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Molecular and Cellular Biology, November 2001, p. 7587-7600, Vol. 21, No. 22
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.22.7587-7600.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Reduced Rates of Gene Loss, Gene Silencing, and Gene Mutation in Dnmt1-Deficient Embryonic Stem Cells

Matilda F. Chan,1 Renée van Amerongen,1,dagger Tarlochan Nijjar,1,Dagger Edwin Cuppen,1,§ Peter A. Jones,1,2 and Peter W. Laird1,3,*

Department of Biochemistry and Molecular Biology,1 Department of Urology,2 and Department of Surgery,3 Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California 90089-9176

Received 7 June 2001/Returned for modification 30 July 2001/Accepted 16 August 2001

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.

* Corresponding author. Mailing address: USC/Norris Cancer Center, Room 6418, 1441 Eastlake Ave., Los Angeles, CA 90089-9176. Phone: (323) 865-0650. Fax: (323) 865-0158. E-mail: plaird{at}hsc.usc.edu.

dagger Present address: Division of Molecular Genetics and Center of Biomedical Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

Dagger Present address: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.

§ Present address: Hubrecht Laboratory/Netherlands Institute for Developmental Biology, 3584 CT Utrecht, The Netherlands.

Molecular and Cellular Biology, November 2001, p. 7587-7600, Vol. 21, No. 22
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.22.7587-7600.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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