Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation.
Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation.
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DOI：
10.1080/15592294.2015.1039220
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Lavelle D
中科院分区：
文献类型：
--
作者：
Ruiz MA;Rivers A;Ibanez V;Vaitkus K;Mahmud N;DeSimone J;Lavelle D
γ-珠蛋白基因表达的发育阶段特异性调控机制涉及DNA甲基化。先前的结果表明，γ-珠蛋白启动子在胎儿肝红系分化期间几乎完全去甲基化，在成人骨髓红系分化期间部分去甲基化。假设5-羟甲基胞嘧啶（5 hmC），一种已知的DNA去甲基化途径的中间体，在红系分化过程中，通过分析流式细胞仪中5′-γ-珠蛋白基因启动子区域内CCGG位点的5-甲基胞嘧啶（5 mC）和5 hmC水平，研究了在γ-珠蛋白基因启动子脱甲基过程中的作用。来自狒狒骨髓和胎肝的纯化细胞富集用于红系分化的不同阶段。我们的研究结果表明，在γ-珠蛋白启动子的5 mC和5 hmC水平在红细胞分化过程中动态调节，5 hmC的峰值水平在去甲基化之前和/或与之一致。催化5 hmC形成的Tet 2和Tet 3双加氧酶在红系分化的早期阶段表达，并且Tet 3表达随着分化的进行而增加。在狒狒CD 34+骨髓源性红系祖细胞培养物中，γ-珠蛋白表达与γ-珠蛋白启动子处的5 hmC呈正相关，与5 mC呈负相关。在培养基中补充维生素C（一种泰特双加氧酶的辅因子），当单独添加和以加和方式与DNA甲基转移酶或LSD 1抑制剂组合添加时，降低γ-珠蛋白启动子DNA甲基化并增加γ-珠蛋白表达。这些结果有力地支持了Tet介导的5 hmC通路通过介导γ-珠蛋白启动子的去甲基化参与γ-珠蛋白表达的发育阶段特异性调节的假设。
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.