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5-AZA-2′-deoxycytidine (5-AZA-CdR) leads to down-regulation of Dnmt1o and gene expression in preimplantation mouse embryos

Published online by Cambridge University Press:  01 May 2009

Jian-Ning Yu ,
Chun-Yang Xue ,
Xu-Guang Wang ,
Fei Lin ,
Chun-Yi Liu ,
Fu-Zeng Lu  and
Hong-Lin Liu
Jian-Ning Yu
Affiliation:
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, P.R China. Institute of Animal Science, Jiangsu Academy of Agri-cultural Sciences, Nanjing, Jiangsu, P.R. China.
Chun-Yang Xue
Affiliation:
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, P.R China.
Xu-Guang Wang
Affiliation:
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, P.R China.
Fei Lin
Affiliation:
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, P.R China.
Chun-Yi Liu
Affiliation:
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, P.R China.
Fu-Zeng Lu
Affiliation:
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, P.R China.
Hong-Lin Liu*
Affiliation:
College of Animal Science and Technology, Nanjing Agriculture University, No. 1 WeiGang Street, Nanjing, Jiangsu, P.R. China210095.
*
All correspondence to Hong-Lin Liu. College of Animal Science and Technology, Nanjing Agriculture University, No. 1 WeiGang Street, Nanjing, Jiangsu, P.R. China 210095. Tel: +86 25 84395106. e-mail: [email protected]
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Summary

5-AZA-2′-deoxycytidine (5-AZA-CdR) is a demethylating, teratogenic agent and a mutagen, which causes defects in the developing mouse and rat after implantation. Our previous data indicated that 5-AZA-CdR (0.2 and 1.0 μM) inhibited the development of mouse preimplantation embryos. Pronuclear embryos exposed to 5-AZA-CdR at the pronuclear stage were unable to form 8-cell embryos, while 2-cell-stage embryos exposed to 5-AZA-CdR only developed into uncompacted 8-cell-stage embryos. And there was no formation of blastocysts when 4-cell embryos cultured in 5-AZA-CdR. In our present study, we detected Dnmt1o protein and some developmental gene expression in order to find the reasons for the developmental arrest. Dnmt1o could not traffic to 8-cell nuclei as control when embryos were exposed to 5-AZA-CdR. Dnmt1o was in cytoplasm at 2-cell and 4-cell stages before and after treated with 5-AZA-CdR. Gene expression changes were also detected in this research. Our data indicated that connexin 31 (Cx31), connexin 43 (Cx43), connexin 45 (Cx45), E-cadherin (Cdh1) and β-catenin (Ctnnb1) were all downregulated by 5-AZA-CdR. Cx31, Cx43 and Cx45 are members of connexins family, which have a central role in gap junctions. Cdh1 and Ctnnb1 are necessary for the foundation of tight junctions. Therefore, developmental arrest induced by 5-AZA-CdR may be caused by the failure of Dnmt1o cytoplasmic–nuclear traffic and the down-regulation of developmental gene expression. Normal compaction and blastocoel cavitation need Dnmt1o traffic to 8-cell nuclei and the right gene expression, especially the correlative genes in gap junctions and tight junctions.

Keywords

5-AZA-CdRDNA methylationDnmt1oGene expressionPreimplantation mouse embryos
Type
Research Article
Information
Zygote , Volume 17 , Issue 2 , May 2009 , pp. 137 - 145
Copyright
Copyright © Cambridge University Press 2009

