Abstract
DNA methylation plays a significant role in the expression of the genetic code and affects early growth and development through its influence on gene expression. DNA methyltransferase 1 (Dnmt1) is the enzyme responsible for maintaining the methylation marks through cell division. However, the de novo methyltransferases, Dnmt3a and Dnmt3b, can also contribute to the maintenance of the methylation pattern. Manipulation of these enzymes, especially Dnmt1, provides a means to alter DNA methylation levels. Manipulation of the DNA methylation pattern of somatic cells will allow a better understanding of the different molecular process associated with chromatin structure and gene expression. Different approaches to artificially manipulate the expression of Dnmt1 in somatic cells include the addition of 5-azacytidine, culture of cells for an extended period of time, and the use of small interfering RNA technologies.
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References
Giraldo, A. M., Lynn, J. W., Purpera, M. N., Vaught, T. D., Ayares, D. L., Godke, R. A., and Bondioli, K. R. (2009). Inhibition of DNA methyltransferase 1 expression in bovine fibroblast cells used for nuclear transfer. Reprod. Fertil. Dev. 21, 785–795.
Koukalova, B., Fojtova, M., Lim, K. Y., Fulnecek, J., Leitch, A. R., and Kovarik, A. (2005). Dedifferentiation of tobacco cells is associated with ribosomal RNA gene hypomethylation, increased transcription, and chromatin alterations. Plant Physiol 139, 275–286.
Yeo, S., Jeong, S., Kim, J., Han, J. S., Han, Y. M., and Kang, Y. K. (2007). Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells. Biochem. Biophys. Res Commun. 359, 536–542.
Chen, T., Ueda, Y., Dodge, J. E., Wang Z., Li, E. (2003). Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol Cell Biol. 23, 5594–5605.
Enright, B. P., Kubota, C., Yang, X., and Tian, X. C. (2003). Epigenetic characteristics and development of embryos cloned from donor cells treated by trichostatin A or 5-aza-2’-deoxycytidine. Biol Reprod 69, 896–901.
Gaudet, F., Hodgson, J. G., Eden, A., Jackson-Grusby, L., Dausman, J., Gray, J. W., Leonhardt, H., and Jaenisch, R. (2003). Induction of tumors in mice by genomic hypomethylation. Science 300, 489–492.
Leu, Y. W., Rahmatpanah, F., Shi, H., Wei, S. H., Liu, J. C., Yan, P. S., and Huang, T. H. (2003). Double RNA interference of DNMT3b and DNMT1 enhances DNA demethylation and gene reactivation. Cancer Res. 63, 6110–6115.
Suzuki, M., Sunaga, N., Shames, D. S., Toyooka, S., Gazdar, A. F., and Minna, J. D. (2004). RNA interference-mediated knockdown of DNA methyltransferase 1 leads to promoter demethylation and gene re-expression in human lung and breast cancer cells. Cancer Res 64, 3137–3143.
Giraldo, A. M., Hylan, D. A., Ballard, C. B., Purpera, M. N., Vaught, T. D., Lynn, J. W., Godke, R. A., and Bondioli, K. R. (2008). Effect of epigenetic modifications of donor somatic cells on the subsequent chromatin remodeling of cloned bovine embryos. Biol. Reprod. 78, 832–840.
Li, E., Bestor, T. H., and Jaenisch, R. (1992). Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926.
Lei, H., Oh, S. P., Okano, M., Juttermann, R., Goss, K. A., Jaenisch, R., and Li, E. (1996). De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development 122, 3195–3205.
Wilson, V. L. and Jones, P. A. (1983). DNA methylation decreases in aging but not in immortal cells. Science 220, 1055–1057.
Moore, K., Wroclawska, E., Kramer, J. M., and Goica, S. L. (2006). Cell type and culture over time effect DNA methyltransferase 1 expression in bovine donor fibroblast cells. Reprod. Fertil. Dev. 19, 151.
Adams, A. M., Pratt, S. L., and Stice, S. L. (2005). Knockdown of the Dnmt1s transcript using small interfering RNA in primary murine and bovine fibroblast cells. Mol. Reprod Dev. 72, 311–319.
Jones, P. A., Taylor, S. M. (1980). Cellular differentiation, cytidine analogs and DNA methylation. Cell. 20, 85–93.
Villar-Garea, A., Fraga, M. F., Espada, J., Esteller, M. (2003). Procaine is a DNA-demethylating agent with growth-inhibitory effects in human cancer cells. Cancer Res. 63, 4984–4989.
Christman, J. K. (2002). 5-Azacytidine and 5-aza-2’-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 21, 5483–5495.
Juttermann, R., Li, E., and Jaenisch, R. (1994). Toxicity of 5-aza-2’-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation. Proc. Natl. Acad. Sci. U. S. A 91, 11797–11801.
Li, L. H., Olin, E. J., Buskirk, H. H., Reineke, L. M. (1970). Cytotoxicity and mode of action of 5-azacytidine on L1210 leukemia. Cancer Res. 30, 2760–2769.
Santos, F., Dean, W. (2006). Using immunofluorescence to observe methylation changes in mammalian preimplantation embryos. Methods Mol Biol. 325, 129–37.
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Giraldo, A.M., Bondioli, K.R. (2011). Inhibition of DNA Methylation in Somatic Cells. In: Tollefsbol, T. (eds) Epigenetics Protocols. Methods in Molecular Biology, vol 791. Humana Press. https://doi.org/10.1007/978-1-61779-316-5_12
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DOI: https://doi.org/10.1007/978-1-61779-316-5_12
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