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. 1999 Dec 1;344(Pt 2):367–374.

Sp1 mediates cell proliferation-dependent regulation of rat DNA topoisomerase IIalpha gene promoter.

J H Yoon 1, J K Kim 1, G B Rha 1, M Oh 1, S H Park 1, R H Seong 1, S H Hong 1, S D Park 1
PMCID: PMC1220652  PMID: 10567217

Abstract

DNA topoisomerase IIalpha (topo IIalpha) is an essential nuclear enzyme required for chromosome segregation during mitosis. Consistent with its critical role in cell division is the fact that the expression of the gene for topo IIalpha is strongly regulated by the proliferation state of cells. Using a transient expression system, we determined the contribution of putative cis-acting elements in its promoter region to its basal level and cell proliferation-dependent transcription. Experiments with 5' and/or 3' serial deletion and site-directed mutation revealed that (1) maximal promoter activity resides in the fragment extending to position -663 bp from the ATG initiation codon, (2) minimal promoter activity is harboured at -195 bp, (3) the defined minimal promoter contains only two putative elements, inverted CCAAT box 4 (ICB4) (-166 to -162 bp) and the most proximal GC-rich box in the promoter (GC2) (-149 to -143 bp), and (4) ICB4 is most important in the basal-level transcription of the gene for rat topo IIalpha. The luciferase activities of the mutated reporter plasmids in G(0)-arrested and exponentially growing cells showed that proliferation-specific regulation is controlled mainly by GC2. Electrophoretic mobility-shift assays indicated that Sp1 binds specifically to the GC2 site. The extent of DNA-protein complex formation increases after the stimulation of cells to proliferate. These results indicate that the increased binding activity of Sp1 to GC2 is important in the up-regulation of the gene for topo IIalpha in growing cells.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adachi N., Kobayashi M., Koyama H. Cell cycle-dependent regulation of the mouse DNA topoisomerase IIalpha gene promoter. Biochem Biophys Res Commun. 1997 Jan 3;230(1):105–109. doi: 10.1006/bbrc.1996.5893. [DOI] [PubMed] [Google Scholar]
  2. Andrews N. C., Faller D. V. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 1991 May 11;19(9):2499–2499. doi: 10.1093/nar/19.9.2499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bird A., Taggart M., Frommer M., Miller O. J., Macleod D. A fraction of the mouse genome that is derived from islands of nonmethylated, CpG-rich DNA. Cell. 1985 Jan;40(1):91–99. doi: 10.1016/0092-8674(85)90312-5. [DOI] [PubMed] [Google Scholar]
  4. Chung T. D., Drake F. H., Tan K. B., Per S. R., Crooke S. T., Mirabelli C. K. Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9431–9435. doi: 10.1073/pnas.86.23.9431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dalton S. Cell cycle regulation of the human cdc2 gene. EMBO J. 1992 May;11(5):1797–1804. doi: 10.1002/j.1460-2075.1992.tb05231.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dorn A., Bollekens J., Staub A., Benoist C., Mathis D. A multiplicity of CCAAT box-binding proteins. Cell. 1987 Sep 11;50(6):863–872. doi: 10.1016/0092-8674(87)90513-7. [DOI] [PubMed] [Google Scholar]
  7. Drake F. H., Hofmann G. A., Bartus H. F., Mattern M. R., Crooke S. T., Mirabelli C. K. Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II. Biochemistry. 1989 Oct 3;28(20):8154–8160. doi: 10.1021/bi00446a029. [DOI] [PubMed] [Google Scholar]