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References

Arai, M., Yokosuka, O., Hirasawa, Y., Fukai, K., Chiba, T., Imazeki, F., Kanda, T., Yatomi, M., Takiguchi, Y., Seki, N., Saisho, H. & Ochiai, T. (2006). Sequential gene expression changes in cancer cell lines after treatment with the demethylation agent 5-aza-2′-deoxycytidine. Cancer 106, 2514–25.Google Scholar
Baylin, S.B. (1997). Tying it all together: epigenetics, genetics, cell cycle and cancer. Science 277, 1948–9.Google Scholar
Becker, D.L., Evans, W.H., Green, C.R. & Warner, A. (1995). Functional analysis of amino acid sequences in connexin43 involved in intercellular communication through gap junctions. J. Cell Sci. 108, 1455–67.Google Scholar
Bevilacqua, A., Erickson, R.P. & Hieber, V. (1988). Antisense RNA inhibits endogenous gene expression in mouse preimplantation embryos: lack of double-stranded RNA ‘melting’ activity. Proc. Natl. Acad. Sci. USA 85, 831–5.Google Scholar
Branch, S., Francis, B.M., Brownie, C.F. & Chernoff, N. (1996). Teratogenic effects of the demethylating agent 5-AZA-2′-deoxycytidine in the Swiss Webster mouse. Toxicology 112, 37–43.Google Scholar
Branch, S., Chernoff, N., Brownie, C. & Francis, B.M. (1999). 5-AZA-2′-deoxycytidine-induced dysmorphogenesis in the rat. Teratog. Carcinog. Mutagen 19, 329–38.Google Scholar
Cardoso, M.C. & Leonhardt, H.DNA methyltransferase is actively retained in the cytoplasm during early development. (1999). J. Cell Biol. 147, 25–32.Google Scholar
Carlson, L.L., Page, A.W. & Bestor, T.H. (1992). Properties and localization of DNA methyltransferase in preimplantation mouse embryos: implications for genomic imprinting. Genes Dev. 6, 2536–41.Google Scholar
Chatot, C.L., Ziomek, C.A., Bavister, B.D., Lewis, J.L. & Torres, I. (1989). An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J. Reprod. Fertil. 86, 679–88.Google Scholar
Chen, Y., Zhang, Y.L. & Zhang, Q.Y. (2002). Gene expression and regulation of blastocyst formation. Dev. Reprod. Biol. 11, 75–81.Google Scholar
Christman, J.K. (2002). 5-AZA-cytidine and 5-AZA-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 21, 5483–95.Google Scholar
Chung, Y.G., Ratnam, S., Chaillet, J.R. & Latham, K.E. (2003). Abnormal regulation of DNA methyltransferase expression in cloned mouse embryos. Biol. Reprod. 69, 146–53.Google Scholar
Cisneros, F.J. & Branch, S. (2003). 5-AZA-2′-Deoxycytidine (5-AZA-CdR): a demethylating agent affecting development and reproductive capacity. J. Appl. Toxicol. 23, 115–20.Google Scholar
Cooney, C.A. (1993). Are somatic cells inherently deficient in methylation metabolism? A proposed mechanism for DNA methylation loss, senescence and aging. Growth Dev. Aging 57, 261–73.Google Scholar
Creusot, F., Acs, G. & Christman, J.K. (1982). Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2′-deoxycytidine. J. Biol. Chem. 257, 2041–8.Google Scholar
Davies, T.C., Barr, K.J., Jones, D.H., Zhu, D. & Kidder, G.M. (1996). Multiple members of the connexin gene family participate in preimplantation development of the mouse. Dev. Genet. 18, 234–43.Google Scholar
De Sousa, P.A., Juneja, S.C., Caveney, S., Houghton, F.D., Davies, T.C., Reaume, A.G, Rossant, J. & Kidder, G.M. (1997). Normal development of preimplantation mouse embryos deficient in gap junctional coupling. J. Cell Sci. 110, 1751–8.Google Scholar
De Vries, W.N., Evsikov, A.V., Haac1, B.E., Fancher, K.S., Holbrook, A.E., Kemler, R., Solter, D. & Knowles, B.B. (2004). Maternal β-catenin and E-cadherin in mouse development. Development 131, 4435–45.Google Scholar
