Elevated Levels Of γ-Globin Gene Promoter 5-Hydroxymethylcytosine are Associated With Increased DNA Hypomethylation and HbF Expression

DNA methylation of the γ-globin gene promoter represses γ-globin expression in adult-stage erythroid cells while high level γ-globin expression in fetal liver erythroid cells is associated with DNA hypomethylation. Previously we showed that DNA demethylation of the γ-globin gene promoter during fetal liver erythroid differentiation is responsible for the nearly complete loss of DNA methylation, while much more limited DNA demethylation during adult bone marrow (BM) erythroid differentiation maintains a relatively high level of γ-globin promoter DNA methylation (Singh et al Exp Hematol 35:48, 2007). As recent studies have shown the importance of 5-hydroxymethylcytosine (5-hmC) as an intermediate in active and passive mechanisms of DNA demethylation that alter epigenetic modifications regulating development and hematopoietic differentiation, experiments were performed to 1) investigate the hypothesis that DNA demethylation of the γ-globin promoter during erythroid differentiation involves 5-hmC, and 2) evaluate the role of 5-hmC in γ-globin gene expression. Levels of 5-hmC and 5-methylcytosine (5-mC) located at a CpG residue within the context of a HpaII site within the 5' γ-globin promoter were measured in 1) baboon BM cells enriched for different stages of erythroid differentiation, and 2) CD34+ BM-derived erythroid progenitors expressing high levels of γ-globin grown in liquid culture or expressing low levels of γ-globin in co-culture with the AFT024 cell line. Analysis of BM cells showed that CD117+CD36+ BM cells enriched in clonogenic late BFUe/CFUe had nearly 3 fold higher levels of γ-globin promoter 5-hmC (6.91+1.41%) compared to BM-derived erythroid precursors (2.57+0.75%; p<0.0001). In erythroid precursors expressing low levels of HbF derived from CD34+ BM cells grown in co-culture with the AFT024 murine fetal liver cell line, the levels of γ-globin promoter 5-hmC (1.72+1.19%) and 5-mC (64.84+9.22%) were not significantly different from BM-derived erythroid precursors. In contrast, the level of γ-globin promoter 5-hmC in erythroid precursors derived from CD34+ BM cells grown in liquid cultures expressing elevated levels of HbF was significantly higher (6.18+1.35%) than terminal erythroid precursors from either adult BM (p<.0001) or AFT024 co-cultures (1.72+1.19%; p<.0001) but was not significantly different than the level in CD117+CD36+ BM cells. Levels of γ-globin promoter 5-hmC were similar in erythroid precursors from liquid cultures on d7 (5.57+1.24%), d11 (6.13+1.02%), d14 (6.73+1.74%), and more primitive d7 gly- basophilic erythroblasts (6.21+1.38%). The level of DNA methylation (5-mC) was significantly less in erythroid precursors derived from liquid cultures (40.37+14.33%) compared to erythroid precursors derived from adult BM (63.10+7.72%; p<0.0005), AFT024 co-cultures (64.84+9.22%; p<0.001) and CD117+CD36+ BM cells (67.71+7.45%; p<0.002). Reduced levels of 5-mC were observed in erythroid precursors from liquid cultures on d14 (34.87+14.67%) compared to d7 (48.64+15.56%; p<0.055) suggesting that the γ-globin gene is progressively demethylated during erythroid differentiation in liquid culture. We conclude that γ-globin promoter 5-hmC levels are modulated during adult BM erythroid differentiation with 3 fold higher levels in CD117+CD36+ cells enriched in late BFUe/CFUe compared to erythroid precursors. Similar levels of γ-globin promoter 5-hmC, 5-mC, and HbF are observed in adult BM erythroid precursors and erythroid precursors derived from AFT024 co-cultures. In contrast, high levels of levels of γ-globin promoter 5-hmC, similar to levels in CD117+CD36+ BM cells, are sustained in erythroid precursors derived from liquid cultures of CD34+ BM cells and are associated with decreased γ-globin promoter 5-mC and increased HbF. Supplementation of cultures with ascorbic acid, a co-factor of the TET oxygenases that catalyze 5-hmC, reduced levels of γ-globin promoter 5-mC (20.94+9.77%) compared to controls (51.24+14.61%; p<.025) and increased γ-globin expression. These results support the hypothesis that DNA demethylation of the γ-globin promoter during erythroid differentiation, resulting in high HbF expression, occurs through a 5-hmC-mediated mechanism subject to developmental regulation by factors in the micro-environment.