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. 2001 Nov 1;359(Pt 3):669–677. doi: 10.1042/0264-6021:3590669

Characterization of the MN/CA 9 promoter proximal region: a role for specificity protein (SP) and activator protein 1 (AP1) factors.

M Kaluzová 1, S Pastoreková 1, E Svastová 1, J Pastorek 1, E J Stanbridge 1, S Kaluz 1
PMCID: PMC1222189  PMID: 11672442

Abstract

MN/CA IX (MN) is a tumour-associated isoenzyme of the carbonic anhydrase family. Previous deletion analysis of the MN promoter established that protected regions (PRs) 1 and 2 are crucial for its transcriptional activity. Computer-assisted searching indicated putative binding sites for activator protein (AP) 2 and specificity protein (SP) 1 transcription factors, plus a CACCC box in PR1 and an AP1 site in PR2. PR1 produced four complexes in electrophoretic mobility-shift assay (EMSA) with HeLa nuclear extracts. Of these, three were completely competed with the SP1 and transforming growth factor-beta retinoblastoma control-element CACCC box (RCE) probes, whereas the AP2 probe competed against the same three complexes partially. Supershift EMSA identified SP1 in the complex 1 and SP3 in the complexes 2 and 4. Point mutations in the SP1 site abrogated the PR1 function, while mutations affecting the overlapping CACCC box/AP2 site in PR1 had minor effect on MN promoter activity. Block-replaced MN promoter mutants that had a consensus binding site (SP1 or AP2) or the RCE in place of PR1 demonstrated the stringent selectivity of the PR1 position as only the SP1 mutant reconstituted the MN promoter activity. The consensus SP1 probe generated the same SP1 and SP3 complexes as PR1 in EMSA; therefore we conclude that SP activity is both necessary and sufficient in the PR1 position. The critical role of AP1 in the PR2 position was confirmed by supershift of the PR2 complex with c-Fos antibody and markedly decreased activity of the construct with a mutated AP1 site. Detailed deletion analysis proved that PR1+PR2 account for 90% of the MN promoter activity, while neither PR1 nor PR2 on their own are sufficient for transactivation. Thus, synergistic co-operation between SP and AP1 factors bound to the adjacent PR1 and PR2, respectively, is necessary for MN transcriptional activity. The PR1+PR2 module also stimulated transcription from a heterologous promoter. The modulation of AP1 activity with PMA stimulated MN expression and activated the MN promoter, whereas inhibition of protein kinase C activity had no effect on MN expression in HeLa 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. Adam E., Kerkhofs P., Mammerickx M., Burny A., Kettmann R., Willems L. The CREB, ATF-1, and ATF-2 transcription factors from bovine leukemia virus-infected B lymphocytes activate viral expression. J Virol. 1996 Mar;70(3):1990–1999. doi: 10.1128/jvi.70.3.1990-1999.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Banks E. B., Crish J. F., Welter J. F., Eckert R. L. Characterization of human involucrin promoter distal regulatory region transcriptional activator elements-a role for Sp1 and AP1 binding sites. Biochem J. 1998 Apr 1;331(Pt 1):61–68. doi: 10.1042/bj3310061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bohmann D., Bos T. J., Admon A., Nishimura T., Vogt P. K., Tjian R. Human proto-oncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor AP-1. Science. 1987 Dec 4;238(4832):1386–1392. doi: 10.1126/science.2825349. [DOI] [PubMed] [Google Scholar]
