURE 1

A. Flow cytometric analysis of GFP content of CID-dependent mouse βYAC BMCs following nucleofection with pMAXGFP. B. Effect of 3 siRNAs targeting DNMT1 on expression of DNMT1. Cells were transfected with s65071, s65072, or s65073 siRNAs targeting DNMT1 or nonsilencing siRNA. Levels of DNMT1 (filled bars), DNMT3A (checked bars), and DNMT3B (unfilled bars) 48 hours post-transfection were analyzed by real time PCR with mouse GAPDH as the standard. C. Western blot analysis showing reduction in DNMT1 protein in CID-dependent mouse βYAC BMCs 72 hours post-transfection with siRNAs targeting DNMT1 compared to untreated control cells, cells subjected to nucleofection procedure in the absence of siRNA, and cells transfected with siRNA targeting G9A and nonsilencing siRNA as negative controls. As positive control for loss of DNMT1, cells were treated with decitabine (0.5 × 10^-7 M) for 72 hours.

FIGURE 2

A. siRNAs targeting DNMT1 increase ε- and γ-globin RNA in CID-dependent mouse βYAC BMCs. Real time PCR analysis of ε-, γ-, and human β-globin expression in untreated parental CID-dependent βYAC BM cells, following transfection with nonsilencing siRNA, and siDNMT1, and following treatment with decitabine (0.5 × 10^-7 M) cells. Results are shown as RNA/total (ε+γ+β) human β-like globin RNA (mean ± SD).

FIGURE 3

Levels of DNA methylation of CpG residues within the 5’ γ-globin promoter region (A) and ε-globin promoter region (B), are decreased following transfection of siDNMT1 and decitabine treatment in CID-dependent βYAC BM cells. Each row represents the sequence analysis of a single pCR4 clone containing an amplicon produced from bisulfite-treated DNA. Methylated sites (red); unmethylated sites (green).

FIGURE 4

A. Flow cytomteric analysis of GFP content of a baboon erythroid progenitor cell culture transfected with the pMAXGFP vector by nucleofection on d8. Cells were analyzed 24 hours post-transfection. Flow cytometric analysis showed that 54.6% of the cells were GFP+. B. Western blot analysis of DNMT1 72 hours following transfection of baboon erythroid progenitor cells with either nonsilencing siRNA (NS), or the s4216 and s4217 siRNAs targeting DNMT1. The blot was reprobed with anti-actin antibody as a loading control. C. siDNMT1 and decitabine increase ε- and γ-globin RNA expression in erythroid progenitor cultures derived from CD34+ baboon BM cells. RNA was analyzed by real time PCR and presented as RNA/total (ε+γ+β) β-globin like RNA (mean ± SD). D. siDNMT1 and decitabine increase the γ/γ+β globin chain synthetic ratio in erythroid progenitor cell cultures derived from CD34+ baboon BM. Cells were incubated with [³H] leucine in leucine-free media 72 hours following transfection with nonsilencing siRNA, siDNMT1, or decitabine treatment. Lysates were separated by HPLC and fractions collected. Radioactivity in fractions was determined by scintillation counting. Results are shown as γ/γ + β ratios (mean ± SD).

FIGURE 5

Representative HPLC chromatograms from two biosynthetic radiolabelling experiments showing the pattern of globin chain synthesis in two baboon erythroid progenitor cell cultures treated with either nonsilencing siRNA or siDNMT1.

FIGURE 6

Levels of DNA methylation of 5 CpG residues within the 5’ γ-globin promoter region in erythroid progenitor cultures following treatment with nonsilencing siRNA, siDNMT1, and decitabine compared to untreated control cultures. Each row represents the sequence analysis of a single pCR4 clone containing an amplicon produced from bisulfite-treated DNA. Both the γ-globin (A) and ε-globin promoter (B) were analyzed. Methylated sites (red); unmethylated sites (green); polymorphic sites where the methylatable CpG residues is not present (yellow).