De novo CpG island methylation in human cancer cells
De novo CpG island methylation in human cancer cells
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DOI：
10.1158/0008-5472.can-05-1980
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发表时间：
2006-01-15
期刊：
影响因子：
11.2
通讯作者：
Schuebel, KE
中科院分区：
文献类型：
--
作者：
Jair, KW;Bachman, KE;Schuebel, KE
了解异常哺乳动物胞嘧啶DNA甲基化模式是如何建立的一个主要障碍是难以量化催化这些反应的DNA甲基转移酶（DNMT）的新生甲基化活性。在这里，我们描述了一种新的方法，使用来自癌症中经常发生高甲基化的基因的天然人类CpG岛底物，产生强大的活性来测量从头CpG甲基化。然后，我们调查了具有不同dnmt基因工程缺陷的结肠癌细胞，发现这些细胞提取物中针对这些底物的主要活性是DNMT1， DNMT3b贡献较小，DNMT3a没有贡献，DNMT3a是唯一已知的真正的从头甲基转移酶。通过将CpG岛底物和DNMT1引入果蝇细胞，进一步检测DNMT1对未甲基化的富含CpG DNA的活性。外源DNMT1甲基化整合哺乳动物的CpG岛，但不甲基化果蝇的DNA。此外，在缺乏DNMT1和DNMT3b并且几乎不存在基因组甲基化的人类癌细胞中，重新引入DNMT1可以启动基因特异性从头甲基化。我们的研究为dnmtts的新生活性提供了一种新的分析方法，数据表明DNMT1在人类癌细胞启动子CpG岛超甲基化的启动过程中可能起作用。
A major obstacle toward understanding how patterns of abnormal mammalian cytosine DNA methylation are established is the difficulty in quantitating the de novo methylation activities of DNA methyltransferases (DNMT) thought to catalyze these reactions. Here, we describe a novel method, using native human CpG island substrates from genes that frequently become hypermethylated in cancer, which generates robust activity for measuring de novo CpG methylation. We then survey colon cancer cells with genetically engineered deficiencies in different DNMTs and find that the major activity against these substrates in extracts of these cells is DNMT1, with minor contribution from DNMT3b and none from DNMT3a, the only known bona fide de novo methyltransferases. The activity of DNMT1 against unmethylated CpG rich DNA was further tested by introducing CpG island substrates and DNMT1 into Drosophila melanogaster cells. The exogenous DNMT1 methylates the integrated mammalian CpG islands but not the Drosophila DNA. Additionally, in human cancer cells lacking DNMT1 and DNMT3b and having nearly absent genomic methylation, gene-specific de novo methylation can be initiated by reintroduction of DNMT1. Our studies provide a new assay for de novo activity of DNMTs and data suggesting a potential role for DNMT1 in the initiation of promoter CpG island hypermethylation in human cancer cells.