Hydroxymethylcytosine and demethylation of the g -globin gene promoter during erythroid differentiation Maria Armila Ruiz 1,2 , Angela Rivers 1,3 , Vinzon Ibanez 1,2 , Kestis Vaitkus 1,2 , Nadim Mahmud 2 , Joseph DeSimone 1,2 , and Donald Lavelle 1,2, * 1 Jesse Brown VA Medical Center; Chicago, IL USA; 2 Department of Medicine; University of Illinois at Chicago; Chicago, IL USA; 3 Department of Pediatrics; University of Illinois at Chicago; Chicago, IL USA Keywords: baboon, decitabine, DNA methylation, erythropoiesis, globin gene switching, hydroxymethylcytosine, tranylcypromine, vitamin C, g-globin gene Abbreviations: 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 The mechanism responsible for developmental stage-specific regulation of g-globin gene expression involves DNA methylation. Previous results have shown that the g-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 g-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5mC) and 5hmC at a CCGG site within the 5 0 g-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 g-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 CD34C bone marrow-derived erythroid progenitor cell cultures, g-globin expression was positively correlated with 5hmC and negatively correlated with 5mC at the g-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced g-globin promoter DNA methylation and increased g-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 g-globin expression by mediating demethylation of the g-globin promoter. Introduction The human b-globin gene complex spans approximately 70 kb on the long arm of chromosome 11 and includes 5 dis- tinct genes, e-, Gg-, Ag-, d-, and b-, that are activated sequen- tially in a highly regulated manner during development. Increased understanding of the mechanism(s) that regulate developmental expression of this cohort of genes, particularly the duplicated g-globin genes, is of critical importance in the development of new therapeutic interventions for sickle cell dis- ease and b-thalassemia because increased levels of fetal hemoglo- bin (HbF; a 2 g 2 ) inhibit polymerization of deoxygenated sickle hemoglobin and are associated with decreased risk of pain crises and death in patients with sickle cell disease. 1,2 Current evidence suggests that the mechanism of developmental globin gene regu- lation involves the targeting of repressive epigenetic modifica- tions to critical regulatory elements by recruitment of co- repressor complexes. 3-5 A mechanistic role for DNA methylation in developmental stage-specific repression of the g-globin gene has long been sup- ported by experimental studies. Previous analysis of the DNA methylation status of the g-globin promoter by Southern blot fol- lowing digestion of DNA with methylation sensitive restriction enzymes established a strong negative correlation between the © Maria Armila Ruiz, Angela Rivers, Vinzon Ibanez, Kestis Vaitkus, Nadim Mahmud, Joseph DeSimone, and Donald Lavelle *Correspondence to: Donald Lavelle; Email: dlavelle@uic.edu Submitted: 01/22/2015; Revised: 03/30/2015; Accepted: 04/05/2015 http://dx.doi.org/10.1080/15592294.2015.1039220 This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted. www.tandfonline.com 397 Epigenetics Epigenetics 10:5, 397--407; May 2015; Published with license by Taylor & Francis Group, LLC RESEARCH PAPER