Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation

The mechanism responsible for developmental stage-specific regulation of gamma-globin gene expression involves DNA methylation. Previous results have shown that the gamma-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the gamma-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5mC) and 5hmC at a CCGG site within the 5 ' gamma-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5mC and 5hmC levels at the gamma-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, gamma-globin expression was positively correlated with 5hmC and negatively correlated with 5mC at the gamma-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced gamma-globin promoter DNA methylation and increased gamma-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5hmC pathway is involved in developmental stage-specific regulation of gamma-globin expression by mediating demethylation of the gamma-globin promoter.