Dynamic Regulation of 5-Hydroxymethylcytosine At the γ-Globin Promoter During Erythroid Differentiation

Abstract 824

DNA methylation is a key element responsible for γ-globin gene repression in adult erythroid cells. Our laboratory previously observed that the γ-globin gene promoter region was demethylated in a progressive manner as γ-globin expression was activated during erythroid differentiation of primary baboon pre-switch fetal liver cells and, to a lesser extent, of adult baboon bone marrow (BM) cells (Singh et al Exp Hematol 35:48-55, 2007). The mechanism responsible for DNA demethylation of the γ-globin promoter during erythroid differentiation remains unknown. Recent studies have shown that DNA demethylation in the early embryo is mediated by the “sixth base” 5-hydroxymethylcytosine (5-hmC) whose formation from 5-methylcytosine is catalyzed by the enzymatic activity of the three TET proteins. To investigate the hypothesis that 5-hmC mediates DNA demethylation of the γ-globin promoter during erythroid differentiation, levels of 5-hmC at Msp I (CCGG) sites within the γ- and ϵ-globin promoters and γ-globin IVS II region in DNA isolated from peripheral WBC, purified terminal erythroid precursors, and FACS-purified adult bone marrow subpopulations enriched for different stages of differentiation (CD117+ CD36-, CD117+ CD36+, and CD117-CD36+), were analyzed using a T4 glucosylase-MspI quantitative real time PCR assay. Levels of 5-hmC associated with the γ-globin promoter MspI site were 11.6 fold higher (p<.0001) in terminal erythroblasts (n=9) compared to peripheral blood WBC (n=4) while 5-hmC levels associated with the ϵ-globin promoter were 11.8 fold higher (p<.0001) in terminal erythroid precursors (n=13) compared to peripheral WBC (n=4). Levels of 5-hmC associated with the γ-globin promoter (n=9) were 9.4 fold higher (p<.0001) than with the IVS II region of the γ-globin gene (n=8) in terminal erythroid precursors suggesting that elevated levels of 5-hmC within the β-globin gene complex in terminal erythroid precursors may be localized to promoter regions. Genomic 5-hmC levels, analyzed by HPLC-MS, were similar in WBC and terminal erythroid precursors. Within purified BM subpopulations enriched for different stages of erythroid differentiation, levels of 5-hmC associated with the γ-globin promoter were 3.2 to 4.1 fold higher in the CD117+CD36+ subpopulation enriched in erythroid colony forming cells (n=4) than in CD117+CD36- (n=3; p<.01), more differentiated CD117-CD36+ (n=3; p<.02), and terminal erythroid precursor (p<.001) subpopulations. Similar differences in levels of 5-hmC associated with the ϵ-globin promoter were also observed between these subpopulations. Enrichment of 5-hmC within the β-globin locus in the CD117+CD36+ and terminal erythroid precursors compared to peripheral WBC was confirmed by 5-hmC affinity selection and PCR analysis. In addition, similar differences in genomic 5-hmC levels between these subpopulations were also observed by HPLC-MS analysis. Bisulfite sequence analysis showed that the changes in 5-hmC levels at the γ-globin promoter were temporally associated with loss of DNA methylation within the γ-globin promoter as erythroid differentiation progressed. TET gene expression analysis showed that TET2 expression was 3 fold less (p<.01) while TET3 expression was >7 fold higher (p<.05) in terminal erythroid precursors compared to the CD117+CD36- subpopulation. These results strongly suggest that differences in 5-hmC associated with the ϵ- and γ-globin promoters are the result of wider dynamic alterations of genomic 5-hmC levels during erythroid differentiation that may be mediated by differences in TET gene expression and support the hypothesis that 5-hmC is involved in the mechanism responsible for DNA demethylation of the γ-globin promoter during erythroid differentiation.