  4. Boshart M., Klüppel M., Schmidt A., Schütz G., Luckow B. Reporter constructs with low background activity utilizing the cat gene. Gene. 1992 Jan 2;110(1):129–130. doi: 10.1016/0378-1119(92)90456-y. [DOI] [PubMed] [Google Scholar]
  5. Gutman A., Wasylyk B. The collagenase gene promoter contains a TPA and oncogene-responsive unit encompassing the PEA3 and AP-1 binding sites. EMBO J. 1990 Jul;9(7):2241–2246. doi: 10.1002/j.1460-2075.1990.tb07394.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hanson R. D., Grisolano J. L., Ley T. J. Consensus AP-1 and CRE motifs upstream from the human cytotoxic serine protease B (CSP-B/CGL-1) gene synergize to activate transcription. Blood. 1993 Nov 1;82(9):2749–2757. [PubMed] [Google Scholar]
  7. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ivanov S. V., Kuzmin I., Wei M. H., Pack S., Geil L., Johnson B. E., Stanbridge E. J., Lerman M. I. Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes. Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12596–12601. doi: 10.1073/pnas.95.21.12596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ivanov S., Liao S. Y., Ivanova A., Danilkovitch-Miagkova A., Tarasova N., Weirich G., Merrill M. J., Proescholdt M. A., Oldfield E. H., Lee J. Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer. Am J Pathol. 2001 Mar;158(3):905–919. doi: 10.1016/S0002-9440(10)64038-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Kaluz S., Kaluzová M., Opavský R., Pastoreková S., Gibadulinová A., Dequiedt F., Kettmann R., Pastorek J. Transcriptional regulation of the MN/CA 9 gene coding for the tumor-associated carbonic anhydrase IX. Identification and characterization of a proximal silencer element. J Biol Chem. 1999 Nov 12;274(46):32588–32595. doi: 10.1074/jbc.274.46.32588. [DOI] [PubMed] [Google Scholar]
  12. Kaluzová M., Pastoreková S., Pastorek J., Kaluz S. P53 tumour suppressor modulates transcription of the TATA-less gene coding for the tumour-associated carbonic anhydrase MN/CA IX in MaTu cells. Biochim Biophys Acta. 2000 Apr 25;1491(1-3):20–26. doi: 10.1016/s0167-4781(00)00015-4. [DOI] [PubMed] [Google Scholar]
  13. Kim S. J., Lee H. D., Robbins P. D., Busam K., Sporn M. B., Roberts A. B. Regulation of transforming growth factor beta 1 gene expression by the product of the retinoblastoma-susceptibility gene. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3052–3056. doi: 10.1073/pnas.88.8.3052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kivelä A. J., Parkkila S., Saarnio J., Karttunen T. J., Kivelä J., Parkkila A. K., Pastoreková S., Pastorek J., Waheed A., Sly W. S. Expression of transmembrane carbonic anhydrase isoenzymes IX and XII in normal human pancreas and pancreatic tumours. Histochem Cell Biol. 2000 Sep;114(3):197–204. doi: 10.1007/s004180000181. [DOI] [PubMed] [Google Scholar]
  15. Kwon H. S., Kim M. S., Edenberg H. J., Hur M. W. Sp3 and Sp4 can repress transcription by competing with Sp1 for the core cis-elements on the human ADH5/FDH minimal promoter. J Biol Chem. 1999 Jan 1;274(1):20–28. doi: 10.1074/jbc.274.1.20. [DOI] [PubMed] [Google Scholar]
  16. Lallemand D., Spyrou G., Yaniv M., Pfarr C. M. Variations in Jun and Fos protein expression and AP-1 activity in cycling, resting and stimulated fibroblasts. Oncogene. 1997 Feb 20;14(7):819–830. doi: 10.1038/sj.onc.1200901. [DOI] [PubMed] [Google Scholar]
  17. Liao S. Y., Aurelio O. N., Jan K., Zavada J., Stanbridge E. J. Identification of the MN/CA9 protein as a reliable diagnostic biomarker of clear cell carcinoma of the kidney. Cancer Res. 1997 Jul 15;57(14):2827–2831. [PubMed] [Google Scholar]
