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

ABSTRACT: The mechanism responsible for developmental stage-specific regulation of ?-globin gene expression involves DNA methylation. Previous results have shown that the ?-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 (5 hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the ?-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5 mC) and 5 hmC at a CCGG site within the 5' ?-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 5 mC and 5 hmC levels at the ?-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5 hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5 hmC 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, ?-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the ?-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced ?-globin promoter DNA methylation and increased ?-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 5 hmC pathway is involved in developmental stage-specific regulation of ?-globin expression by mediating demethylation of the ?-globin promoter.

SUBMITTER: Ruiz MA 

PROVIDER: S-EPMC4622718 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature