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. 1997 May 15;25(10):2012–2019. doi: 10.1093/nar/25.10.2012

Transcription factor Sp3 antagonizes activation of the ornithine decarboxylase promoter by Sp1.

A P Kumar 1, A P Butler 1
PMCID: PMC146696  PMID: 9115370

Abstract

Ornithine decarboxylase (ODC) expression is important for proliferation and is elevated in many tumor cells. We previously showed that Sp1 is a major positive regulator of ODC transcription. In this paper we have investigated transcriptional regulation of rat ODC by the closely related factor Sp3. While over-expression of Sp1 caused a dramatic activation of the ODC promoter, over-expression of Sp3 caused little or no activation in either Drosophila SL2 cells (lacking endogenous Sp1 or Sp3) or in H35 rat hepatoma cells. Furthermore, co-transfection studies demonstrated that Sp3 abolished trans -activation of the ODC promoter by Sp1. DNase I footprint studies and electrophoretic mobility shift assays demonstrated that both recombinant Sp1 and Sp3 bind specifically to several sites within the ODC promoter also protected by nuclear extracts, including overlapping GC and CT motifs located between -116 and -104. This CT element is a site of negative ODC regulation. Mutation of either element reduced binding, but mutation of both sites was required to eliminate binding of either Sp1 or Sp3. These results demonstrate that ODC is positively regulated by Sp1 and negatively regulated by Sp3, suggesting that the ratio of these transcription factors may be an important determinant of ODC expression during development or transformation.

