Rupon Jeremy W, Wang Shou Zhen, Gaensler Karin, Lloyd Joyce, Ginder Gordon D
Massey Cancer Center and Departments of Internal Medicine, Microbiology and Immunology, and Human Genetics, Virginia Commonwealth University, Richmond, VA 23298-0037, USA.
Proc Natl Acad Sci U S A. 2006 Apr 25;103(17):6617-22. doi: 10.1073/pnas.0509322103. Epub 2006 Apr 11.
The genes of the vertebrate beta-globin locus undergo a switch in expression during erythroid development whereby embryonic/fetal genes of the cluster are sequentially silenced and adult genes are activated. We describe here a role for DNA methylation and MBD2 in the silencing of the human fetal gamma-globin gene. The gamma-globin gene is reactivated upon treatment with the DNA methyltransferase inhibitor 5-azacytidine in the context of a mouse containing the entire human beta-globin locus as a yeast artificial chromosome (betaYAC) transgene. To elucidate the mechanism through which DNA methylation represses the gamma-globin gene in adult erythroid cells, betaYAC/MBD2-/- mice were generated by breeding betaYAC mice with MBD2-/- mice. Adult betaYAC/MBD2-/- mice continue to express the gamma-globin gene at a level commensurate with 5-azacytidine treatment, 10- to 20-fold over that observed with 1-acetyl-2-phenylhydrazine treatment alone. In addition, the level of gamma-globin expression is consistently higher in MBD2-/- mice in 14.5- and 16.5-days postcoitus fetal liver erythroblasts suggesting a role for MBD2 in embryonic/fetal erythroid development. DNA methylation levels are modestly decreased in MBD2-/- mice. MBD2 does not bind to the gamma-globin promoter region to maintain gamma-globin silencing. Finally, treatment of MBD2-null mice with 5-azacytidine induces only a small, nonadditive induction of gamma-globin mRNA, signifying that DNA methylation acts primarily through MBD2 to maintain gamma-globin suppression in adult erythroid cells.
脊椎动物β-珠蛋白基因座的基因在红细胞发育过程中会发生表达转换，即该基因簇的胚胎/胎儿基因会依次沉默，而成人基因则被激活。我们在此描述了DNA甲基化和MBD2在人类胎儿γ-珠蛋白基因沉默中的作用。在含有整个人类β-珠蛋白基因座作为酵母人工染色体（βYAC）转基因的小鼠中，用DNA甲基转移酶抑制剂5-氮杂胞苷处理后，γ-珠蛋白基因会重新激活。为了阐明DNA甲基化在成人红细胞中抑制γ-珠蛋白基因的机制，通过将βYAC小鼠与MBD2基因敲除小鼠杂交，培育出了βYAC/MBD2基因敲除小鼠。成年βYAC/MBD2基因敲除小鼠继续以与5-氮杂胞苷处理相当的水平表达γ-珠蛋白基因，比单独用1-乙酰-2-苯基肼处理时观察到的水平高10至20倍。此外，在交配后14.5天和16.5天的胎儿肝脏成红细胞中，MBD2基因敲除小鼠的γ-珠蛋白表达水平始终较高，这表明MBD2在胚胎/胎儿红细胞发育中起作用。MBD2基因敲除小鼠的DNA甲基化水平略有降低。MBD2不与γ-珠蛋白启动子区域结合以维持γ-珠蛋白沉默。最后，用5-氮杂胞苷处理MBD2基因缺失小鼠仅诱导γ-珠蛋白mRNA产生少量的、非累加性的诱导，这表明DNA甲基化主要通过MBD2起作用，以维持成人红细胞中γ-珠蛋白的抑制。
