Molecules and Cells

, Volume 27, Issue 6, pp 635–640 | Cite as

H19 gene is epigenetically stable in mouse multipotent germline stem cells

  • Shin Hye Oh
  • Yoon Hee Jung
  • Mukesh Kumar Gupta
  • Sang Jun Uhm
  • Hoon Taek Lee


Testis-derived germline stem (GS) cells can undergo re-programming to acquire multipotency when cultured under appropriate culture conditions. These multipotent GS (mGS) cells have been known to differ from GS cells in their DNA methylation pattern. In this study, we examined the DNA methylation status of the H19 imprinting control region (ICR) in multipotent adult germline stem (maGS) cells to elucidate how epigenetic imprints are altered by culture conditions. DNA methylation was analyzed by bisulfite sequencing PCR of established maGS cells cultured in the presence of glial cell line-derived neurotrophic factor (GDNF) alone or both GDNF and leukemia inhibitory factor (LIF). The results showed that the H19 ICR in maGS cells of both groups was hypermethylated and had an androge-netic pattern similar to that of GS cells. In line with these data, the relative abundance of the Igf2 mRNA transcript was two-fold higher and that of H19 was three fold lower than in control embryonic stem cells. The androgenetic DNA methylation pattern of the H19 ICR was maintained even after 54 passages. Furthermore, differentiating maGS cells from retinoic acid-treated embryoid bodies maintained the androgenetic imprinting pattern of the H19 ICR. Taken together these data suggest that our maGS cells are epigenetically stable for the H19 gene during in vitro modifications. Further studies on the epigenetic regulation and chromatin structure of maGS cells are therefore necessary before their full potential can be utilized in regenerative medicine.


