Kaneda Masahiro, Okano Masaki, Hata Kenichiro, Sado Takashi, Tsujimoto Naomi, Li En, Sasaki Hiroyuki
Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Research Organization of Information and Systems, Mishima 411-8540, Japan.
Nature. 2004 Jun 24;429(6994):900-3. doi: 10.1038/nature02633.
Imprinted genes are epigenetically marked during gametogenesis so that they are exclusively expressed from either the paternal or the maternal allele in offspring. Imprinting prevents parthenogenesis in mammals and is often disrupted in congenital malformation syndromes, tumours and cloned animals. Although de novo DNA methyltransferases of the Dnmt3 family are implicated in maternal imprinting, the lethality of Dnmt3a and Dnmt3b knockout mice has precluded further studies. We here report the disruption of Dnmt3a and Dnmt3b in germ cells, with their preservation in somatic cells, by conditional knockout technology. Offspring from Dnmt3a conditional mutant females die in utero and lack methylation and allele-specific expression at all maternally imprinted loci examined. Dnmt3a conditional mutant males show impaired spermatogenesis and lack methylation at two of three paternally imprinted loci examined in spermatogonia. By contrast, Dnmt3b conditional mutants and their offspring show no apparent phenotype. The phenotype of Dnmt3a conditional mutants is indistinguishable from that of Dnmt3L knockout mice, except for the discrepancy in methylation at one locus. These results indicate that both Dnmt3a and Dnmt3L are required for methylation of most imprinted loci in germ cells, but also suggest the involvement of other factors.
印记基因在配子发生过程中会发生表观遗传标记，从而使其在后代中仅从父本或母本等位基因中表达。印记作用可防止哺乳动物孤雌生殖，并且在先天性畸形综合征、肿瘤和克隆动物中常常受到破坏。尽管Dnmt3家族的从头DNA甲基转移酶与母本印记有关，但Dnmt3a和Dnmt3b基因敲除小鼠的致死性阻碍了进一步研究。我们在此报告通过条件性基因敲除技术在生殖细胞中破坏Dnmt3a和Dnmt3b，同时在体细胞中保留它们。来自Dnmt3a条件性突变雌性的后代在子宫内死亡，在所检测的所有母本印记位点均缺乏甲基化和等位基因特异性表达。Dnmt3a条件性突变雄性的精子发生受损，在所检测的精原细胞中的三个父本印记位点中有两个缺乏甲基化。相比之下，Dnmt3b条件性突变体及其后代没有明显的表型。Dnmt3a条件性突变体的表型与Dnmt3L基因敲除小鼠的表型无法区分，除了一个位点的甲基化存在差异。这些结果表明，Dnmt3a和Dnmt3L对于生殖细胞中大多数印记位点的甲基化都是必需的，但也表明还涉及其他因素。