  18. Liao S. Y., Brewer C., Závada J., Pastorek J., Pastorekova S., Manetta A., Berman M. L., DiSaia P. J., Stanbridge E. J. Identification of the MN antigen as a diagnostic biomarker of cervical intraepithelial squamous and glandular neoplasia and cervical carcinomas. Am J Pathol. 1994 Sep;145(3):598–609. [PMC free article] [PubMed] [Google Scholar]
  19. Lieskovská J., Kaluzova M., Opavsky R., Kaluz S., Pastorek J., Kettmann R., Pastoreková S. Up-regulation of p53 by antisense expression of HPV18 E6 oncogene does not influence the level of MN/CA IX tumor-associated protein in HeLa cervical carcinoma cells. Int J Oncol. 1998 Nov;13(5):1081–1086. doi: 10.3892/ijo.13.5.1081. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Mukhopadhyay D., Knebelmann B., Cohen H. T., Ananth S., Sukhatme V. P. The von Hippel-Lindau tumor suppressor gene product interacts with Sp1 to repress vascular endothelial growth factor promoter activity. Mol Cell Biol. 1997 Sep;17(9):5629–5639. doi: 10.1128/mcb.17.9.5629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Opavský R., Pastoreková S., Zelník V., Gibadulinová A., Stanbridge E. J., Závada J., Kettmann R., Pastorek J. Human MN/CA9 gene, a novel member of the carbonic anhydrase family: structure and exon to protein domain relationships. Genomics. 1996 May 1;33(3):480–487. doi: 10.1006/geno.1996.0223. [DOI] [PubMed] [Google Scholar]
  23. Parkkila S., Rajaniemi H., Parkkila A. K., Kivela J., Waheed A., Pastorekova S., Pastorek J., Sly W. S. Carbonic anhydrase inhibitor suppresses invasion of renal cancer cells in vitro. Proc Natl Acad Sci U S A. 2000 Feb 29;97(5):2220–2224. doi: 10.1073/pnas.040554897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pastorek J., Pastoreková S., Callebaut I., Mornon J. P., Zelník V., Opavský R., Zat'ovicová M., Liao S., Portetelle D., Stanbridge E. J. Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment. Oncogene. 1994 Oct;9(10):2877–2888. [PubMed] [Google Scholar]
  25. Pastoreková S., Parkkila S., Parkkila A. K., Opavský R., Zelník V., Saarnio J., Pastorek J. Carbonic anhydrase IX, MN/CA IX: analysis of stomach complementary DNA sequence and expression in human and rat alimentary tracts. Gastroenterology. 1997 Feb;112(2):398–408. doi: 10.1053/gast.1997.v112.pm9024293. [DOI] [PubMed] [Google Scholar]
  26. Prestridge D. S. SIGNAL SCAN: a computer program that scans DNA sequences for eukaryotic transcriptional elements. Comput Appl Biosci. 1991 Apr;7(2):203–206. doi: 10.1093/bioinformatics/7.2.203. [DOI] [PubMed] [Google Scholar]
  27. Ptashne M., Gann A. Transcriptional activation by recruitment. Nature. 1997 Apr 10;386(6625):569–577. doi: 10.1038/386569a0. [DOI] [PubMed] [Google Scholar]
  28. Riabowol K., Schiff J., Gilman M. Z. Transcription factor AP-1 activity is required for initiation of DNA synthesis and is lost during cellular aging. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):157–161. doi: 10.1073/pnas.89.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Saarnio J., Parkkila S., Parkkila A. K., Haukipuro K., Pastoreková S., Pastorek J., Kairaluoma M. I., Karttunen T. J. Immunohistochemical study of colorectal tumors for expression of a novel transmembrane carbonic anhydrase, MN/CA IX, with potential value as a marker of cell proliferation. Am J Pathol. 1998 Jul;153(1):279–285. doi: 10.1016/S0002-9440(10)65569-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shi Q., Le X., Abbruzzese J. L., Peng Z., Qian C. N., Tang H., Xiong Q., Wang B., Li X. C., Xie K. Constitutive Sp1 activity is essential for differential constitutive expression of vascular endothelial growth factor in human pancreatic adenocarcinoma. Cancer Res. 2001 May 15;61(10):4143–4154. [PubMed] [Google Scholar]
