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. 1997 Nov 1;25(21):4314–4322. doi: 10.1093/nar/25.21.4314

Overlapping DNA recognition motifs between Sp1 and a novel trans-acting factor within the wt1 tumour suppressor gene promoter.

M T Discenza 1, M Dehbi 1, J Pelletier 1
PMCID: PMC147031  PMID: 9336463

Abstract

The Wilms' tumor suppressor gene, wt1 , encodes a zinc finger transcription factor which has been shown to regulate the expression of several genes involved in cellular proliferation and differentiation. Expression of wt1 is developmentally regulated and restricted to a small set of tissues which include the fetal urogenital system, mesothelium and spleen. A highly conserved motif within the wt1 promoter, located between nucleotides -34 and -71 relative to the first transcription start site in the murine promoter, harbors consensus binding sites for Sp1 and members of the paired-box transcription factor family. Pax-2 and Pax-8 are known to enhance expression of wt1 through this conserved regulatory element. In this report, we demonstrate that Sp1 is able to bind to two sites within the 38 bp conserved region (CR). By electrophoretic mobility shift assays (EMSAs), we have identified a novel binding activity, referred to as complex D, which recognizes sequences overlapping one of the Sp1 sites in the CR. EMSA competition experiments indicate that binding of complex D and Sp1 to the CR is mutually exclusive and Sp1 is able to displace complex D binding. In situ UV crosslinking and molecular mass determinations indicate that complex D is a complex of approximately 130 kDa, consisting of at least two proteins of approximately 62 and approximately 70 kDa. Transient transfections suggest that complex D may function as an activator.

