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.
Similar content being viewed by others
References
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.
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.
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.
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.
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.
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.
Fonatsch C, Duchrow G, Rieder H. Assignment of the human Ki67 gene (NKI67) to 10q25-qter. Genomics. 1991;11:467–77.
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.
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.
Safe S, Kim K. Nuclear receptor-mediated transactivation through interaction with Sp proteins. Prog Nucleic Acid Re. 2004;77:1–36.
Mounier C, Posner BI. Transcriptional regulation by insulin: from the receptor to the gene. Can J Physiol Pharmacol. 2006;84:713–24.
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.
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.
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.
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.
Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000;182:311–22.
Safe S, Abdelrahim M. Sp transcription factor family and its role in cancer. Eur J Cancer. 2005;41:2438–48.
Taguchi A, White MF. Insulin-like signaling, nutrient homeostasis and life span. Annu Rev Physiol. 2008;70:191–212.
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.
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.
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.
Bouwman P, Philipsen S. Regulation of the activity of Sp1-related transcription factors. Mol Cell Endocrinol. 2002;195:27–38.
Wade PA. Methyl CpG-binding proteins and transcriptional repression. BioEssays. 2007;23:1131–7.
Lee TF, Zhai J, Meyers BC. Conservation and divergence in eukaryotic DNA methylation. Proc Natl Acad Sci U S A. 2010;107:9027–8.
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.
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.
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.
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
Author information
Authors and Affiliations
Corresponding author
Additional information
Dong-Sheng Pei and Guo-Wei Qian contributed equally to this work.
Rights and permissions
About this article
Cite this article
Pei, DS., Qian, GW., Tian, H. et al. Analysis of human Ki-67 gene promoter and identification of the Sp1 binding sites for Ki-67 transcription. Tumor Biol. 33, 257–266 (2012). https://doi.org/10.1007/s13277-011-0277-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13277-011-0277-z