Hydroxymethylcytosine and demethylation of theγ-globingene promoter during erythroid differentiation

Abstract

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 (5hmC), 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 (5mC) and 5hmC 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 5mC and 5hmC levels at the γ-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, γ-globin expression was positively correlated with 5hmC and negatively correlated with 5mC 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 5hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter.

MIR29B mediates epigenetic mechanisms of HBG gene activation

“…Using in silico analysis, we discovered that MIR29B has a consensus sequence complimentary to the 3′‐untranslated region of DNMT3A and DNMT3B (Starlard‐Davenport et al , ). Since HBG is silenced via DNA hypermethylation in adult erythroid cells (Lavelle et al , ; Ruiz et al , ) and MIR29B silences MYB (Martinez et al , ), a known HBG repressor protein, we determined whether MIR29B alters DNMT and MYB expression as a mechanism of HbF induction. Using KU812 cells, which express the HBG and HBB genes, we confirmed efficient transfection of MIR29B mimic into KU812 cells after 48 h by RT‐qPCR analysis.…”

“…DNA methylation is an epigenetic event that can be reversed by targeting key genes, such as DNMT s and MECP2 , which are involved in mechanisms of gene silencing (Wu & Zhang, ). Elevated levels of 5‐methylcytosine at the −54 methylation site in the HBG promoter during differentiation of erythroid progenitors is associated with HBG methylation and transcriptional silencing (Ruiz et al , ). In our current study, we observed significantly lower HBG promoter methylation following MIR210 overexpression in normal human erythroid progenitors mediated by decreased 5‐methylcytosine and MECP2 expression.…”

“…Levels of 5‐methylcytosine and unmodified cytosine at the −54 CCGG site 5′ to the HBG gene cap site were quantitated by Epimark assays as previously described (Ruiz et al , ). DNA was purified from cell pellets using QIAGEN AllPrep DNA/RNA/Protein Minikits (Qiagen catalogue number 80234).…”

“…DNA was purified from cell pellets using QIAGEN AllPrep DNA/RNA/Protein Minikits (Qiagen catalogue number 80234). DNA was analysed by qPCR using primers that target the HBG promoter and spanned the −54 CCGG site as published (Ruiz et al , ). The percentage of 5‐methylcytosine and unmodified cytosine was determined using the comparative Ct method according to the manufacturer's instructions (EpiMark 5‐hmC and 5‐mC Analysis Kit).…”

Cell signaling pathways involved in drug-mediated fetal hemoglobin induction: Strategies to treat sickle cell disease

“…The therapeutic activity of the DNMT1 inhibitors 5-azacytidine and decitabine produces demethylation at the g-globin promoters and HbF induction. 33,34 Decitabine is currently in clinical trials for the treatment of SCD (ClinicalTrials.gov Identifier: NCT01685515). Another chromatin modifying protein LSD1 demethylates mono-and dimethyl histone H3 lysine 4, an active histone mark.…”