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Selected References

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  1. Ackerman S. L., Minden A. G., Williams G. T., Bobonis C., Yeung C. Y. Functional significance of an overlapping consensus binding motif for Sp1 and Zif268 in the murine adenosine deaminase gene promoter. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7523–7527. doi: 10.1073/pnas.88.17.7523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ammendola R., Mesuraca M., Russo T., Cimino F. Sp1 DNA binding efficiency is highly reduced in nuclear extracts from aged rat tissues. J Biol Chem. 1992 Sep 5;267(25):17944–17948. [PubMed] [Google Scholar]
  3. Baeuerle P. A., Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor. Cell. 1988 Apr 22;53(2):211–217. doi: 10.1016/0092-8674(88)90382-0. [DOI] [PubMed] [Google Scholar]
  4. Bryan J. K. Molecular weights of protein multimers from polyacrylamide gel electrophoresis. Anal Biochem. 1977 Apr;78(2):513–519. doi: 10.1016/0003-2697(77)90111-7. [DOI] [PubMed] [Google Scholar]
  5. Chen L. I., Nishinaka T., Kwan K., Kitabayashi I., Yokoyama K., Fu Y. H., Grünwald S., Chiu R. The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator. Mol Cell Biol. 1994 Jul;14(7):4380–4389. doi: 10.1128/mcb.14.7.4380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chung D. C., Brand S. J., Tillotson L. G. Mutually exclusive interactions between factors binding to adjacent Sp1 and AT-rich elements regulate gastrin gene transcription in insulinoma cells. J Biol Chem. 1995 Apr 14;270(15):8829–8836. doi: 10.1074/jbc.270.15.8829. [DOI] [PubMed] [Google Scholar]
  7. Cohen H. T., Bossone S. A., Zhu G., McDonald G. A., Sukhatme V. P. Sp1 is a critical regulator of the Wilms' tumor-1 gene. J Biol Chem. 1997 Jan 31;272(5):2901–2913. doi: 10.1074/jbc.272.5.2901. [DOI] [PubMed] [Google Scholar]
  8. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  9. Dehbi M., Ghahremani M., Lechner M., Dressler G., Pelletier J. The paired-box transcription factor, PAX2, positively modulates expression of the Wilms' tumor suppressor gene (WT1). Oncogene. 1996 Aug 1;13(3):447–453. [PubMed] [Google Scholar]
  10. Dehbi M., Pelletier J. PAX8-mediated activation of the wt1 tumor suppressor gene. EMBO J. 1996 Aug 15;15(16):4297–4306. [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Fraizer G. C., Wu Y. J., Hewitt S. M., Maity T., Ton C. C., Huff V., Saunders G. F. Transcriptional regulation of the human Wilms' tumor gene (WT1). Cell type-specific enhancer and promiscuous promoter. J Biol Chem. 1994 Mar 25;269(12):8892–8900. [PubMed] [Google Scholar]
  13. 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]
  14. Hofmann W., Royer H. D., Drechsler M., Schneider S., Royer-Pokora B. Characterization of the transcriptional regulatory region of the human WT1 gene. Oncogene. 1993 Nov;8(11):3123–3132. [PubMed] [Google Scholar]
  15. Huang H. C., Sundseth R., Hansen U. Transcription factor LSF binds two variant bipartite sites within the SV40 late promoter. Genes Dev. 1990 Feb;4(2):287–298. doi: 10.1101/gad.4.2.287. [DOI] [PubMed] [Google Scholar]
  16. Jackson S. P., MacDonald J. J., Lees-Miller S., Tjian R. GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase. Cell. 1990 Oct 5;63(1):155–165. doi: 10.1016/0092-8674(90)90296-q. [DOI] [PubMed] [Google Scholar]
  17. Jackson S. P., Tjian R. O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation. Cell. 1988 Oct 7;55(1):125–133. doi: 10.1016/0092-8674(88)90015-3. [DOI] [PubMed] [Google Scholar]
  18. Khachigian L. M., Williams A. J., Collins T. Interplay of Sp1 and Egr-1 in the proximal platelet-derived growth factor A-chain promoter in cultured vascular endothelial cells. J Biol Chem. 1995 Nov 17;270(46):27679–27686. doi: 10.1074/jbc.270.46.27679. [DOI] [PubMed] [Google Scholar]
  19. Kreidberg J. A., Sariola H., Loring J. M., Maeda M., Pelletier J., Housman D., Jaenisch R. WT-1 is required for early kidney development. Cell. 1993 Aug 27;74(4):679–691. doi: 10.1016/0092-8674(93)90515-r. [DOI] [PubMed] [Google Scholar]
  20. Kriwacki R. W., Schultz S. C., Steitz T. A., Caradonna J. P. Sequence-specific recognition of DNA by zinc-finger peptides derived from the transcription factor Sp1. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9759–9763. doi: 10.1073/pnas.89.20.9759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leggett R. W., Armstrong S. A., Barry D., Mueller C. R. Sp1 is phosphorylated and its DNA binding activity down-regulated upon terminal differentiation of the liver. J Biol Chem. 1995 Oct 27;270(43):25879–25884. doi: 10.1074/jbc.270.43.25879. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Morris J. F., Madden S. L., Tournay O. E., Cook D. M., Sukhatme V. P., Rauscher F. J., 3rd Characterization of the zinc finger protein encoded by the WT1 Wilms' tumor locus. Oncogene. 1991 Dec;6(12):2339–2348. [PubMed] [Google Scholar]
  24. Mundlos S., Pelletier J., Darveau A., Bachmann M., Winterpacht A., Zabel B. Nuclear localization of the protein encoded by the Wilms' tumor gene WT1 in embryonic and adult tissues. Development. 1993 Dec;119(4):1329–1341. doi: 10.1242/dev.119.4.1329. [DOI] [PubMed] [Google Scholar]
  25. Murata Y., Kim H. G., Rogers K. T., Udvadia A. J., Horowitz J. M. Negative regulation of Sp1 trans-activation is correlated with the binding of cellular proteins to the amino terminus of the Sp1 trans-activation domain. J Biol Chem. 1994 Aug 12;269(32):20674–20681. [PubMed] [Google Scholar]
  26. Nehls M. C., Rippe R. A., Veloz L., Brenner D. A. Transcription factors nuclear factor I and Sp1 interact with the murine collagen alpha 1 (I) promoter. Mol Cell Biol. 1991 Aug;11(8):4065–4073. doi: 10.1128/mcb.11.8.4065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pelletier J., Bruening W., Kashtan C. E., Mauer S. M., Manivel J. C., Striegel J. E., Houghton D. C., Junien C., Habib R., Fouser L. Germline mutations in the Wilms' tumor suppressor gene are associated with abnormal urogenital development in Denys-Drash syndrome. Cell. 1991 Oct 18;67(2):437–447. doi: 10.1016/0092-8674(91)90194-4. [DOI] [PubMed] [Google Scholar]
  28. Pelletier J., Schalling M., Buckler A. J., Rogers A., Haber D. A., Housman D. Expression of the Wilms' tumor gene WT1 in the murine urogenital system. Genes Dev. 1991 Aug;5(8):1345–1356. doi: 10.1101/gad.5.8.1345. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Pritchard-Jones K., Fleming S., Davidson D., Bickmore W., Porteous D., Gosden C., Bard J., Buckler A., Pelletier J., Housman D. The candidate Wilms' tumour gene is involved in genitourinary development. Nature. 1990 Jul 12;346(6280):194–197. doi: 10.1038/346194a0. [DOI] [PubMed] [Google Scholar]
  31. Reddy J. C., Licht J. D. The WT1 Wilms' tumor suppressor gene: how much do we really know? Biochim Biophys Acta. 1996 May 16;1287(1):1–28. doi: 10.1016/0304-419x(95)00014-7. [DOI] [PubMed] [Google Scholar]
  32. Saffer J. D., Jackson S. P., Annarella M. B. Developmental expression of Sp1 in the mouse. Mol Cell Biol. 1991 Apr;11(4):2189–2199. doi: 10.1128/mcb.11.4.2189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schaufele F., West B. L., Reudelhuber T. L. Overlapping Pit-1 and Sp1 binding sites are both essential to full rat growth hormone gene promoter activity despite mutually exclusive Pit-1 and Sp1 binding. J Biol Chem. 1990 Oct 5;265(28):17189–17196. [PubMed] [Google Scholar]
  34. Shirra M. K., Zhu Q., Huang H. C., Pallas D., Hansen U. One exon of the human LSF gene includes conserved regions involved in novel DNA-binding and dimerization motifs. Mol Cell Biol. 1994 Aug;14(8):5076–5087. doi: 10.1128/mcb.14.8.5076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wu Y., Fraizer G. C., Saunders G. F. GATA-1 transactivates the WT1 hematopoietic specific enhancer. J Biol Chem. 1995 Mar 17;270(11):5944–5949. doi: 10.1074/jbc.270.11.5944. [DOI] [PubMed] [Google Scholar]

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