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Tumor Biology

, Volume 33, Issue 1, pp 257–266 | Cite as

Analysis of human Ki-67 gene promoter and identification of the Sp1 binding sites for Ki-67 transcription

  • Dong-Sheng Pei
  • Guo-Wei Qian
  • Hui Tian
  • Jie Mou
  • Wang Li
  • Jun-Nian Zheng
Research Article

Abstract

Ki-67 as a cell proliferation marker is tightly associated with maintenance and regulation of the cell division. To understand the mechanism of Ki-67 gene expression and regulation, we first cloned 5′-flanking region and identified the Ki-67 core promoter. The deletion analysis and the dual luciferase reporter assay were used to locate the Ki-67 core promoter from −223 to +12 nt relative to the transcriptional initiation site, which is the TATA less, GC rich region comprised of several putative Sp1 binding sites. Compared with the hTERT promoter and Survivin promoter, the Ki-67 core promoter possessed higher transcription activity and more desirable tumor selectivity. In order to further demonstrate the contribution of transcription factor Sp1 on regulating the Ki-67 gene transcription, we confirmed three Sp1 binding sites from −170 to −145 nt, from −63 to −38 nt, and from −14 to +12 nt existed in the Ki-67 core promoter by the supershift assay. The deletion mutagenesis, together with the dual luciferase reporter assay, indicated that these Sp1 binding sites, particularly the region from −170 to −145 nt, were involved in positive regulation of the Ki-67 gene expression. Collectively, it was demonstrated that the region from −223 to +12 nt could drive the transcription of the Ki-67 gene, and the Sp1 binding site is essential to transcriptional regulation of the Ki-67 gene.

Keywords

Ki-67 Core promoter Sp1 Supershift assay 

Notes

Acknowledgments

This project is supported by grants from the National Natural Science Foundation of China (No. 30972976 and 81071854), the Program for New Century Excellent Talents in University (NCET-08-0700), and the Science and Technology Department of Jiangsu Province (No. BK2009089, BK2009091, and BK2010177).