  8. Everett R. D., Baty D., Chambon P. The repeated GC-rich motifs upstream from the TATA box are important elements of the SV40 early promoter. Nucleic Acids Res. 1983 Apr 25;11(8):2447–2464. doi: 10.1093/nar/11.8.2447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Filatov D., Thelander L. Role of a proximal NF-Y binding promoter element in S phase-specific expression of mouse ribonucleotide reductase R2 gene. J Biol Chem. 1995 Oct 20;270(42):25239–25243. doi: 10.1074/jbc.270.42.25239. [DOI] [PubMed] [Google Scholar]
  10. Garlatti M., Tchesnokov V., Daheshia M., Feilleux-Duché S., Hanoune J., Aggerbeck M., Barouki R. CCAAT/enhancer-binding protein-related proteins bind to the unusual promoter of the aspartate aminotransferase housekeeping gene. J Biol Chem. 1993 Mar 25;268(9):6567–6574. [PubMed] [Google Scholar]
  11. Gidoni D., Kadonaga J. T., Barrera-Saldaña H., Takahashi K., Chambon P., Tjian R. Bidirectional SV40 transcription mediated by tandem Sp1 binding interactions. Science. 1985 Nov 1;230(4725):511–517. doi: 10.1126/science.2996137. [DOI] [PubMed] [Google Scholar]
  12. Goswami P. C., Roti Roti J. L., Hunt C. R. The cell cycle-coupled expression of topoisomerase IIalpha during S phase is regulated by mRNA stability and is disrupted by heat shock or ionizing radiation. Mol Cell Biol. 1996 Apr;16(4):1500–1508. doi: 10.1128/mcb.16.4.1500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Graves B. J., Johnson P. F., McKnight S. L. Homologous recognition of a promoter domain common to the MSV LTR and the HSV tk gene. Cell. 1986 Feb 28;44(4):565–576. doi: 10.1016/0092-8674(86)90266-7. [DOI] [PubMed] [Google Scholar]
  14. Hatamochi A., Golumbek P. T., Van Schaftingen E., de Crombrugghe B. A CCAAT DNA binding factor consisting of two different components that are both required for DNA binding. J Biol Chem. 1988 Apr 25;263(12):5940–5947. [PubMed] [Google Scholar]
  15. Henglein B., Chenivesse X., Wang J., Eick D., Bréchot C. Structure and cell cycle-regulated transcription of the human cyclin A gene. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5490–5494. doi: 10.1073/pnas.91.12.5490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Herzog C. E., Zwelling L. A. Evaluation of a potential regulatory role for inverted CCAAT boxes in the human topoisomerase II alpha promoter. Biochem Biophys Res Commun. 1997 Mar 27;232(3):608–612. doi: 10.1006/bbrc.1997.6267. [DOI] [PubMed] [Google Scholar]
  17. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  18. Hochhauser D., Stanway C. A., Harris A. L., Hickson I. D. Cloning and characterization of the 5'-flanking region of the human topoisomerase II alpha gene. J Biol Chem. 1992 Sep 15;267(26):18961–18965. [PubMed] [Google Scholar]
  19. Holm C., Goto T., Wang J. C., Botstein D. DNA topoisomerase II is required at the time of mitosis in yeast. Cell. 1985 Jun;41(2):553–563. doi: 10.1016/s0092-8674(85)80028-3. [DOI] [PubMed] [Google Scholar]
  20. Isaacs R. J., Davies S. L., Sandri M. I., Redwood C., Wells N. J., Hickson I. D. Physiological regulation of eukaryotic topoisomerase II. Biochim Biophys Acta. 1998 Oct 1;1400(1-3):121–137. doi: 10.1016/s0167-4781(98)00131-6. [DOI] [PubMed] [Google Scholar]
  21. Isaacs R. J., Harris A. L., Hickson I. D. Regulation of the human topoisomerase IIalpha gene promoter in confluence-arrested cells. J Biol Chem. 1996 Jul 12;271(28):16741–16747. doi: 10.1074/jbc.271.28.16741. [DOI] [PubMed] [Google Scholar]
  22. Jenkins J. R., Ayton P., Jones T., Davies S. L., Simmons D. L., Harris A. L., Sheer D., Hickson I. D. Isolation of cDNA clones encoding the beta isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24. Nucleic Acids Res. 1992 Nov 11;20(21):5587–5592. doi: 10.1093/nar/20.21.5587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jones K. A., Kadonaga J. T., Rosenfeld P. J., Kelly T. J., Tjian R. A cellular DNA-binding protein that activates eukaryotic transcription and DNA replication. Cell. 1987 Jan 16;48(1):79–89. doi: 10.1016/0092-8674(87)90358-8. [DOI] [PubMed] [Google Scholar]
