Epigenetic Analysis of Beta-Globin Gene Cluster during Hematopoiesis.

Hemoglobin (Hb) switching is described as temporal, tissue- and stage-specific patterns of globin gene expression; from embryonic to fetal and adult Hb in parallel to developmental stages of erythropoiesis. DNA methylation, one of the epigenetic mechanisms, was associated with inactivated chromatin domain and repressive transcription. To study the role of the DNA methylation on the beta (β)-globin genes, we analyzed CpG dinucleotides in 87 kb regions around β-globin gene cluster, including 5’upstream locus control regions (LCR; DNAse I Hypersensitive site (HS) 1–5), 3’HS1, the promoter regions of the G-and A-gamma (^Gγ and ^Aγ), and β-globin genes, in several representative cells. These cells were

1. primary adult erythroid cells culture (three different stages: early, intermediate, and late),

2. fetal cord blood DNA, and

3. neutrophil cell line (non-erythroid).

Using bisulphite modification, followed by nested PCR and in vitro translation, the cleavage products were analysed by MALDI-TOF Mass Spectrometry to quantify the DNA methylation level. The results were consistent with bisulphite sequencing. We found that the promoters of ^Gγ and ^Aγ-globin genes were significantly hypomethylated in fetal cells (44% and 47% global methylation), when γ-globin genes were fully expressed, while they were heavily methylated in non-erythroid (86% and 95%). There was also a decreasing trend of the DNA methylation level at ^Gγ and ^Aγ-globin genes during adult erythroid differentiation from 80% and 82%, in early stage, to 67% and 66% in late stage (p=0.12 and 0.04). At β-globin promoter, the global methylation level changed from 90% in non-erythroid to 81%, 42%, and 26% in fetal, early and late adult erythroid cells, respectively. Moreover, we found the significant changes at 5’HS4, 3, and 1 as all erythroid cells were hypomethylated compare to non-erythroid. While at the insulators, 5’HS5 and 3’HS1, all tested CpG dinucleotides were heavily methylated in all cells. This is the first report that demonstrates the differences in DNA methylation at β-globin LCR between erythroid and non-erythroid cells. These epigenetic marks were associated with globin genes expression and might be useful to predict clinical severity in patients with β-thalassemia intermedia.