DNA methylation genomic imprinting germline stem cells H19 Igf2 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Andollo, N., Boyano, M.D., Andrade, R., and Arechaga, J.M. (2006). Epigenetic regulation of the imprinted U2af1-rs1 gene during retinoic acid-induced differentiation of embryonic stem cells. Dev. Growth Differ. 48, 349–360.PubMedCrossRefGoogle Scholar
  2. Bain, G., Ray, W.J., Yao, M., and Gottlieb, D.I. (1996). Retinoic acid promotes neural and represses mesodermal gene expression in mouse embryonic stem cells in culture. Biochem. Biophys. Res. Commun. 223, 691–694.PubMedCrossRefGoogle Scholar
  3. Bernstein, B.E., Meissner, A., and Lander, E.S. (2007). The mammalian epigenome. Cell 128, 669–681.PubMedCrossRefGoogle Scholar
  4. Carr, M.S., Yevtodiyenko, A., Schmidt, C.L., and Schmidt, J.V. (2007). Allele-specific histone modifications regulate expression of the Dlk1-Gtl2 imprinted domain. Genomics 89,280–290.PubMedCrossRefGoogle Scholar
  5. Chang, G., Liu, S., Wang, F., Zhang, Y., Kou, Z., Chen, D., and Gao, S. (2009). Differential methylation status of imprinted genes in nuclear transfer derived ES (NT-ES) cells. Genomics 03,112–119.CrossRefGoogle Scholar
  6. Dean, W., Bowden, L., Aitchison, A., Klose, J., Moore, T., Meneses, J.J., Reik, W., and Feil, R. (1998). Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes. Development 125,2273–2282.PubMedGoogle Scholar
  7. Deng, T., Kuang, Y., Zhang, D., Wang, L., Sun, R., Xu, G., Wang, Z., and Fei, J. (2007). Disruption of imprinting and aberrant embryo development in completely inbred embryonic stem cell-derived mice. Dev. Growth Differ. 49, 603–610.PubMedGoogle Scholar
  8. Doherty, A.S., Mann, M.R., Tremblay, K.D., Bartolomei, M.S., and Schultz, R.M. (2000). Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol. Re-prod. 62, 1526–1535.CrossRefGoogle Scholar
  9. Eckardt, S., Leu, N.A., Bradley, H.L., Kato, H., Bunting, K.D., and McLaughlin, K.J. (2007). Hematopoietic reconstitution with andro-genetic and gynogenetic stem cells. Genes Dev. 21,409–419.PubMedCrossRefGoogle Scholar
  10. Grandjean, V., O’Neill, L., Sado, T., Turner, B., and Ferguson-Smith, A. (2001). Relationship between DNA methylation, histone H4 acetylation and gene expression in the mouse imprinted Igf2-H19 domain. FEBS Lett. 488,165–169.PubMedCrossRefGoogle Scholar
  11. Guan, K., Nayernia, K., Maier, L.S., Wagner, S., Dressel, R., Lee, J.H., Nolte, J., Wolf, F., Li, M., Engel, W., et al. (2006). Pluripo-tency of spermatogonial stem cells from adult mouse testis. Nature 440, 1199–1203PubMedCrossRefGoogle Scholar
  12. Guan, K., Wagner, S., Unsold, B., Maier, L.S., Kaiser, D., Hemmer-lein, B., Nayernia, K., Engel, W., and Hasenfuss, G. (2007). Generation of functional cardiomyocytes from adult mouse sper-matogonial stem cells. Circ. Res. 100,1615–1625.PubMedCrossRefGoogle Scholar
  13. Han, D.W., Im, Y.B., Do, J.T., Gupta, M.K., Uhm, S.J., Kim, J.H., Scholer, H.R., and Lee, H.T. (2008). Methylation status of putative differentially methylated regions of porcine IGF2 and H19. Mol. Reprod. Dev. 75, 777–784.PubMedCrossRefGoogle Scholar
  14. Hernandez, L., Kozlov, S., Piras, G., and Stewart, C.L. (2003). Paternal and maternal genomes confer opposite effects on proliferation, cell-cycle length, senescence, and tumor formation. Proc. Natl. Acad. Sci. USA 100,13344–13349.PubMedCrossRefGoogle Scholar
  15. Horii, T., Kimura, M., Morita, S., Nagao, Y., and Hatada, I. (2008). Loss of genomic imprinting in mouse parthenogenetic embryonic stem cells. Stem Cells 26,79–88.PubMedCrossRefGoogle Scholar
  16. Humpherys, D., Eggan, K., Akutsu, H., Hochedlinger, K., Rideout, W.M., 3rd., Biniszkiewicz, D., Yanagimachi, R., and Jaenisch, R. (2001). Epigenetic instability in ES cells and cloned mice. Science 293, 95–97.PubMedCrossRefGoogle Scholar
  17. Izadyar, F., Pau, F., Marh, J., Slepko, N., Wang, T., Gonzalez, R., Ramos, T., Howerton, K., Sayre, C., and Silva, F. (2008). Generation of multipotent cell lines from a distinct population of male germ line stem cells. Reproduction 135, 771–784.PubMedCrossRefGoogle Scholar
  18. Kanatsu-Shinohara, M., Inoue, K., Lee, J., Yoshimoto, M., Ogonuki, N., Miki, H., Baba, S., Kato, T., Kazuki, Y., Toyokuni, S., (2004). Generation of pluripotent stem cells from neonatal mouse testis. Cell 119,1001–1012.PubMedCrossRefGoogle Scholar
