Reduced rates of gene loss, gene silencing, and gene mutation in Dnmt1-deficient embryonic stem cells

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 ea ch of these different pathways varies among tumor suppressor genes and by c ancer type. The factors that influence the relative utilization of gene ina ctivation pathways are poorly understood. In this study, we describe a deta iled quantitative analysis of the three major gene inactivation mechanisms for a model gene at two different genomic integration sites in mouse embryo nic stem (ES) cells. In addition, we targeted the major DNA methyltransfera se 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 reduc ed in Dnmt1-deficient cells. Gene silencing by promoter methylation require s Dnmt1, suggesting that the expression of Dnmt3a and Dnmt3b alone in ES ce lls is insufficient to achieve effective gene silencing. We used a novel as say to show that missense mutation rates are also substantially reduced in Dnmt1-deficient cells. This is the first direct demonstration that DNA meth ylation affects point mutation rates in mammalian cells. Surprisingly, the fraction of CpG transition mutations was not reduced in Dnmt1-deficient cel ls. Finally, we show that methyl group-deficient growth conditions do not c ause an increase in missense mutation rates in Dnmt1-proficient cells, as p redicted by methyltransferase-mediated mutagenesis models. We conclude that Dnmt1 deficiency and the accompanying genomic DNA hypomethylation result i n a reduction of three major pathways of gene inactivation in our model sys tem.