Decitabine increases fetal hemoglobin in Papio anubis by increasing γ-globin gene transcription

Abstract

Objective: The mechanism responsible for increased fetal hemoglobin levels following decitabine treatment remains controversial. These experiments were performed to evaluate the role of transcriptional vs. translational mechanisms in the ability of decitabine to increase fetal hemoglobin levels in vivo.

Materials and methods: Three normal, nonanemic baboons were treated with decitabine subcutaneously (0.5 mg/kg/d) for 10 days. The effect of decitabine on globin chain synthesis and globin messenger RNA levels was measured in pre- and posttreatment bone marrow aspirates by biosynthetic radiolabeling with [(3)H] leucine followed by separation of globin chains by high-performance liquid chromatography, and real-time polymerase chain reaction, respectively. The effect on DNA methylation of the ɛ- and γ-globin gene promoters was determined by bisulfite sequence analysis.

Results: Decitabine treatment of normal, nonanemic baboons induced similar increases in the γ/γ+β chain synthetic ratio and the γ/total β-like globin RNA ratio and also increased expression of ɛ-globin transcripts. Increased expression of ɛ- and γ-globin was associated with decreased DNA methylation of the ɛ- and γ-globin gene promoters.

Conclusions: Decitabine increases fetal hemoglobin in vivo by transcriptional activation of the γ-globin gene.

Figure 1

Effect of decitabine on DNA methylation of ε- and γ-globin promoter regions. Results of bisulfite sequence analysis of DNA methylation of CpG residues within the 5′ ε- and γ-globin promoter regions in the BM erythroid precursor cells isolated from two baboons pre- and post-treatment. Each row depicts the results of sequence analysis of a PCR amplicon cloned in pCR4. Methylated residues (Red), unmethylated residues (green), polymorphic sites in the baboon γ-globin promoter where the CpG site is absent (yellow).