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Selected References

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  1. Abrahamsen M. S., Li R. S., Dietrich-Goetz W., Morris D. R. Multiple DNA elements responsible for transcriptional regulation of the ornithine decarboxylase gene by protein kinase A. J Biol Chem. 1992 Sep 15;267(26):18866–18873. [PubMed] [Google Scholar]
  2. Auvinen M., Paasinen A., Andersson L. C., Hölttä E. Ornithine decarboxylase activity is critical for cell transformation. Nature. 1992 Nov 26;360(6402):355–358. doi: 10.1038/360355a0. [DOI] [PubMed] [Google Scholar]
  3. Bello-Fernandez C., Cleveland J. L. c-myc transactivates the ornithine decarboxylase gene. Curr Top Microbiol Immunol. 1992;182:445–452. doi: 10.1007/978-3-642-77633-5_56. [DOI] [PubMed] [Google Scholar]
  4. Brabant M., McConlogue L., van Daalen Wetters T., Coffino P. Mouse ornithine decarboxylase gene: cloning, structure, and expression. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2200–2204. doi: 10.1073/pnas.85.7.2200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Butler A. P., Mar P. K., McDonald F. F., Ramsay R. L. Involvement of protein kinase C in the regulation of ornithine decarboxylase mRNA by phorbol esters in rat hepatoma cells. Exp Cell Res. 1991 May;194(1):56–61. doi: 10.1016/0014-4827(91)90129-i. [DOI] [PubMed] [Google Scholar]
  7. Courey A. J., Tjian R. Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell. 1988 Dec 2;55(5):887–898. doi: 10.1016/0092-8674(88)90144-4. [DOI] [PubMed] [Google Scholar]
  8. Dennig J., Beato M., Suske G. An inhibitor domain in Sp3 regulates its glutamine-rich activation domains. EMBO J. 1996 Oct 15;15(20):5659–5667. [PMC free article] [PubMed] [Google Scholar]
  9. Dennig J., Hagen G., Beato M., Suske G. Members of the Sp transcription factor family control transcription from the uteroglobin promoter. J Biol Chem. 1995 May 26;270(21):12737–12744. doi: 10.1074/jbc.270.21.12737. [DOI] [PubMed] [Google Scholar]
  10. DesJardins E., Hay N. Repeated CT elements bound by zinc finger proteins control the absolute and relative activities of the two principal human c-myc promoters. Mol Cell Biol. 1993 Sep;13(9):5710–5724. doi: 10.1128/mcb.13.9.5710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Desjarlais J. R., Berg J. M. Toward rules relating zinc finger protein sequences and DNA binding site preferences. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7345–7349. doi: 10.1073/pnas.89.16.7345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Di Nocera P. P., Dawid I. B. Transient expression of genes introduced into cultured cells of Drosophila. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7095–7098. doi: 10.1073/pnas.80.23.7095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feinstein S. C., Dana S. L., McConlogue L., Shooter E. M., Coffino P. Nerve growth factor rapidly induces ornithine decarboxylase mRNA in PC12 rat pheochromocytoma cells. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5761–5765. doi: 10.1073/pnas.82.17.5761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gilmour S. K., Avdalovic N., Madara T., O'Brien T. G. Induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol 13-acetate in hamster fibroblasts. Relationship between levels of enzyme activity, immunoreactive protein, and RNA during the induction process. J Biol Chem. 1985 Dec 25;260(30):16439–16444. [PubMed] [Google Scholar]
  17. Gilmour S. K., Verma A. K., Madara T., O'Brien T. G. Regulation of ornithine decarboxylase gene expression in mouse epidermis and epidermal tumors during two-stage tumorigenesis. Cancer Res. 1987 Mar 1;47(5):1221–1225. [PubMed] [Google Scholar]
  18. Greenberg M. E., Ziff E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. doi: 10.1038/311433a0. [DOI] [PubMed] [Google Scholar]
  19. Grens A., Steglich C., Pilz R., Scheffler I. E. Nucleotide sequence of the Chinese hamster ornithine decarboxylase gene. Nucleic Acids Res. 1989 Dec 25;17(24):10497–10497. doi: 10.1093/nar/17.24.10497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hagen G., Müller S., Beato M., Suske G. Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes. Nucleic Acids Res. 1992 Nov 11;20(21):5519–5525. doi: 10.1093/nar/20.21.5519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hagen G., Müller S., Beato M., Suske G. Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J. 1994 Aug 15;13(16):3843–3851. doi: 10.1002/j.1460-2075.1994.tb06695.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Heby O., Persson L. Molecular genetics of polyamine synthesis in eukaryotic cells. Trends Biochem Sci. 1990 Apr;15(4):153–158. doi: 10.1016/0968-0004(90)90216-x. [DOI] [PubMed] [Google Scholar]
  23. Hoey T., Weinzierl R. O., Gill G., Chen J. L., Dynlacht B. D., Tjian R. Molecular cloning and functional analysis of Drosophila TAF110 reveal properties expected of coactivators. Cell. 1993 Jan 29;72(2):247–260. doi: 10.1016/0092-8674(93)90664-c. [DOI] [PubMed] [Google Scholar]
