This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chan, M. F.
Right arrow Articles by Laird, P. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chan, M. F.
Right arrow Articles by Laird, P. W.

 Previous Article  |  Next Article 

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.



This article has been cited by other articles:

  • Wang, S., Zhao, Y., Leiby, M. A., Zhu, J. (2009). Studying human telomerase gene transcription by a chromatinized reporter generated by recombinase-mediated targeting of a bacterial artificial chromosome. Nucleic Acids Res 0: gkp511v1-gkp511 [Abstract] [Full Text]  
  • Damelin, M., Bestor, T. H. (2007). Biological Functions of DNA Methyltransferase 1 Require Its Methyltransferase Activity. Mol. Cell. Biol. 27: 3891-3899 [Abstract] [Full Text]  
  • Evans, D G R, Maher, E R, Baser, M E (2005). Age related shift in the mutation spectra of germline and somatic NF2 mutations: hypothetical role of DNA repair mechanisms. J. Med. Genet. 42: 630-632 [Abstract] [Full Text]  
  • Mathers, J C (2005). Reversal of DNA hypomethylation by folic acid supplements: possible role in colorectal cancer prevention. Gut 54: 579-581 [Full Text]  
  • Laird, P. W. (2005). Cancer epigenetics. Hum Mol Genet 14: R65-R76 [Abstract] [Full Text]  
  • Cook, W. D., McCaw, B. J., Herring, C., John, D. L., Foote, S. J., Nutt, S. L., Adams, J. M. (2004). PU.1 is a suppressor of myeloid leukemia, inactivated in mice by gene deletion and mutation of its DNA binding domain. Blood 104: 3437-3444 [Abstract] [Full Text]  
  • Kim, M., Trinh, B. N., Long, T. I., Oghamian, S., Laird, P. W. (2004). Dnmt1 deficiency leads to enhanced microsatellite instability in mouse embryonic stem cells. Nucleic Acids Res 32: 5742-5749 [Abstract] [Full Text]  
  • Trinh, B. N., Long, T. I., Nickel, A. E., Shibata, D., Laird, P. W. (2002). DNA Methyltransferase Deficiency Modifies Cancer Susceptibility in Mice Lacking DNA Mismatch Repair. Mol. Cell. Biol. 22: 2906-2917 [Abstract] [Full Text]  
  • Ordway, J. M., Curran, T. (2002). Methylation Matters: Modeling a Manageable Genome. Cell Growth Differ. 13: 149-162 [Full Text]  
  • Kashkush, K., Feldman, M., Levy, A. A. (2002). Gene Loss, Silencing and Activation in a Newly Synthesized Wheat Allotetraploid. Genetics 160: 1651-1659 [Abstract] [Full Text]  
  • Eads, C. A., Nickel, A. E., Laird, P. W. (2002). Complete Genetic Suppression of Polyp Formation and Reduction of CpG-Island Hypermethylation in ApcMin/+Dnmt1-Hypomorphic Mice. Cancer Res. 62: 1296-1299 [Abstract] [Full Text]  
  • Bird, A. (2002). DNA methylation patterns and epigenetic memory. Genes Dev. 16: 6-21 [Full Text]