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. 1994 Jul;14(7):4380–4389. doi: 10.1128/mcb.14.7.4380

The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator.

L I Chen 1, T Nishinaka 1, K Kwan 1, I Kitabayashi 1, K Yokoyama 1, Y H Fu 1, S Grünwald 1, R Chiu 1
PMCID: PMC358809  PMID: 8007947

Abstract

Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the Sp1 consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Sp1-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Sp1 complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of SP1. These results suggest that RB is directly or indirectly involved in Sp1-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Sp1 negative regulator(s) (Sp1-I) that specifically inhibits Sp1 binding to a c-jun Sp1 site. This inhibition is reversed by addition of recombinant RB proteins, suggesting that RB stimulates Sp1-mediated transactivation by liberating Sp1 from Sp1-I. Additional evidence for Sp1-I involvement in Sp1-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Sp1, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Sp1-I, a approximately 20-kDa protein(s) that inhibits the Sp1 complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.

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Selected References

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

  1. Angel P., Baumann I., Stein B., Delius H., Rahmsdorf H. J., Herrlich P. 12-O-tetradecanoyl-phorbol-13-acetate induction of the human collagenase gene is mediated by an inducible enhancer element located in the 5'-flanking region. Mol Cell Biol. 1987 Jun;7(6):2256–2266. doi: 10.1128/mcb.7.6.2256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Angel P., Imagawa M., Chiu R., Stein B., Imbra R. J., Rahmsdorf H. J., Jonat C., Herrlich P., Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987 Jun 19;49(6):729–739. doi: 10.1016/0092-8674(87)90611-8. [DOI] [PubMed] [Google Scholar]
  3. Bagchi S., Weinmann R., Raychaudhuri P. The retinoblastoma protein copurifies with E2F-I, an E1A-regulated inhibitor of the transcription factor E2F. Cell. 1991 Jun 14;65(6):1063–1072. doi: 10.1016/0092-8674(91)90558-g. [DOI] [PubMed] [Google Scholar]
  4. Bernards R., Shackleford G. M., Schackleford G. M., Gerber M. R., Horowitz J. M., Friend S. H., Schartl M., Bogenmann E., Rapaport J. M., McGee T. Structure and expression of the murine retinoblastoma gene and characterization of its encoded protein. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6474–6478. doi: 10.1073/pnas.86.17.6474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bookstein R., Rio P., Madreperla S. A., Hong F., Allred C., Grizzle W. E., Lee W. H. Promoter deletion and loss of retinoblastoma gene expression in human prostate carcinoma. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7762–7766. doi: 10.1073/pnas.87.19.7762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brenner D. A., O'Hara M., Angel P., Chojkier M., Karin M. Prolonged activation of jun and collagenase genes by tumour necrosis factor-alpha. Nature. 1989 Feb 16;337(6208):661–663. doi: 10.1038/337661a0. [DOI] [PubMed] [Google Scholar]
  7. Buchkovich K., Duffy L. A., Harlow E. The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell. 1989 Sep 22;58(6):1097–1105. doi: 10.1016/0092-8674(89)90508-4. [DOI] [PubMed] [Google Scholar]
  8. Chellappan S. P., Hiebert S., Mudryj M., Horowitz J. M., Nevins J. R. The E2F transcription factor is a cellular target for the RB protein. Cell. 1991 Jun 14;65(6):1053–1061. doi: 10.1016/0092-8674(91)90557-f. [DOI] [PubMed] [Google Scholar]
  9. Chen P. L., Chen Y., Shan B., Bookstein R., Lee W. H. Stability of retinoblastoma gene expression determines the tumorigenicity of reconstituted retinoblastoma cells. Cell Growth Differ. 1992 Feb;3(2):119–125. [PubMed] [Google Scholar]
  10. Chen P. L., Scully P., Shew J. Y., Wang J. Y., Lee W. H. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell. 1989 Sep 22;58(6):1193–1198. doi: 10.1016/0092-8674(89)90517-5. [DOI] [PubMed] [Google Scholar]
  11. Chiu R., Boyle W. J., Meek J., Smeal T., Hunter T., Karin M. The c-Fos protein interacts with c-Jun/AP-1 to stimulate transcription of AP-1 responsive genes. Cell. 1988 Aug 12;54(4):541–552. doi: 10.1016/0092-8674(88)90076-1. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. DeCaprio J. A., Ludlow J. W., Lynch D., Furukawa Y., Griffin J., Piwnica-Worms H., Huang C. M., Livingston D. M. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell. 1989 Sep 22;58(6):1085–1095. doi: 10.1016/0092-8674(89)90507-2. [DOI] [PubMed] [Google Scholar]