  24. Kim D. S., Ahn S. K., Yoon J. H., Hong S. H., Kim K. E., Maurer R. A., Park S. D. Involvement of a cAMP-responsive DNA element in mediating TRH responsiveness of the human thyrotropin alpha-subunit gene. Mol Endocrinol. 1994 Apr;8(4):528–536. doi: 10.1210/mend.8.4.7519724. [DOI] [PubMed] [Google Scholar]
  25. Kimura K., Saijo M., Ui M., Enomoto T. Growth state- and cell cycle-dependent fluctuation in the expression of two forms of DNA topoisomerase II and possible specific modification of the higher molecular weight form in the M phase. J Biol Chem. 1994 Jan 14;269(2):1173–1176. [PubMed] [Google Scholar]
  26. Linhoff M. W., Wright K. L., Ting J. P. CCAAT-binding factor NF-Y and RFX are required for in vivo assembly of a nucleoprotein complex that spans 250 base pairs: the invariant chain promoter as a model. Mol Cell Biol. 1997 Aug;17(8):4589–4596. doi: 10.1128/mcb.17.8.4589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maity S. N., Sinha S., Ruteshouser E. C., de Crombrugghe B. Three different polypeptides are necessary for DNA binding of the mammalian heteromeric CCAAT binding factor. J Biol Chem. 1992 Aug 15;267(23):16574–16580. [PubMed] [Google Scholar]
  28. McKnight S., Tjian R. Transcriptional selectivity of viral genes in mammalian cells. Cell. 1986 Sep 12;46(6):795–805. doi: 10.1016/0092-8674(86)90061-9. [DOI] [PubMed] [Google Scholar]
  29. Negri C., Chiesa R., Cerino A., Bestagno M., Sala C., Zini N., Maraldi N. M., Astaldi Ricotti G. C. Monoclonal antibodies to human DNA topoisomerase I and the two isoforms of DNA topoisomerase II: 170- and 180-kDa isozymes. Exp Cell Res. 1992 Jun;200(2):452–459. doi: 10.1016/0014-4827(92)90195-e. [DOI] [PubMed] [Google Scholar]
  30. Ng S. W., Eder J. P., Schnipper L. E., Chan V. T. Molecular cloning and characterization of the promoter for the Chinese hamster DNA topoisomerase II alpha gene. J Biol Chem. 1995 Oct 27;270(43):25850–25858. doi: 10.1074/jbc.270.43.25850. [DOI] [PubMed] [Google Scholar]
  31. Okazaki K., Nishizawa M., Furuno N., Yasuda H., Sagata N. Differential occurrence of CSF-like activity and transforming activity of Mos during the cell cycle in fibroblasts. EMBO J. 1992 Jul;11(7):2447–2456. doi: 10.1002/j.1460-2075.1992.tb05309.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Papazafiri P., Ogami K., Ramji D. P., Nicosia A., Monaci P., Cladaras C., Zannis V. I. Promoter elements and factors involved in hepatic transcription of the human ApoA-I gene positive and negative regulators bind to overlapping sites. J Biol Chem. 1991 Mar 25;266(9):5790–5797. [PubMed] [Google Scholar]
  33. Park S. H., Yoon J. H., Cho H. A., Kwon Y. D., Seong R. H., Hong S. H., Park S. D. Isolation and characterization of the promoter region of the rat DNA topoisomerase II alpha gene. J Biochem. 1995 Oct;118(4):725–733. doi: 10.1093/oxfordjournals.jbchem.a124972. [DOI] [PubMed] [Google Scholar]
  34. Raymondjean M., Cereghini S., Yaniv M. Several distinct "CCAAT" box binding proteins coexist in eukaryotic cells. Proc Natl Acad Sci U S A. 1988 Feb;85(3):757–761. doi: 10.1073/pnas.85.3.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Raymondjean M., Pichard A. L., Gregori C., Ginot F., Kahn A. Interplay of an original combination of factors: C/EBP, NFY, HNF3, and HNF1 in the rat aldolase B gene promoter. Nucleic Acids Res. 1991 Nov 25;19(22):6145–6153. doi: 10.1093/nar/19.22.6145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Roca J. The mechanisms of DNA topoisomerases. Trends Biochem Sci. 1995 Apr;20(4):156–160. doi: 10.1016/s0968-0004(00)88993-8. [DOI] [PubMed] [Google Scholar]
