Reduced rates of gene loss, gene silencing, and gene mutation in Dnmt1-deficient embryonic stem cells
Reduced rates of gene loss, gene silencing, and gene mutation in Dnmt1-deficient embryonic stem cells
复制标题
DOI：
10.1128/mcb.21.22.7587-7600.2001
复制
发表时间：
2001-11-01
影响因子：
5.3
通讯作者：
Laird, PW
中科院分区：
文献类型：
--
作者：
Chan, MF;van Amerongen, R;Laird, PW
抑癌基因失活是肿瘤发生的关键事件。基因失活机制包括导致杂合性缺失（洛）、基因突变和转录沉默的事件。这些不同途径中的每一种的贡献在肿瘤抑制基因和癌症类型之间是不同的。影响基因失活途径相对利用的因素知之甚少。在这项研究中，我们描述了一个模型基因在两个不同的基因组整合位点在小鼠胚胎干细胞（ES）的三个主要基因失活机制的详细定量分析。此外，我们针对主要的DNA甲基转移酶基因，Dnmt 1，研究DNA甲基化对这些不同的竞争基因失活途径的相对贡献。我们的数据表明，基因丢失是一个单纯疱疹病毒胸苷激酶新霉素磷酸转移酶报告基因（HSV TKNeo）在两个整合位点测试失活的主要模式，这一事件是显着减少Dnmt 1缺陷细胞。通过启动子甲基化的基因沉默需要Dnmt1，这表明在ES细胞中单独表达Dnmt3a和Dnmt3b不足以实现有效的基因沉默。我们使用了一种新的检测方法来表明，在Dnmt 1缺陷细胞中，错义突变率也大大降低。这是第一次直接证明DNA甲基化影响哺乳动物细胞中的点突变率。令人惊讶的是，CpG转换突变的分数在Dnmt 1缺陷细胞中没有减少。最后，我们表明，甲基缺陷的生长条件不会导致错义突变率增加Dnmt1精通细胞，甲基转移酶介导的诱变模型预测。我们的结论是，Dnmt 1缺陷和伴随的基因组DNA低甲基化导致在我们的模型系统中的基因失活的三个主要途径的减少。
Tumor suppressor gene inactivation is a crucial event in oncogenesis. Gene inactivation mechanisms include events resulting in loss of heterozygosity (LOH), gene mutation, and transcriptional silencing. The contribution of each of these different pathways varies among tumor suppressor genes and by cancer type. The factors that influence the relative utilization of gene inactivation pathways are poorly understood. In this study, we describe a detailed quantitative analysis of the three major gene inactivation mechanisms for a model gene at two different genomic integration sites in mouse embryonic stem (ES) cells. In addition, we targeted the major DNA methyltransferase gene, Dnmt1, to investigate the relative contribution of DNA methylation to these various competing gene inactivation pathways. Our data show that gene loss is the predominant mode of inactivation of a herpes simplex virus thymidine kinase neomycin phosphotransferase reporter gene (HSV TKNeo) at the two integration sites tested and that this event is significantly reduced in Dnmt1-deficient cells. Gene silencing by promoter methylation requires Dnmt1, suggesting that the expression of Dnmt3a and Dnmt3b alone in ES cells is insufficient to achieve effective gene silencing. We used a novel assay to show that missense mutation rates are also substantially reduced in Dnmt1-deficient cells. This is the first direct demonstration that DNA methylation affects point mutation rates in mammalian cells. Surprisingly, the fraction of CpG transition mutations was not reduced in Dnmt1-deficient cells. Finally, we show that methyl group-deficient growth conditions do not cause an increase in missense mutation rates in Dnmt1-proficient cells, as predicted by methyltransferase-mediated mutagenesis models. We conclude that Dnmt1 deficiency and the accompanying genomic DNA hypomethylation result in a reduction of three major pathways of gene inactivation in our model system.