  31. Sif S., Capobianco A. J., Gilmore T. D. The v-Rel oncoprotein increases expression from Sp1 site-containing promoters in chicken embryo fibroblasts. Oncogene. 1993 Sep;8(9):2501–2509. [PubMed] [Google Scholar]
  32. Sirum-Connolly K., Brinckerhoff C. E. Interleukin-1 or phorbol induction of the stromelysin promoter requires an element that cooperates with AP-1. Nucleic Acids Res. 1991 Jan 25;19(2):335–341. doi: 10.1093/nar/19.2.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Suske G. The Sp-family of transcription factors. Gene. 1999 Oct 1;238(2):291–300. doi: 10.1016/s0378-1119(99)00357-1. [DOI] [PubMed] [Google Scholar]
  34. Thomas R. S., Tymms M. J., McKinlay L. H., Shannon M. F., Seth A., Kola I. ETS1, NFkappaB and AP1 synergistically transactivate the human GM-CSF promoter. Oncogene. 1997 Jun 12;14(23):2845–2855. doi: 10.1038/sj.onc.1201125. [DOI] [PubMed] [Google Scholar]
  35. Tischer E., Mitchell R., Hartman T., Silva M., Gospodarowicz D., Fiddes J. C., Abraham J. A. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem. 1991 Jun 25;266(18):11947–11954. [PubMed] [Google Scholar]
  36. Toullec D., Pianetti P., Coste H., Bellevergue P., Grand-Perret T., Ajakane M., Baudet V., Boissin P., Boursier E., Loriolle F. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991 Aug 25;266(24):15771–15781. [PubMed] [Google Scholar]
  37. Turner J. R., Odze R. D., Crum C. P., Resnick M. B. MN antigen expression in normal, preneoplastic, and neoplastic esophagus: a clinicopathological study of a new cancer-associated biomarker. Hum Pathol. 1997 Jun;28(6):740–744. doi: 10.1016/s0046-8177(97)90185-4. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Vermylen P., Roufosse C., Burny A., Verhest A., Bosschaerts T., Pastorekova S., Ninane V., Sculier J. P. Carbonic anhydrase IX antigen differentiates between preneoplastic malignant lesions in non-small cell lung carcinoma. Eur Respir J. 1999 Oct;14(4):806–811. doi: 10.1034/j.1399-3003.1999.14d14.x. [DOI] [PubMed] [Google Scholar]
  40. Wang W. D., Gralla J. D. Differential ability of proximal and remote element pairs to cooperate in activating RNA polymerase II transcription. Mol Cell Biol. 1991 Sep;11(9):4561–4571. doi: 10.1128/mcb.11.9.4561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Williams T., Admon A., Lüscher B., Tjian R. Cloning and expression of AP-2, a cell-type-specific transcription factor that activates inducible enhancer elements. Genes Dev. 1988 Dec;2(12A):1557–1569. doi: 10.1101/gad.2.12a.1557. [DOI] [PubMed] [Google Scholar]
  42. Wykoff C. C., Beasley N. J., Watson P. H., Turner K. J., Pastorek J., Sibtain A., Wilson G. D., Turley H., Talks K. L., Maxwell P. H. Hypoxia-inducible expression of tumor-associated carbonic anhydrases. Cancer Res. 2000 Dec 15;60(24):7075–7083. [PubMed] [Google Scholar]
  43. Závada J., Závadová Z., Pastoreková S., Ciampor F., Pastorek J., Zelník V. Expression of MaTu-MN protein in human tumor cultures and in clinical specimens. Int J Cancer. 1993 May 8;54(2):268–274. doi: 10.1002/ijc.2910540218. [DOI] [PubMed] [Google Scholar]

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