Lei H, Oh S P, Okano M, Jüttermann R, Goss K A, Jaenisch R, Li E
Cardiovascular Research Center, Massachusetts General Hospital-East, Charlestown 02129, USA.
Development. 1996 Oct;122(10):3195-205. doi: 10.1242/dev.122.10.3195.
It has been a controversial issue as to how many DNA cytosine methyltransferase mammalian cells have and whether de novo methylation and maintenance methylation activities are encoded by a single gene or two different genes. To address these questions, we have generated a null mutation of the only known mammalian DNA methyltransferase gene through homologous recombination in mouse embryonic stem cells and found that the development of the homozygous embryos is arrested prior to the 8-somite stage. Surprisingly, the null mutant embryonic stem cells are viable and contain low but stable levels of methyl cytosine and methyltransferase activity, suggesting the existence of a second DNA methyltransferase in mammalian cells. Further studies indicate that de novo methylation activity is not impaired by the mutation as integrated provirus DNA in MoMuLV-infected homozygous embryonic stem cells become methylated at a similar rate as in wild-type cells. Differentiation of mutant cells results in further reduction of methyl cytosine levels, consistent with the de novo methylation activity being down regulated in differentiated cells. These results provide the first evidence that an independently encoded DNA methyltransferase is present in mammalian cells which is capable of de novo methylating cellular and viral DNA in vivo.
哺乳动物细胞中有多少种DNA胞嘧啶甲基转移酶，以及从头甲基化和维持甲基化活性是由单个基因还是两个不同基因编码，一直是个有争议的问题。为了解决这些问题，我们通过在小鼠胚胎干细胞中进行同源重组，产生了唯一已知的哺乳动物DNA甲基转移酶基因的无效突变，发现纯合胚胎的发育在8体节阶段之前就停止了。令人惊讶的是，无效突变胚胎干细胞是有活力的，并且含有低但稳定水平的甲基胞嘧啶和甲基转移酶活性，这表明哺乳动物细胞中存在第二种DNA甲基转移酶。进一步的研究表明，由于莫洛尼鼠白血病病毒（MoMuLV）感染的纯合胚胎干细胞中的整合前病毒DNA以与野生型细胞相似的速率甲基化，因此突变不会损害从头甲基化活性。突变细胞的分化导致甲基胞嘧啶水平进一步降低，这与分化细胞中从头甲基化活性下调一致。这些结果提供了第一个证据，表明哺乳动物细胞中存在一种独立编码的DNA甲基转移酶，它能够在体内对细胞和病毒DNA进行从头甲基化。
