A Dnmt2-1ike protein mediates DNA methylation in Drosophila
A Dnmt2-1ike protein mediates DNA methylation in Drosophila
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DOI：
10.1242/dev.00716
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发表时间：
2003-11-01
期刊：
影响因子：
4.6
通讯作者：
Lyko, F
中科院分区：
文献类型：
--
作者：
Kunert, N;Marhold, J;Lyko, F
长期以来，果蝇DNA的甲基化状态一直备受争议。最近的证据有力地支持了5-甲基胞嘧啶在果蝇胚胎发育阶段DNA制备中的存在。果蝇基因组包含一个单一的候选DNA甲基转移酶基因，被称为Dnmt2。该基因属于一个广泛保守的假定DNA甲基转移酶家族。然而，目前还没有发现任何dnmt2样蛋白的催化活性。我们现在已经建立了一个方案的免疫检测甲基化胞嘧啶在苍蝇胚胎。免疫染色胚胎的共聚焦分析提供了胚胎DNA甲基化的直接证据。为了分析Dnmt2在DNA甲基化中的功能，我们通过RNA干扰去除该蛋白。Dnmt2的缺失对胚胎发育没有可检测到的影响，并导致DNA甲基化的完全丧失。同样，诱导转基因的Dnmt2过表达导致CpT和CpA二核苷酸显著的基因组高甲基化。这些结果表明，Dnmt2是果蝇DNA甲基化的必要条件和充分条件，并表明Dnmt2蛋白具有新的CpT/ a特异性DNA甲基转移酶活性。
The methylation status of Drosophila DNA has been discussed controversially over a long time. Recent evidence has provided strong support for the existence of 5-methylcytosine in DNA preparations from embryonic stages of fly development. The Drosophila genome contains a single candidate DNA methyltransferase gene that has been termed Dnmt2. This gene belongs to a widely conserved family of putative DNA methyltransferases. However, no catalytic activity has been demonstrated for any Dnmt2-like protein yet. We have now established a protocol for the immunological detection of methylated cytosine in fly embryos. Confocal analysis of immunostained embryos provided direct evidence for the methylation of embryonic DNA. In order to analyse the function of Dnmt2 in DNA methylation, we depleted the protein by RNA interference. Depletion of Dnmt2 had no detectable effect on embryonic development and resulted in a complete loss of DNA methylation. Consistently, overexpression of Dnmt2 from an inducible transgene resulted in significant genomic hypermethylation at CpT and CpA dinucleotides. These results demonstrate that Dnmt2 is both necessary and sufficient for DNA methylation in Drosophila and suggest a novel CpT/A-specific DNA methyltransferase activity for Dnmt2 proteins.