  14. Emami K. H., Carey M. A synergistic increase in potency of a multimerized VP16 transcriptional activation domain. EMBO J. 1992 Dec;11(13):5005–5012. doi: 10.1002/j.1460-2075.1992.tb05607.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Friend S. H., Bernards R., Rogelj S., Weinberg R. A., Rapaport J. M., Albert D. M., Dryja T. P. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature. 1986 Oct 16;323(6089):643–646. doi: 10.1038/323643a0. [DOI] [PubMed] [Google Scholar]
  16. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harbour J. W., Lai S. L., Whang-Peng J., Gazdar A. F., Minna J. D., Kaye F. J. Abnormalities in structure and expression of the human retinoblastoma gene in SCLC. Science. 1988 Jul 15;241(4863):353–357. doi: 10.1126/science.2838909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huang H. J., Yee J. K., Shew J. Y., Chen P. L., Bookstein R., Friedmann T., Lee E. Y., Lee W. H. Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science. 1988 Dec 16;242(4885):1563–1566. doi: 10.1126/science.3201247. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Kim S. J., Onwuta U. S., Lee Y. I., Li R., Botchan M. R., Robbins P. D. The retinoblastoma gene product regulates Sp1-mediated transcription. Mol Cell Biol. 1992 Jun;12(6):2455–2463. doi: 10.1128/mcb.12.6.2455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kim S. J., Wagner S., Liu F., O'Reilly M. A., Robbins P. D., Green M. R. Retinoblastoma gene product activates expression of the human TGF-beta 2 gene through transcription factor ATF-2. Nature. 1992 Jul 23;358(6384):331–334. doi: 10.1038/358331a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Kitabayashi I., Chiu R., Gachelin G., Yokoyama K. E1A dependent up-regulation of c-jun/AP-1 activity. Nucleic Acids Res. 1991 Feb 11;19(3):649–655. doi: 10.1093/nar/19.3.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kitabayashi I., Kawakami Z., Chiu R., Ozawa K., Matsuoka T., Toyoshima S., Umesono K., Evans R. M., Gachelin G., Yokoyama K. Transcriptional regulation of the c-jun gene by retinoic acid and E1A during differentiation of F9 cells. EMBO J. 1992 Jan;11(1):167–175. doi: 10.1002/j.1460-2075.1992.tb05039.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lamph W. W., Wamsley P., Sassone-Corsi P., Verma I. M. Induction of proto-oncogene JUN/AP-1 by serum and TPA. Nature. 1988 Aug 18;334(6183):629–631. doi: 10.1038/334629a0. [DOI] [PubMed] [Google Scholar]
  27. Li R., Knight J. D., Jackson S. P., Tjian R., Botchan M. R. Direct interaction between Sp1 and the BPV enhancer E2 protein mediates synergistic activation of transcription. Cell. 1991 May 3;65(3):493–505. doi: 10.1016/0092-8674(91)90467-d. [DOI] [PubMed] [Google Scholar]
  28. Lin B. T., Gruenwald S., Morla A. O., Lee W. H., Wang J. Y. Retinoblastoma cancer suppressor gene product is a substrate of the cell cycle regulator cdc2 kinase. EMBO J. 1991 Apr;10(4):857–864. doi: 10.1002/j.1460-2075.1991.tb08018.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ludlow J. W., Shon J., Pipas J. M., Livingston D. M., DeCaprio J. A. The retinoblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation and binding to and release from SV40 large T. Cell. 1990 Feb 9;60(3):387–396. doi: 10.1016/0092-8674(90)90590-b. [DOI] [PubMed] [Google Scholar]
  30. Muegge K., Williams T. M., Kant J., Karin M., Chiu R., Schmidt A., Siebenlist U., Young H. A., Durum S. K. Interleukin-1 costimulatory activity on the interleukin-2 promoter via AP-1. Science. 1989 Oct 13;246(4927):249–251. doi: 10.1126/science.2799385. [DOI] [PubMed] [Google Scholar]
  31. Pietenpol J. A., Münger K., Howley P. M., Stein R. W., Moses H. L. Factor-binding element in the human c-myc promoter involved in transcriptional regulation by transforming growth factor beta 1 and by the retinoblastoma gene product. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10227–10231. doi: 10.1073/pnas.88.22.10227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rauscher F. J., 3rd, Sambucetti L. C., Curran T., Distel R. J., Spiegelman B. M. Common DNA binding site for Fos protein complexes and transcription factor AP-1. Cell. 1988 Feb 12;52(3):471–480. doi: 10.1016/s0092-8674(88)80039-4. [DOI] [PubMed] [Google Scholar]