  19. Kanatsu-Shinohara, M., Ogonuki, N., Iwano, T., Lee, J., Kazuki, Y., Inoue, K., Miki, H., Takehashi, M., Toyokuni, S., Shinkai, Y., et al. (2005). Genetic and epigenetic properties of mouse male germ-line stem cells during long-term culture. Development 132, 4155–4163.PubMedCrossRefGoogle Scholar
  20. Khosla, S., Dean, W., Brown, D., Reik, W., and Feil, R. (2001). Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes. Biol. Reprod. 64, 918–926.PubMedCrossRefGoogle Scholar
  21. Kubota, H., Avarbock, M.R., and Brinster, R.L. (2004). Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc. Natl. Acad. Sci. USA 101, 16489–16494.PubMedCrossRefGoogle Scholar
  22. Lee, J., Kanatsu-Shinohara, M., Ogonuki, N., Miki, H., Inoue, K., Morimoto, T., Morimoto, H., Ogura, A., and Shinohara, T. (2009). Heritable imprinting defect caused by epigenetic abnormalities in mouse spermatcgonial stem cells. Biol. Reprod. 80, 518–527.PubMedCrossRefGoogle Scholar
  23. Lucifero, D., Mertineit, C., Clarke, H.J., Bestor, T.H., and Trasler, J.M. (2002). Methylation dynamics of imprinted genes in mouse germ cells. Genomics 79, 530–538.PubMedCrossRefGoogle Scholar
  24. Murphy, S.K., Wylie, A.A., and Jirtle, R.L. (2001). Imprinting of PEG3, the human homologue of a mouse gene involved in nurturing behavior. Genomics 71,110–117.PubMedCrossRefGoogle Scholar
  25. Nakamura, T., Arai, Y., Umehara, H., Masuhara, M., Kimura, T., Taniguchi, H., Sekimoto, T., Ikawa, M., Yoneda, Y., Okabe, M., et al. (2007). PGC7/Stella protects against DNA demethylation in early embryogenesis. Nat. Cell Biol. 9,64–71.PubMedCrossRefGoogle Scholar
  26. Okita, K., and Yamanaka, S. (2006). Intracellular signaling pathways regulating pluripotency of embryonic stem cells. Curr. Stem Cell Res. Ther. /, 103-111.Google Scholar
  27. Reik, W. (2007). Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447,425–432.PubMedCrossRefGoogle Scholar
  28. Reik, W., and Walter, J. (2001). Genomic imprinting: parental influence on the genome. Nat. Rev. Genet. 2,21–32.PubMedCrossRefGoogle Scholar
  29. Reik, W., Constancia, M., Dean, W., Davies, K., Bowden, L., Murrell, A., Feil, R., Walter, J., and Kelsey, G. (2000). Igf2 imprinting in development and disease. Int. J. Dev. Biol. 44,145–150.PubMedGoogle Scholar
  30. Rugg-Gunn, P.J., Ferguson-Smith, AC., and Pedersen, R.A. (2005). Epigenetic status of human embryonic stem cells. Nat. Genet. 37, 585–587.PubMedCrossRefGoogle Scholar
  31. Sasaki, H., Ishihara, K., and Kato, R. (2000). Mechanisms of Igf2/H19 imprinting: DNA methylation, chromatin and longdistance gene regulation. J. Biochem. 127, 711–715.PubMedGoogle Scholar
  32. Shovlin, T.C., Durcova-Hills, G., Surani, A., and McLaren, A. (2008). Heterogeneity in imprinted methylation patterns of pluripotent embryonic germ cells derived from pre-migratory mouse germ cells. Dev. Biol. 313, 674–681.PubMedCrossRefGoogle Scholar
  33. Thorvaldsen, J.L., Duran, K.L., and Bartolomei, M.S. (1998). Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2. Genes Dev. 12, 3693–3702.PubMedCrossRefGoogle Scholar
  34. Yamagata, K., Yamazaki, T., Miki, H., Ogonuki, N., Inoue, K., Ogura, A., and Baba, T. (2007). Centromeric DNA hypomethylation as an epigenetic signature discriminates between germ and somatic cell lineages. Dev. Biol. 3/2,419–426.CrossRefGoogle Scholar
  35. Yang, Y., Hu, J.F., Ulaner, G.A., Li, T., Yao, X., Vu, T.H., and Hoffman, A.R. (2003). Epigenetic regulation of Igf2/H19 imprinting at CTCF insulator binding sites. J. Cell Biochem. 90,1038–1055.PubMedCrossRefGoogle Scholar
  36. Zhu, J.Q., Liu, J.H., Liang, X.W., Xu, B.Z., Hou, Y., Zhao, X.X., and Sun, Q.Y. (2008). Heat stress causes aberrant DNA methylation of H19 and lgf-2r in mouse blastocysts. Mol. Cells 25,211–215.PubMedGoogle Scholar
  37. Zovoilis, A., Nolte, J., Drusenheimer, N., Zechner, U., Hada, H., Guan, K., Hasenfuss, G., Nayernia, K., and Engel, W. (2008). Multipotent adult germline stem cells and embryonic stem cells have similar microRNA profiles. Mol. Hum. Reprod. 14, 521–529.PubMedCrossRefGoogle Scholar

Copyright information

© The Korean Society for Molecular and Cellular Biology and Springer Netherlands 2009

Authors and Affiliations

  • Shin Hye Oh
    • 1
  • Yoon Hee Jung
    • 1
  • Mukesh Kumar Gupta
    • 1
  • Sang Jun Uhm
    • 1
  • Hoon Taek Lee
    • 1
  1. 1.Department of Bioscience and Biotechnology, Bio-Organ Research CenterKonkuk UniversitySeoulKorea

Personalised recommendations