  37. Roman C., Platero J. S., Shuman J., Calame K. Ig/EBP-1: a ubiquitously expressed immunoglobulin enhancer binding protein that is similar to C/EBP and heterodimerizes with C/EBP. Genes Dev. 1990 Aug;4(8):1404–1415. doi: 10.1101/gad.4.8.1404. [DOI] [PubMed] [Google Scholar]
  38. Ronchi A. E., Bottardi S., Mazzucchelli C., Ottolenghi S., Santoro C. Differential binding of the NFE3 and CP1/NFY transcription factors to the human gamma- and epsilon-globin CCAAT boxes. J Biol Chem. 1995 Sep 15;270(37):21934–21941. doi: 10.1074/jbc.270.37.21934. [DOI] [PubMed] [Google Scholar]
  39. Sinha S., Maity S. N., Lu J., de Crombrugghe B. Recombinant rat CBF-C, the third subunit of CBF/NFY, allows formation of a protein-DNA complex with CBF-A and CBF-B and with yeast HAP2 and HAP3. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1624–1628. doi: 10.1073/pnas.92.5.1624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tronche F., Rollier A., Sourdive D., Cereghini S., Yaniv M. NFY or a related CCAAT binding factor can be replaced by other transcriptional activators for co-operation with HNF1 in driving the rat albumin promoter in vivo. J Mol Biol. 1991 Nov 5;222(1):31–43. doi: 10.1016/0022-2836(91)90735-o. [DOI] [PubMed] [Google Scholar]
  41. Tsai-Pflugfelder M., Liu L. F., Liu A. A., Tewey K. M., Whang-Peng J., Knutsen T., Huebner K., Croce C. M., Wang J. C. Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7177–7181. doi: 10.1073/pnas.85.19.7177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tsutsui K., Tsutsui K., Okada S., Watanabe M., Shohmori T., Seki S., Inoue Y. Molecular cloning of partial cDNAs for rat DNA topoisomerase II isoforms and their differential expression in brain development. J Biol Chem. 1993 Sep 5;268(25):19076–19083. [PubMed] [Google Scholar]
  43. Uemura T., Ohkura H., Adachi Y., Morino K., Shiozaki K., Yanagida M. DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe. Cell. 1987 Sep 11;50(6):917–925. doi: 10.1016/0092-8674(87)90518-6. [DOI] [PubMed] [Google Scholar]
  44. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1996;65:635–692. doi: 10.1146/annurev.bi.65.070196.003223. [DOI] [PubMed] [Google Scholar]
  45. Woessner R. D., Mattern M. R., Mirabelli C. K., Johnson R. K., Drake F. H. Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells. Cell Growth Differ. 1991 Apr;2(4):209–214. [PubMed] [Google Scholar]
  46. Wright K. L., Moore T. L., Vilen B. J., Brown A. M., Ting J. P. Major histocompatibility complex class II-associated invariant chain gene expression is up-regulated by cooperative interactions of Sp1 and NF-Y. J Biol Chem. 1995 Sep 8;270(36):20978–20986. doi: 10.1074/jbc.270.36.20978. [DOI] [PubMed] [Google Scholar]
  47. Zwicker J., Gross C., Lucibello F. C., Truss M., Ehlert F., Engeland K., Müller R. Cell cycle regulation of cdc25C transcription is mediated by the periodic repression of the glutamine-rich activators NF-Y and Sp1. Nucleic Acids Res. 1995 Oct 11;23(19):3822–3830. doi: 10.1093/nar/23.19.3822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zwicker J., Lucibello F. C., Wolfraim L. A., Gross C., Truss M., Engeland K., Müller R. Cell cycle regulation of the cyclin A, cdc25C and cdc2 genes is based on a common mechanism of transcriptional repression. EMBO J. 1995 Sep 15;14(18):4514–4522. doi: 10.1002/j.1460-2075.1995.tb00130.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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