  24. Hölttä E., Sistonen L., Alitalo K. The mechanisms of ornithine decarboxylase deregulation in c-Ha-ras oncogene-transformed NIH 3T3 cells. J Biol Chem. 1988 Mar 25;263(9):4500–4507. [PubMed] [Google Scholar]
  25. Imataka H., Sogawa K., Yasumoto K., Kikuchi Y., Sasano K., Kobayashi A., Hayami M., Fujii-Kuriyama Y. Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P-4501A1 gene. EMBO J. 1992 Oct;11(10):3663–3671. doi: 10.1002/j.1460-2075.1992.tb05451.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Johnson A. D. The price of repression. Cell. 1995 Jun 2;81(5):655–658. doi: 10.1016/0092-8674(95)90524-3. [DOI] [PubMed] [Google Scholar]
  27. Kadonaga J. T., Carner K. R., Masiarz F. R., Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell. 1987 Dec 24;51(6):1079–1090. doi: 10.1016/0092-8674(87)90594-0. [DOI] [PubMed] [Google Scholar]
  28. Kahana C., Nathans D. Translational regulation of mammalian ornithine decarboxylase by polyamines. J Biol Chem. 1985 Dec 15;260(29):15390–15393. [PubMed] [Google Scholar]
  29. Katz A., Kahana C. Transcriptional activation of mammalian ornithine decarboxylase during stimulated growth. Mol Cell Biol. 1987 Jul;7(7):2641–2643. doi: 10.1128/mcb.7.7.2641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kim Y. J., Pan H., Verma A. K. Non-AP-1 tumor promoter 12-O-tetradecanoylphorbol-13-acetate-responsive sequences in the human ornithine decarboxylase gene. Mol Carcinog. 1994 Jul;10(3):169–179. doi: 10.1002/mc.2940100308. [DOI] [PubMed] [Google Scholar]
  31. Kingsley C., Winoto A. Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression. Mol Cell Biol. 1992 Oct;12(10):4251–4261. doi: 10.1128/mcb.12.10.4251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kumar A. P., Mar P. K., Zhao B., Montgomery R. L., Kang D. C., Butler A. P. Regulation of rat ornithine decarboxylase promoter activity by binding of transcription factor Sp1. J Biol Chem. 1995 Mar 3;270(9):4341–4348. doi: 10.1074/jbc.270.9.4341. [DOI] [PubMed] [Google Scholar]
  33. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  34. Levine M., Manley J. L. Transcriptional repression of eukaryotic promoters. Cell. 1989 Nov 3;59(3):405–408. doi: 10.1016/0092-8674(89)90024-x. [DOI] [PubMed] [Google Scholar]
  35. Li R. S., Abrahamsen M. S., Johnson R. R., Morris D. R. Complex interactions at a GC-rich domain regulate cell type-dependent activity of the ornithine decarboxylase promoter. J Biol Chem. 1994 Mar 18;269(11):7941–7949. [PubMed] [Google Scholar]
  36. Li X., Coffino P. Degradation of ornithine decarboxylase: exposure of the C-terminal target by a polyamine-inducible inhibitory protein. Mol Cell Biol. 1993 Apr;13(4):2377–2383. doi: 10.1128/mcb.13.4.2377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Liang Y., Robinson D. F., Dennig J., Suske G., Fahl W. E. Transcriptional regulation of the SIS/PDGF-B gene in human osteosarcoma cells by the Sp family of transcription factors. J Biol Chem. 1996 May 17;271(20):11792–11797. doi: 10.1074/jbc.271.20.11792. [DOI] [PubMed] [Google Scholar]
  38. Majello B., De Luca P., Hagen G., Suske G., Lania L. Different members of the Sp1 multigene family exert opposite transcriptional regulation of the long terminal repeat of HIV-1. Nucleic Acids Res. 1994 Nov 25;22(23):4914–4921. doi: 10.1093/nar/22.23.4914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Majello B., De Luca P., Lania L. Sp3 is a bifunctional transcription regulator with modular independent activation and repression domains. J Biol Chem. 1997 Feb 14;272(7):4021–4026. doi: 10.1074/jbc.272.7.4021. [DOI] [PubMed] [Google Scholar]
  40. Majello B., De Luca P., Suske G., Lania L. Differential transcriptional regulation of c-myc promoter through the same DNA binding sites targeted by Sp1-like proteins. Oncogene. 1995 May 4;10(9):1841–1848. [PubMed] [Google Scholar]
  41. Manzella J. M., Blackshear P. J. Regulation of rat ornithine decarboxylase mRNA translation by its 5'-untranslated region. J Biol Chem. 1990 Jul 15;265(20):11817–11822. [PubMed] [Google Scholar]
  42. Mar P. K., Kumar A. P., Kang D. C., Zhao B., Martinez L. A., Montgomery R. L., Anderson L., Butler A. P. Characterization of novel phorbol ester- and serum-responsive sequences of the rat ornithine decarboxylase gene promoter. Mol Carcinog. 1995 Dec;14(4):240–250. doi: 10.1002/mc.2940140404. [DOI] [PubMed] [Google Scholar]
  43. Miller I. J., Bieker J. J. A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Krüppel family of nuclear proteins. Mol Cell Biol. 1993 May;13(5):2776–2786. doi: 10.1128/mcb.13.5.2776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Moshier J. A., Dosescu J., Skunca M., Luk G. D. Transformation of NIH/3T3 cells by ornithine decarboxylase overexpression. Cancer Res. 1993 Jun 1;53(11):2618–2622. [PubMed] [Google Scholar]
