Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene
Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene
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DOI：
10.1073/pnas.091097298
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发表时间：
2001-04-24
影响因子：
11.1
通讯作者：
Jost, JP
中科院分区：
文献类型：
--
作者：
Zhu, B;Benjamin, D;Jost, JP
我们已经证明，从鸡胚中分离的DNA去甲基化复合物具有G T错配DNA糖基化酶，也具有5-甲基胞嘧啶DNA糖基化酶（5-MCDG）活性。在此，我们表明，人胚肾细胞稳定转染5-MCDG cDNA连接到一个巨细胞病毒启动子过表达5-MCDG，一个15- 20倍的5-MCDG过表达的结果在一个稳定整合的蜕皮激素-视黄酸响应增强子-启动子连接到一个β-半乳糖苷酶报告基因的特异性去甲基化。去甲基化在不存在配体泊那甾酮（蜕皮激素的类似物）的情况下发生。通过Southern印迹分析和亚硫酸氢盐基因组测序反应研究转基因的甲基化状态。去甲基化发生在激素反应元件的下游。未观察到全基因组去甲基化。5-MCDG的失活突变体或空载体的表达不引起报告基因的启动子-增强子的任何去甲基化。当在体外用半甲基化底物检测时，5-MCDG活性的增加不影响DNA甲基转移酶的活性。在用抗生素选择克隆的过程中，转基因拷贝数没有变化。免疫沉淀结合蛋白质印迹分析表明，针对5-MCDG的抗体沉淀含有类维生素A X受体a的复合物。维甲酸受体与5-MCDG之间的结合不依赖于配体。这些结果表明，激素受体与5-MCDG的复合物可以靶向该系统中转基因的去甲基化。
We have shown that the DNA demethylation complex isolated from chicken embryos has a G T mismatch DNA glycosylase that also possesses 5-methylcytosine DNA glycosylase (5-MCDG) activity. Herein we show that human embryonic kidney cells stably transfected with 5-MCDG cDNA linked to a cytomegalovirus promoter overexpress 5-MCDG, A 15- to 20-fold overexpression of 5-MCDG results in the specific demethylation of a stably integrated ecdysone-retinoic acid responsive enhancer-promoter linked to a p-galactosidase reporter gene. Demethylation occurs in the absence of the ligand ponasterone A tan analogue of ecdysone). The state of methylation of the transgene was investigated by Southern blot analysis and by the bisulfite genomic sequencing reaction. Demethylation occurs downstream of the hormone response elements. No genome-wide demethylation was observed. The expression of an inactive mutant of 5-MCDG or the empty vector does not elicit any demethylation of the promoter-enhancer of the reporter gene. An increase in 5-MCDG activity does not influence the activity of DNA methyltransferase(s) when tested in vitro with a hemimethylated substrate. There is no change in the transgene copy number during selection of the clones with antibiotics. Immunoprecipitation combined with Western blot analysis showed that an antibody directed against 5-MCDG precipitates a complex containing the retinoid X receptor a. The association between retinoid receptor and 5-MCDG is not ligand dependent. These results suggest that a complex of the hormone receptor with 5-MCDG may target demethylation of the transgene in this system.