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首页> 外文期刊>Molecular and Cellular Biology >In Vivo Activity of Murine De Novo Methyltransferases, Dnmt3a and Dnmt3b
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In Vivo Activity of Murine De Novo Methyltransferases, Dnmt3a and Dnmt3b

机译：鼠新甲基转移酶Dnmt3a和Dnmt3b的体内活性

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The putative de novo methyltransferases, Dnmt3a and Dnmt3b, were reported to have weak methyltransferase activity in methylating the 3′ long terminal repeat of Moloney murine leukemia virus in vitro. The activity of these enzymes was evaluated in vivo, using a stable episomal system that employs plasmids as targets for DNA methylation in human cells. De novo methylation of a subset of the CpG sites on the stable episomes is detected in human cells overexpressing the murine Dnmt3a or Dnmt3b1 protein. This de novo methylation activity is abolished when the cysteine in the P-C motif, which is the catalytic site of cytosine methyltransferases, is replaced by a serine. The pattern of methylation on the episome is nonrandom, and different regions of the episome are methylated to different extents. Furthermore, Dnmt3a also methylates the sequence methylated by Dnmt3a on the stable episome in the corresponding chromosomal target. Overexpression of human DNMT1 or murine Dnmt3b does not lead to the same pattern or degree of de novo methylation on the episome as overexpression of murine Dnmt3a. This finding suggests that these three enzymes may have different targets or requirements, despite the fact that weak de novo methyltransferase activity has been demonstrated in vitro for all three enzymes. It is also noteworthy that both Dnmt3a and Dnmt3b proteins coat the metaphase chromosomes while displaying a more uniform pattern in the nucleus. This is the first evidence that Dnmt3a and Dnmt3b have de novo methyltransferase function in vivo and the first indication that the Dnmt3a and Dnmt3b proteins may have preferred target sites.
机译：据报道，假定的从头甲基转移酶Dnmt3a和Dnmt3b在体外使莫洛尼鼠白血病病毒的3'长末端重复序列甲基化时具有弱的甲基转移酶活性。使用稳定的游离系统，在体内评估了这些酶的活性，该系统采用质粒作为人类细胞中DNA甲基化的靶标。在过量表达鼠Dnmt3a或Dnmt3b1蛋白的人细胞中检测到了稳定附加体上CpG位点的子集的从头甲基化。当P-C基序中的半胱氨酸（是胞嘧啶甲基转移酶的催化位点）被丝氨酸取代时，这种从头甲基化活性被消除。附加体上的甲基化模式是非随机的，附加体的不同区域甲基化的程度不同。此外，Dnmt3a还使相应染色体靶标中稳定附加体上被Dnmt3a甲基化的序列甲基化。人DNMT1或鼠Dnmt3b的过表达不会导致与鼠Dnmt3a的过表达相同的附加体上的从头甲基化模式或程度。这一发现表明，尽管已在体外对所有三种酶证明了从头甲基转移酶活性较弱，但这三种酶可能具有不同的靶标或要求。还值得注意的是，Dnmt3a和Dnmt3b蛋白都覆盖中期染色体，同时在细胞核中显示出更均匀的模式。这是Dnmt3a和Dnmt3b在体内具有从头甲基转移酶功能的第一个证据，也是Dnmt3a和Dnmt3b蛋白可能具有优选靶位点的第一个迹象。

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