Murine de novo methyltransferase Dnmt3a demonstrates strand asymmetry and site preference in the methylation of DNA in vitro
Murine de novo methyltransferase Dnmt3a demonstrates strand asymmetry and site preference in the methylation of DNA in vitro
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DOI：
10.1128/mcb.22.3.704-723.2002
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发表时间：
2002-02-01
影响因子：
5.3
通讯作者：
Hsieh, CL
中科院分区：
文献类型：
--
作者：
Lin, IG;Han, L;Hsieh, CL
CpG甲基化参与脊椎动物以及植物和真菌中的广泛生物过程。迄今为止，已知三种酶Dnmt1、Dnmt3a和Dnmt3b在小鼠和人类中具有DNA甲基转移酶活性。已经提出，在早期胚胎中观察到的从头甲基化主要由Dnmt3a和Dnmt3b甲基转移酶进行，而Dntm1被认为是负责维持复制时建立的甲基化模式。使用亚硫酸氢盐基因组测序方法对体内甲基化位点的分析证实了先前的发现，即质粒的一些区域比同一质粒上的其他区域被Dnmt3a甲基化得多。然而，由于存在其他甲基化酶、DNA结合蛋白和染色质结构，不能确定酶的优选靶点。为了辨别没有这些复合因子的Dnmt3a的DNA靶标，分析了体外被Dnmt3a甲基化的位点。这些分析揭示了两条cDNA链具有明显不同的甲基化模式。在体外，Dnmt3a优选侧接嘧啶的链上的CpG位点，而不是侧接嘌呤的CpG位点。这些发现表明，与Dnmt 1不同，Dnmt 3a最有可能甲基化DNA的一条链，而不同时甲基化互补链上的CpG位点。这些发现还表明，Dnmt3a可能甲基化一些CpG位点比其他更频繁，这取决于序列背景。在已知的DNA甲基转移酶中，每条DNA链独立地甲基化并具有可能的序列偏好性是一个新的特征。
CpG methylation is involved in a wide range of biological processes in vertebrates as well as in plants and fungi. To date, three enzymes, Dnmt1, Dnmt3a, and Dnmt3b, are known to have DNA methyltransferase activity in mouse and human. It has been proposed that de novo methylation observed in early embryos is predominantly carried out by the Dnmt3a and Dnmt3b methyltransferases, while Dntm1 is believed to be responsible for maintaining the established methylation patterns upon replication. Analysis of the sites methylated in vivo using the bisulfite genomic sequencing method confirms the previous finding that some regions of the plasmid are much more methylated by Dnmt3a than other regions on the same plasmid. However, the preferred targets of the enzyme cannot be determined due to the presence of other methylases, DNA binding proteins, and chromatin structure. To discern the DNA targets of Dnmt3a without these compounding factors, sites methylated by Dnmt3a in vitro were analyzed. These analyses revealed that the two cDNA strands have distinctly different methylation patterns. Dnmt3a prefers CpG sites on a strand in which it is flanked by pyrimidines over CpG sites flanked by, purines in vitro. These findings indicate that, unlike Dnmt1, Dnmt3a most likely methylates one strand of DNA without concurrent methylation of the CpG site on the complementary strand. These findings also indicate that Dnmt3a may methylate some CpG sites more frequently than others, depending on the sequence context. Methylation of each DNA strand independently and with possible sequence preference is a novel feature among the known DNA methyltransferases.