DOI QR코드

DOI QR Code

Characterization of the Nanog 5'-flanking Region in Bovine

  • Choi, Don-Ho (The Institute of Hankook Life Science) ;
  • Kim, Duk-Jung (The Institute of Hankook Life Science) ;
  • Song, Ki-Duk (Department of Animal Biotechnology, Chonbuk National University) ;
  • Park, Hwan-Hee (Department of Biomedical Sciences, School of Medicine, Inha University) ;
  • Ko, Tae Hyun (Department of Biomedical Sciences, School of Medicine, Inha University) ;
  • Pyao, Yuliya (Department of Biomedical Sciences, School of Medicine, Inha University) ;
  • Chung, Ku-Min (The Institute of Hankook Life Science) ;
  • Cha, Seok Ho (Department of Parasitology and Tropical Medicine, School of Medicine, Inha University) ;
  • Sin, Young-Su (Department of Animal Science, Singu University) ;
  • Kim, Nam-Hyung (Department of Animal Science, Chungbuk National University) ;
  • Lee, Woon-Kyu (Department of Biomedical Sciences, School of Medicine, Inha University)
  • Received : 2016.01.12
  • Accepted : 2016.04.07
  • Published : 2016.10.01

Abstract

Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5'-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the -134 to -19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.

Keywords

Bovine;Embryonic Stem Cells;Nanog;Sp1;Transcription Factors

Acknowledgement

Supported by : Inha University

References

  1. Black, A. R., J. D. Black, and J. Azizkhan-Clifford. 2001. Sp1 and kruppel-like factor family of transcription factors in cell growth regulation and cancer. J. Cell. Physiol. 188:143-160. https://doi.org/10.1002/jcp.1111
  2. Bouwman, P. and S. Philipsen. 2002. Regulation of the activity of Sp1-related transcription factors. Mol. Cell. Endocrinol. 195:27-38. https://doi.org/10.1016/S0303-7207(02)00221-6
  3. Chambers, I., D. Colby, M. Robertson, J. Nichols, S. Lee, S. Tweedie, and A. Smith. 2003. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113:643-655. https://doi.org/10.1016/S0092-8674(03)00392-1
  4. Chan, K. K., J. Zhang, N. Y. Chia, Y. S. Chan, H. S. Sim, K. S. Tan, S. K. Oh, H. H. Ng, and A. B. Choo. 2009. KLF4 and PBX1 directly regulate NANOG expression in human embryonic stem cells. Stem Cells 27:2114-2125. https://doi.org/10.1002/stem.143
  5. Ginis, I., Y. Luo, T. Miura, S. Thies, R. Brandenberger, S. Gerecht-Nir, M. Amit, A. Hoke, M. K. Carpenter, J. Itskovitz-Eldor, and M. S. Rao. 2004. Differences between human and mouse embryonic stem cells. Dev. Biol. 269:360-380. https://doi.org/10.1016/j.ydbio.2003.12.034
  6. Hart, A. H., L. Hartley, M. Ibrahim, and L. Robb. 2004. Identification, cloning and expression analysis of the pluripotency promoting Nanog genes in mouse and human. Dev. Dyn. 230:187-198. https://doi.org/10.1002/dvdy.20034
  7. Jiang, J., Y. S. Chan, Y. H. Loh, J. Cai, G. Q. Tong, C. A. Lim, P. Robson, S. Zhong, and H. H. Ng. 2008. A core Klf circuitry regulates self-renewal of embryonic stem cells. Nat. Cell Biol. 10:353-360. https://doi.org/10.1038/ncb1698
  8. Kaczynski, J., T. Cook, and R. Urrutia. 2003. Sp1-and Kruppel-like transcription factors. Genome Biol. 4:206. https://doi.org/10.1186/gb-2003-4-2-206
  9. Keefer, C. L., D. Pant, L. Blomberg, and N. C. Talbot. 2007. Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates. Anim. Reprod. Sci. 98:147-168. https://doi.org/10.1016/j.anireprosci.2006.10.009