  45. Moshier J. A., Gilbert J. D., Skunca M., Dosescu J., Almodovar K. M., Luk G. D. Isolation and expression of a human ornithine decarboxylase gene. J Biol Chem. 1990 Mar 25;265(9):4884–4892. [PubMed] [Google Scholar]
  46. Moshier J. A., Skunca M., Wu W., Boppana S. M., Rauscher F. J., 3rd, Dosescu J. Regulation of ornithine decarboxylase gene expression by the Wilms' tumor suppressor WT1. Nucleic Acids Res. 1996 Mar 15;24(6):1149–1157. doi: 10.1093/nar/24.6.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Palvimo J. J., Eisenberg L. M., Jänne O. A. Protein-DNA interactions in the cAMP responsive promoter region of the murine ornithine decarboxylase gene. Nucleic Acids Res. 1991 Jul 25;19(14):3921–3927. doi: 10.1093/nar/19.14.3921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Pascal E., Tjian R. Different activation domains of Sp1 govern formation of multimers and mediate transcriptional synergism. Genes Dev. 1991 Sep;5(9):1646–1656. doi: 10.1101/gad.5.9.1646. [DOI] [PubMed] [Google Scholar]
  49. Pegg A. E. Recent advances in the biochemistry of polyamines in eukaryotes. Biochem J. 1986 Mar 1;234(2):249–262. doi: 10.1042/bj2340249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Persengiev S. P., Saffer J. D., Kilpatrick D. L. An alternatively spliced form of the transcription factor Sp1 containing only a single glutamine-rich transactivation domain. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9107–9111. doi: 10.1073/pnas.92.20.9107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Peña A., Reddy C. D., Wu S., Hickok N. J., Reddy E. P., Yumet G., Soprano D. R., Soprano K. J. Regulation of human ornithine decarboxylase expression by the c-Myc.Max protein complex. J Biol Chem. 1993 Dec 25;268(36):27277–27285. [PubMed] [Google Scholar]
  52. Schneider I. Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol. 1972 Apr;27(2):353–365. [PubMed] [Google Scholar]
  53. Sistonen L., Hölttä E., Mäkelä T. P., Keski-Oja J., Alitalo K. The cellular response to induction of the p21 c-Ha-ras oncoprotein includes stimulation of jun gene expression. EMBO J. 1989 Mar;8(3):815–822. doi: 10.1002/j.1460-2075.1989.tb03442.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sistonen L., Keski-Oja J., Ulmanen I., Hölttä E., Wikgren B. J., Alitalo K. Dose effects of transfected c-Ha-rasVal 12 oncogene in transformed cell clones. Exp Cell Res. 1987 Feb;168(2):518–530. doi: 10.1016/0014-4827(87)90024-3. [DOI] [PubMed] [Google Scholar]
  55. Sogawa K., Imataka H., Yamasaki Y., Kusume H., Abe H., Fujii-Kuriyama Y. cDNA cloning and transcriptional properties of a novel GC box-binding protein, BTEB2. Nucleic Acids Res. 1993 Apr 11;21(7):1527–1532. doi: 10.1093/nar/21.7.1527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tabor C. W., Tabor H. Polyamines. Annu Rev Biochem. 1984;53:749–790. doi: 10.1146/annurev.bi.53.070184.003533. [DOI] [PubMed] [Google Scholar]
  57. Tobias K. E., Shor J., Kahana C. c-Myc and Max transregulate the mouse ornithine decarboxylase promoter through interaction with two downstream CACGTG motifs. Oncogene. 1995 Nov 2;11(9):1721–1727. [PubMed] [Google Scholar]
  58. Udvadia A. J., Rogers K. T., Higgins P. D., Murata Y., Martin K. H., Humphrey P. A., Horowitz J. M. Sp-1 binds promoter elements regulated by the RB protein and Sp-1-mediated transcription is stimulated by RB coexpression. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3265–3269. doi: 10.1073/pnas.90.8.3265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Udvadia A. J., Templeton D. J., Horowitz J. M. Functional interactions between the retinoblastoma (Rb) protein and Sp-family members: superactivation by Rb requires amino acids necessary for growth suppression. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3953–3957. doi: 10.1073/pnas.92.9.3953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wrighton C., Busslinger M. Direct transcriptional stimulation of the ornithine decarboxylase gene by Fos in PC12 cells but not in fibroblasts. Mol Cell Biol. 1993 Aug;13(8):4657–4669. doi: 10.1128/mcb.13.8.4657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. al-Asadi R., Yi E. C., Merchant J. L. Sp1 affinity for GC-rich elements correlates with ornithine decarboxylase promoter activity. Biochem Biophys Res Commun. 1995 Sep 14;214(2):324–330. doi: 10.1006/bbrc.1995.2291. [DOI] [PubMed] [Google Scholar]
  62. van Steeg H., van Oostrom C. T., Hodemaekers H. M., van Kreyl C. F. Cloning and functional analysis of the rat ornithine decarboxylase-encoding gene. Gene. 1990 Sep 14;93(2):249–256. doi: 10.1016/0378-1119(90)90232-g. [DOI] [PubMed] [Google Scholar]
  63. van Steeg H., van Oostrom C. T., Martens J. W., van Kreyl C., Schepens J., Wieringa B. Nucleotide sequence of the human ornithine decarboxylase gene. Nucleic Acids Res. 1989 Nov 11;17(21):8855–8856. doi: 10.1093/nar/17.21.8855. [DOI] [PMC free article] [PubMed] [Google Scholar]

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