Doherty, A.S., Bartolomei, M.S. & Schultz, R.M. (2002). Regulation of stage-specific nuclear translocation of Dnmt1o during preimplantation mouse development. Dev. Biol. 242, 255–66.Google Scholar
Gattei, V., Aldinucci, D., Petti, M.C., Da Ponte, A., Zagonel, V. & Pinto, A. (1993). In vitro and in vivo effects of 5-aza-2´-deoxycytidine (decitabine) on clonogenic cells from acute myeloid leukemia patients. Leukemia 7, 42–8.Google Scholar
Ghoshal, K., Datta, J., Majumder, S., Bai, S., Kutay, H., Motiwala, T. & Jacob, S.T. (2005). 5-AZA-deoxycytidine induces selective degradation of DNA methyltransferase 1 by a proteasomal pathway that requires the KEN box, bromo-adjacent homology domain and nuclear localization signal. Mol. Cell Biol. 25, 4727–41.Google Scholar
Gius, D., Cui, H., Bradbury, C.M., Cook, J., Smart, D.K., Zhao, S., Young, L., Brandenburg, S.A., Hu, Y., Bisht, K.S., Ho, A.S., Mattson, D., Sun, L., Munson, P.J., Chuang, E.Y., Mitchell, J.B. & Feinberg, A.P. (2004). Distinct effects on gene expression of chemical and genetic manipulation of the cancer epigenome revealed by a multimodality approach. Cancer Cell 6, 361–71.Google Scholar
Gumbiner, B.M. (1996). Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84, 345–57.Google Scholar
Haegel, H., Larue, L., Ohsugi, M., Fedorov, L., Herrenknecht, K. & Kemler, R. (1995). Lack of beta-catenin affects mouse development at gastrulation. Development 121, 3529–37.Google Scholar
Heard, E., Clerc, P. & Avner, P. (1997). X-chromosome inactivation in mammals. Annu. Rev. Genet. 31, 571–610.Google Scholar
Houghton, F.D. (2005). Role of gap junctions during early embryo development. Reproduction 129, 129–35.Google Scholar
Howell, C.Y., Bestor, T.H., Ding, F., Latham, K.E., Mertineit, C., Trasler, J.M. & Chaillet, J.R. (2001). Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene. Cell 104, 829–38.Google Scholar
Huelsken, J., Vogel, R., Brinkmann, V., Erdmann, B., Birchmeier, C. & Birchmeier, W. (2000). Requirement for beta-catenin in anterior–posterior axis formation in mice. J. Cell Biol. 148, 567–78.Google Scholar
Ikeda, K., Iyama, K., Ishikawa, N., Egami, H., Nakao, M., Sado, Y., Ninomiya, Y. & Baba, H. (2006). Loss of expression of type IV collagen alpha5 and alpha6 chains in colorectal cancer associated with the hypermethylation of their promoter region. Am. J. Pathol. 168, 856–65.Google Scholar
Issa, J.P. (2000). CpG-island methylation in aging and cancer. Curr. Top. Microbiol. Immunol. 249, 101–18.Google Scholar
Issa, J.P., Garcia-Manero, G., Giles, F.J., Mannari, R., Thomas, D., Faderl, S., Bayar, E., Lyons, J., Rosenfeld, C.S., Cortes, J. & Kantarjian, H.M. (2004). Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2′-deoxycytidine (decitabine) in hematopoietic malignancies. Blood 103, 1635–40.Google Scholar
Issa, J.P., Gharibyan, V., Cortes, J., Jelinek, J., Morris, G., Verstovsek, S., Talpaz, M., Garcia-Manero, G. & Kantarjian, H.M. (2005). Phase II study of low-dose decitabine in patients with chronic myelogenous leukemia resistant to imatinib mesylate. J. Clin. Oncol. 23, 3948–56.Google Scholar
Jackson-Grusby, L., Beard, C., Possemato, R., Tudor, M., Fambrough, D., Csankovszki, G., Dausman, J., Lee, P., Wilson, C., Lander, E. & Jaenisch, R. (2001). Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation. Nat. Genet. 27, 31–9.Google Scholar
