Okano M, Xie S, Li E
Cardiovascular Research Center, Massachusetts General Hospital, Department of Medicine,Harvard Medical School, 149, 13th Street, Charlestown, MA 02129, USA.
Nucleic Acids Res. 1998 Jun 1;26(11):2536-40. doi: 10.1093/nar/26.11.2536.
We have shown previously that de novo methylation activities persist in mouse embryonic stem (ES) cells homozygous for a null mutation of Dnmt1 that encodes the major DNA cytosine methyltransferase. In this study, we have cloned a putative mammalian DNA methyltransferase gene, termed Dnmt2 , that is homologous to pmt1 of fission yeast. Different from pmt1 in which the catalytic Pro-Pro-Cys (PPC) motif is 'mutated' to Pro-Ser-Cys, Dnmt2 contains all the conserved methyltransferase motifs, thus likely encoding a functional cytosine methyltransferase. However, baculovirus-expressed Dnmt2 protein failed to methylate DNA in vitro . To investigate whether Dnmt2 functions as a DNA methyltransferase in vivo , we inactivated the Dnmt2 gene by targeted deletion of the putative catalytic PPC motif in ES cells. We showed that endogenous virus was fully methylated in Dnmt2 -deficient mutant ES cells. Furthermore, newly integrated retrovirus DNA was methylated de novo in infected mutant ES cells as efficiently as in wild-type cells. These results indicate that Dnmt2 is not essential for global de novo or maintenance methylation of DNA in ES cells.
我们之前已经表明，编码主要DNA胞嘧啶甲基转移酶的Dnmt1基因发生无效突变的纯合小鼠胚胎干细胞（ES细胞）中存在从头甲基化活性。在本研究中，我们克隆了一个推定的哺乳动物DNA甲基转移酶基因，命名为Dnmt2，它与裂殖酵母的pmt1同源。与pmt1不同，pmt1中的催化脯氨酸-脯氨酸-半胱氨酸（PPC）基序“突变”为脯氨酸-丝氨酸-半胱氨酸，而Dnmt2包含所有保守的甲基转移酶基序，因此可能编码一种功能性胞嘧啶甲基转移酶。然而，杆状病毒表达的Dnmt2蛋白在体外不能使DNA甲基化。为了研究Dnmt2在体内是否作为DNA甲基转移酶发挥作用，我们通过靶向缺失ES细胞中推定的催化PPC基序使Dnmt2基因失活。我们发现，在缺乏Dnmt2的突变ES细胞中，内源性病毒完全甲基化。此外，新整合的逆转录病毒DNA在感染的突变ES细胞中从头甲基化的效率与在野生型细胞中一样高。这些结果表明，Dnmt2对于ES细胞中DNA的整体从头甲基化或维持甲基化并非必不可少。