  10. Kuijk, E. W., L. Du Puy, H. T. Van-Tol, C. H. Oei, H. P. Haagsman, B. Colenbrander, and B. A. Roelen. 2008. Differences in early lineage segregation between mammals. Dev. Dyn. 237:918-927. https://doi.org/10.1002/dvdy.21480
  11. Kuroda, T., M. Tada, H. Kubota, H. Kimura, S. Y. Hatano, H. Suemori, N. Nakatsuji, and T. Tada. 2005. Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Mol. Cell. Biol. 25:2475-2485. https://doi.org/10.1128/MCB.25.6.2475-2485.2005
  12. Lee, W. K., Y. M. Kim, N. Malik, C. Ma, and H. Westphal. 2006. Cloning and characterization of the 5'-flanking region of the Ehox gene. Biochem. Biophys. Res. Commun. 341:225-231. https://doi.org/10.1016/j.bbrc.2005.12.176
  13. Mitsui, K., Y. Tokuzawa, H. Itoh, K. Segawa, M. Murakami, K. Takahashi, M. Maruyama, M. Maeda, and S. Yamanaka. 2003. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113:631-642. https://doi.org/10.1016/S0092-8674(03)00393-3
  14. Munoz, M., A. Rodriguez, C. De Frutos, J. N. Caamano, C. Diez, N. Facal, and E. Gomez. 2008. Conventional pluripotency markers are unspecific for bovine embryonic-derived cell-lines. Theriogenology 69:1159-1164. https://doi.org/10.1016/j.theriogenology.2008.02.014
  15. Pan, G. and J. A. Thomson. 2007. Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res. 17:42-49. https://doi.org/10.1038/sj.cr.7310125
  16. Pesce, M., M. Marin-Gomez, S. Philipsen, and H. R. Scholer. 1999.Binding of Sp1 and Sp3 transcription factors to the Oct-4 gene promoter. Cell. Mol. Biol. 45:709-716.
  17. Rodda, D. J., J. L. Chew, L. H. Lim, Y. H. Loh, B. Wang, H. H. Ng, and P. Robson. 2005. Transcriptional regulation of Nanog by OCT4 and SOX2. J. Biol. Chem. 280:24731-24737. https://doi.org/10.1074/jbc.M502573200
  18. Torres, J. and F. M. Watt. 2008. Nanog maintains pluripotency of mouse embryonic stem cells by inhibiting NFkappaB and cooperating with Stat3. Nat. Cell Biol. 10:194-201. https://doi.org/10.1038/ncb1680
  19. Wei, Z., Y. Yang, P. Zhang, R. Andrianakos, K. Hasegawa, J. Lyu, X. Chen, G. Bai, C. Liu, M. Pera, and W. Lu. 2009. Klf4 interacts directly with Oct4 and Sox2 to promote reprogramming. Stem Cells 27:2969-2978.
  20. Wu, D. Y. and Z. Yao. 2005. Isolation and characterization of the murine Nanog gene promoter. Cell Res. 15:317-324. https://doi.org/10.1038/sj.cr.7290300
  21. Wu, D. Y. and Z. Yao. 2006. Functional analysis of two Sp1/Sp3 binding sites in murine Nanog gene promoter. Cell Res. 16:319-322. https://doi.org/10.1038/sj.cr.7310040
  22. Xie, D., C. C. Chen, L. M. Ptaszek, S. Xiao, X. Cao, F. Fang, H. H. Ng, H. A. Lewin, C. Cowan, and S. Zhong. 2010. Rewirable gene regulatory networks in the preimplantation embryonic development of three mammalian species. Genome Res. 20:804-815. https://doi.org/10.1101/gr.100594.109
  23. Xu, R. H., T. L. Sampsell-Barron, F. Gu, S. Root, R. M. Peck, G. Pan, J. Yu, J. Antosiewicz-Bourget, S. Tian, R. Stewart, and J. A. Thomson. 2008. NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling in human ESCs. Cell Stem Cell 3:196-206. https://doi.org/10.1016/j.stem.2008.07.001
  24. Yang, C., S. P. Atkinson, F. Vilella, M. Lloret, L. Armstrong, D. A. Mann, and M. Lako. 2010. Opposing putative roles for canonical and noncanonical NFkappaB signaling on the survival, proliferation, and differentiation potential of human embryonic stem cells. Stem. Cells 28:1970-1980. https://doi.org/10.1002/stem.528