Jost, J.P., Oakeley, E.J., Zhu, B., Benjamin, D., Thiry, S., Siegmann, M. & Jost, Y.C. (2001). 5-Methylcytosine DNA glycosylase participates in the genome-wide loss of DNA methylation occurring during mouse myoblast differentiation. Nucleic Acids Res. 29, 4452–61.CrossRefGoogle Scholar
Jüttermann, R., Li, E. & 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. USA 91, 11797–801.Google Scholar
Larue, L., Ohsugi, M., Hirchenhain, J. & Kemler, R. (1994). E-cadherin null mutant embryos fail to form a trophectoderm epithelium. Proc. Natl. Acad. Sci. USA 91, 8263–7.Google Scholar
Lee, S., Gilula, N.B. & Warner, A.E. (1987). Gap junctional communication and compaction during preimplantation stages of mouse development. Cell 51, 851–60.Google Scholar
Manejwala, F.M., Logan, C.Y. & Schultz, R.M. (1991). Regulation of hsp70 mRNA levels during oocyte maturation and zygotic gene activation in the mouse. Dev. Biol. 144, 301–8.Google Scholar
Mertineit, C., Yoder, J.A., Taketo, T., Laird, D.W., Trasler, J.M. & Bestor, T.H. (1998). Sex-specific exons control DNA methyltransferase in mammalian germ cells. Development 125, 889–97.Google Scholar
Michalowsky, L.A. & Jones, P.A. (1987). Differential nuclear protein binding to 5-azacytosine-containing DNA as a potential mechanism for 5-aza-2′-deoxycytidine resistance. Mol. Cell Biol. 7, 3076–83.Google Scholar
Nagafuchi, A. & Takeichi, M. (1989). Transmembrane control of cadherin-mediated cell adhesion: a 94 kDa protein functionally associated with a specific region of the cytoplasmic domain of E-cadherin. Cell Regul. 1, 37–44.Google Scholar
Okano, M., Bell, D.W., Haber, D.A. & Li, E. (1999). DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247–57.Google Scholar
Ozawa, M., Baribault, H. & Kemler, R. (1989). The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO J. 8, 1711–7.Google Scholar
Piskala, A. & Sorm, F. (1964). Nucleic acids components and their analogues. LI. Synthesis of L-glycosyl derivatives of 5-azauracil and 5-azacytosine. Coll. Czeck. Chem. Commun. 29, 2060–76.Google Scholar
Ratnam, S., Mertineit, C., Ding, F., Howell, C.Y., Clarke, H.J., Bestor, T.H., Chaillet, J.R. & Trasler, J.M. (2002). Dynamics of Dnmt1 methyltransferase expression and intracellular localization during oogenesis and preimplantation development. Dev. Biol. 245, 304–14.Google Scholar
Reik, W. & Dean, W. (2001). DNA methylation and mammalian epigenetics. Electrophoresis 22, 2838–43.Google Scholar
Reik, W., Dean, W. & Walter, J. (2001). Epigenetic reprogra-mming in mammalian development. Science 293, 1089–93.Google Scholar
Reuss, B., Hellmann, P., Traub, O., Butterweck, A. & Winterhager, E. (1997). Expression of connexin31 and connexin43 genes in early rat embryos. Dev. Genet. 21, 82–90.Google Scholar
Rogers, J.M., Francis, B.M., Sulik, K.K., Alles, A.J., Massaro, E.J., Zucker, R.M., Elstein, K.H., Rosen, M.B. & Chernoff, N. (1994). Cell death and cell cycle perturbation in the developmental toxicity of the demethylating agent, 5-AZA-2′-deoxycytidine. Teratology 50, 332–9.Google Scholar
Walsh, C.P. & Bestor, T.H. (1999). Cytosine methylation and mammalian development. Genes Dev. 13, 26–34.Google Scholar
Wei, C.J., Xu, X. & Lo, C.W. (2004). Connexins and cell signaling in development and disease. Annu. Rev. Cell Dev. Biol. 20, 811–38.Google Scholar
Whitten, W.K. (1971). Nutrient requirement for the culture of preimplantation embryos. Adv. Biosci. 6, 129–39.Google Scholar