Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation.
The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-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 γ-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' γ-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 γ-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, γ-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-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 γ-globin expression by mediating demethylation of the γ-globin promoter.
a Jesse Brown VA Medical Center ; Chicago , IL , USA.
Maria Armila
MA
Ruiz
Angela
A
Rivers
Vinzon
V
Ibanez
Kestis
K
Vaitkus
Nadim
N
Mahmud
Joseph
J
DeSimone
Donald
D
Lavelle
D044503:5-Methylcytosine / Q000378:metabolism / Q000031:analogs & derivatives; D000818:Animals; D000831:Animals, Newborn; D000970:Antineoplastic Agents / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001854:Bone Marrow Cells; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D019175:DNA Methylation / Q000187:drug effects*; D000077209:Decitabine; D049308:Dioxygenases / Q000378:metabolism; D041905:Erythroid Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D056466:Histone Demethylases / Q000378:metabolism; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects; D048530:Papio anubis; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D014191:Tranylcypromine / Q000494:pharmacology; D055545:gamma-Globins / Q000378:metabolism*
D000970:Antineoplastic Agents; D055545:gamma-Globins; C011865:5-hydroxymethylcytosine; D014191:Tranylcypromine; D044503:5-Methylcytosine; D000077209:Decitabine; D003596:Cytosine; C578792:TET3 protein, human; D049308:Dioxygenases; D056466:Histone Demethylases; C495195:KDM1A protein, human; D001374:Azacitidine; D001205:Ascorbic Acid; D006918:Hydroxyurea
5hmC, 5-hydroxymethylcytosine; 5mC, 5-methylcytosine; HbF, fetal hemoglobin; DNMT, DNA methyltransferase; BM, bone marrow; FL, fetal liver; LSD1, lysine specific demethylase 1; TC, tranylcypromine; DNA methylation; baboon; decitabine; erythropoiesis; globin gene switching; hydroxymethylcytosine; tranylcypromine; vitamin C; γ-globin gene