Conflicts of interest

None

References

  1. 1.
    Duchrow M, Schlueter C, Wohlenberg C, Flad HD, Gerdes J. Molecular characterization of the gene locus of the human cell proliferation-associated nuclear protein defined by monoclonal antibody Ki-67. Cell Prolif. 1996;29:1–12.PubMedCrossRefGoogle Scholar
  2. 2.
    MacCallum DE, Hall PA. Biochemical characterization of pKi67 with the identification of a mitotic-specific form associated with hyperphosphorylation and altered DNA binding. Exp Cell Res. 1999;252:186–98.PubMedCrossRefGoogle Scholar
  3. 3.
    Scholzen T, Endl E, Wohlenberg C, van der Sar S, Cowell IG, Gerdes J, et al. The Ki-67 protein interacts with members of the heterochromatin protein 1(HP1) family: a potential role in the regulation of higher-order chromatin structure. J Pathol. 2002;196:135–44.PubMedCrossRefGoogle Scholar
  4. 4.
    Bullwinkel J, Baron-Lühr B, Lüdemann A, Wohlenberg C, Gerdes J, Scholzen T. Ki-67 protein is associated with RNA transcription in quiescent and proliferating cells. J Cell Physiol. 2006;206:624–35.PubMedCrossRefGoogle Scholar
  5. 5.
    MacCallum DE, Hall PA. The location of pKi67 in the outer dense fibrillary compartment of the nucleolus points to a role in ribosome biogenesis during the cell division cycle. J Pathol. 2000;190:537–44.PubMedCrossRefGoogle Scholar
  6. 6.
    Rahmanzadeh R, Hüttmann G, Gerdes J, Scholzen T. Chromophore-assisted light inactivation of pKi-67 leads to inhibition of ribosomal RNA synthesis. Cell Prolif. 2007;40:422–30.PubMedCrossRefGoogle Scholar
  7. 7.
    Fonatsch C, Duchrow G, Rieder H. Assignment of the human Ki67 gene (NKI67) to 10q25-qter. Genomics. 1991;11:467–77.CrossRefGoogle Scholar
  8. 8.
    Endl E, Gerdes J. Posttranslational modifications of the Ki-67 protein coincide with two major checkpoints during mitosis. J Cell Physiol. 2000;182:371–80.PubMedCrossRefGoogle Scholar
  9. 9.
    Kausch I, Lingnau A, Endl E, Sellmann K, Deinert I, Ratliff TL, et al. Antisense treatment against Ki-67 mRNA inhibits proliferation and tumor growth in vitro and in vivo. Int J Cancer. 2003;105:710–6.PubMedCrossRefGoogle Scholar
  10. 10.
    Safe S, Kim K. Nuclear receptor-mediated transactivation through interaction with Sp proteins. Prog Nucleic Acid Re. 2004;77:1–36.CrossRefGoogle Scholar
  11. 11.
    Mounier C, Posner BI. Transcriptional regulation by insulin: from the receptor to the gene. Can J Physiol Pharmacol. 2006;84:713–24.PubMedCrossRefGoogle Scholar
  12. 12.
    Lee TH, Chang HC, Chuang LY, Hung WC. Involvement of PKA and Sp1 in the induction of p27(Kip) by tamoxifen. Biochem Pharmacol. 2003;66:371–7.PubMedCrossRefGoogle Scholar
  13. 13.
    Solomon SS, Majumdar G, Martinez-Hernandez A, Raghow R. A critical role of Sp1 transcription factor in regulating gene expression in response to insulin and other hormones. Life Sci. 2008;83:305–12.PubMedCrossRefGoogle Scholar
  14. 14.
    Hwang CK, D’Souza UM, Eisch AJ, Yajima S, Lammers CH, Yang Y, Lee SH, Kim YM, Nestler EJ, Mouradian MM. Dopamine receptor regulating factor, DRRF: a zinc finger transcription factor. Proc Natl Acad Sci U S A.; 98:7558–7563.Google Scholar
  15. 15.
    Schlüter C, Duchrow M, Wohlenberg C, Becker MH, Key G, Flad HD, et al. The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J Cell Biol. 1993;123:513–22.PubMedCrossRefGoogle Scholar
  16. 16.
    Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000;182:311–22.PubMedCrossRefGoogle Scholar
  17. 17.
    Safe S, Abdelrahim M. Sp transcription factor family and its role in cancer. Eur J Cancer. 2005;41:2438–48.PubMedCrossRefGoogle Scholar
  18. 18.
    Taguchi A, White MF. Insulin-like signaling, nutrient homeostasis and life span. Annu Rev Physiol. 2008;70:191–212.PubMedCrossRefGoogle Scholar
  19. 19.
    Geng Y, Tsai-Morris CH, Zhang Y, Dufau ML. The human luteinizing hormone receptor gene promoter: activation by Sp1 and Sp3 and inhibitory regulation. Biochem Bioph Res Co. 1999;263:366–71.CrossRefGoogle Scholar
  20. 20.
    Goldberg HJ, Whiteside CI, Hart GW, Fantus IG. Posttranslational, reversible Ois stimulated by high glucose and mediates plasminogen activator inhibitor-1 gene expression and Sp1 transcriptional activity in glomerular mesangial cells. Endocrinology. 2006;147:222–31.PubMedCrossRefGoogle Scholar
  21. 21.
    Liu WL, Coleman RA, Grob P, King DS, Florens L, Washburn MP, et al. Structural changes in TAF4b-TFIID correlate with promoter selectivity. Mol Cell. 2008;29:81–91.PubMedCrossRefGoogle Scholar
  22. 22.
    Bouwman P, Philipsen S. Regulation of the activity of Sp1-related transcription factors. Mol Cell Endocrinol. 2002;195:27–38.PubMedCrossRefGoogle Scholar
  23. 23.
    Wade PA. Methyl CpG-binding proteins and transcriptional repression. BioEssays. 2007;23:1131–7.CrossRefGoogle Scholar
  24. 24.
    Lee TF, Zhai J, Meyers BC. Conservation and divergence in eukaryotic DNA methylation. Proc Natl Acad Sci U S A. 2010;107:9027–8.PubMedCrossRefGoogle Scholar
  25. 25.
    Brandeis M, Frank D, Keshet I, Siegfried Z, Mendelsohn M, Nemes A, et al. Sp1 elements protect a CpG island from de novo methylation. Nature. 1994;371:435–8.PubMedCrossRefGoogle Scholar
  26. 26.
    Macleod D, Charlton J, Mullins J, Bird AP. Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. Genes Dev. 1994;8:2282–92.PubMedCrossRefGoogle Scholar
  27. 27.
    Zhu WG, Srinivasan K, Dai Z, Duan W, Druhan LJ, Ding H, et al. Methylation of adjacent CpG sites affects Sp1/Sp3 binding and activity in the p21(Cip1) promoter. Mol Cell Biol. 2003;23:4056–65.PubMedCrossRefGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2011

Authors and Affiliations

  • Dong-Sheng Pei
    • 1
  • Guo-Wei Qian
    • 1
  • Hui Tian
    • 1
  • Jie Mou
    • 3
  • Wang Li
    • 1
  • Jun-Nian Zheng
    • 1
    • 2
  1. 1.Jiangsu Key Laboratory of Biological Cancer TherapyXuzhou Medical CollegeXuzhouChina
  2. 2.Center of Clinical OncologyAffiliated Hospital of Xuzhou Medical CollegeXuzhouChina
  3. 3.School of PharmacyXuzhou Medical CollegeXuzhouChina

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