  33. Robbins P. D., Horowitz J. M., Mulligan R. C. Negative regulation of human c-fos expression by the retinoblastoma gene product. Nature. 1990 Aug 16;346(6285):668–671. doi: 10.1038/346668a0. [DOI] [PubMed] [Google Scholar]
  34. Ryder K., Nathans D. Induction of protooncogene c-jun by serum growth factors. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8464–8467. doi: 10.1073/pnas.85.22.8464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ryseck R. P., Hirai S. I., Yaniv M., Bravo R. Transcriptional activation of c-jun during the G0/G1 transition in mouse fibroblasts. Nature. 1988 Aug 11;334(6182):535–537. doi: 10.1038/334535a0. [DOI] [PubMed] [Google Scholar]
  36. Sadowski I., Ma J., Triezenberg S., Ptashne M. GAL4-VP16 is an unusually potent transcriptional activator. Nature. 1988 Oct 6;335(6190):563–564. doi: 10.1038/335563a0. [DOI] [PubMed] [Google Scholar]
  37. Sadowski I., Ptashne M. A vector for expressing GAL4(1-147) fusions in mammalian cells. Nucleic Acids Res. 1989 Sep 25;17(18):7539–7539. doi: 10.1093/nar/17.18.7539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Saffer J. D., Jackson S. P., Thurston S. J. SV40 stimulates expression of the transacting factor Sp1 at the mRNA level. Genes Dev. 1990 Apr;4(4):659–666. doi: 10.1101/gad.4.4.659. [DOI] [PubMed] [Google Scholar]
  39. Sakai T., Ohtani N., McGee T. L., Robbins P. D., Dryja T. P. Oncogenic germ-line mutations in Sp1 and ATF sites in the human retinoblastoma gene. Nature. 1991 Sep 5;353(6339):83–86. doi: 10.1038/353083a0. [DOI] [PubMed] [Google Scholar]
  40. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schütte J., Minna J. D., Birrer M. J. Deregulated expression of human c-jun transforms primary rat embryo cells in cooperation with an activated c-Ha-ras gene and transforms rat-1a cells as a single gene. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2257–2261. doi: 10.1073/pnas.86.7.2257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schütte J., Viallet J., Nau M., Segal S., Fedorko J., Minna J. jun-B inhibits and c-fos stimulates the transforming and trans-activating activities of c-jun. Cell. 1989 Dec 22;59(6):987–997. doi: 10.1016/0092-8674(89)90755-1. [DOI] [PubMed] [Google Scholar]
  43. T'Ang A., Varley J. M., Chakraborty S., Murphree A. L., Fung Y. K. Structural rearrangement of the retinoblastoma gene in human breast carcinoma. Science. 1988 Oct 14;242(4876):263–266. doi: 10.1126/science.3175651. [DOI] [PubMed] [Google Scholar]
  44. Toguchida J., Ishizaki K., Sasaki M. S., Ikenaga M., Sugimoto M., Kotoura Y., Yamamuro T. Chromosomal reorganization for the expression of recessive mutation of retinoblastoma susceptibility gene in the development of osteosarcoma. Cancer Res. 1988 Jul 15;48(14):3939–3943. [PubMed] [Google Scholar]
  45. 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]
  46. Udvadia A. J., Rogers K. T., Horowitz J. M. A common set of nuclear factors bind to promoter elements regulated by the retinoblastoma protein. Cell Growth Differ. 1992 Sep;3(9):597–608. [PubMed] [Google Scholar]
  47. Wang C. Y., Petryniak B., Thompson C. B., Kaelin W. G., Leiden J. M. Regulation of the Ets-related transcription factor Elf-1 by binding to the retinoblastoma protein. Science. 1993 May 28;260(5112):1330–1335. doi: 10.1126/science.8493578. [DOI] [PubMed] [Google Scholar]
  48. Weichselbaum R. R., Beckett M., Diamond A. Some retinoblastomas, osteosarcomas, and soft tissue sarcomas may share a common etiology. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2106–2109. doi: 10.1073/pnas.85.7.2106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yokota J., Akiyama T., Fung Y. K., Benedict W. F., Namba Y., Hanaoka M., Wada M., Terasaki T., Shimosato Y., Sugimura T. Altered expression of the retinoblastoma (RB) gene in small-cell carcinoma of the lung. Oncogene. 1988 Oct;3(4):471–475. [PubMed] [Google Scholar]
  50. Yu D., Matin A., Hung M. C. The retinoblastoma gene product suppresses neu oncogene-induced transformation via transcriptional repression of neu. J Biol Chem. 1992 May 25;267(15):10203–10206. [PubMed] [Google Scholar]

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