Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Oct;15(10):5423–5433. doi: 10.1128/mcb.15.10.5423

G10BP, an E1A-inducible negative regulator of Sp1, represses transcription of the rat fibronectin gene.

M Suzuki 1, C Kuroda 1, E Oda 1, S Tsunoda 1, T Nakamura 1, T Nakajima 1, K Oda 1
PMCID: PMC230792  PMID: 7565693

Abstract

Downregulation of the fibronectin (FN) gene in a rat 3Y1 derivative cell line, XhoC, transformed by the adenovirus E1A and E1B genes seems to be caused by the induction of a negative regulator, G10BP, which binds to three G-rich sequences in the promoter (T. Nakamura, T. Nakajima, S. Tsunoda, S. Nakada, K. Oda, H. Tsurui, and A. Wada, J. Virol. 66:6436-6450, 1992). These are the G10 stretch and two GC boxes consisting of the G10 stretch with one internal C residue insertion. The recognition sequences of G10BP and Sp1 (GGGCGG) overlap in these GC boxes. To analyze the mechanism of the downregulation, G10BP was purified by DNA affinity chromatography, and its molecular mass was estimated to be about 30 kDa. The promoter was modified by substituting the sequence GGGG with ATCC or CTTA in these G-rich sequences, leaving the Sp1 motif intact, and by replacing the Sp1 motif by the T stretch. Transcription of FN promoter-chloramphenicol acetyltransferase fusion genes carrying the base substitution in one or more of these G-rich sequences both in vivo and in vitro revealed that the base substitution in any G-rich sequence results in reduction of promoter activity, although the downstream GC box (GCd) plays a primary role. The addition of G10BP severely inhibited the activities of the FN promoters carrying the wild-type GCd in vitro, while the promoters carrying the mutant GCd were unaffected. The binding affinity of G10BP and Sp1 to each of the G-rich sequences, analyzed by gel shift assays, indicated that G10BP binds strongly to the GCd, moderately to the G10 stretch, and weakly to GCu, while Sp1 binds strongly to GCu, moderately to GCd, and weakly to the G10 stretch. Sp1 binding to GCd and the G10 stretch was inhibited by G10BP, while binding to GCu was unaffected. These results indicate that FN gene transcription is inhibited in XhoC cells primarily by exclusion of Sp1 binding to GCd by G10BP and that G10BP is a new class of Sp1 negative regulator.

Full Text

The Full Text of this article is available as a PDF (727.8 KB).

Selected References

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

  1. Briggs M. R., Kadonaga J. T., Bell S. P., Tjian R. Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. Science. 1986 Oct 3;234(4772):47–52. doi: 10.1126/science.3529394. [DOI] [PubMed] [Google Scholar]
  2. Buck C. A., Horwitz A. F. Cell surface receptors for extracellular matrix molecules. Annu Rev Cell Biol. 1987;3:179–205. doi: 10.1146/annurev.cb.03.110187.001143. [DOI] [PubMed] [Google Scholar]
  3. Burger M. M. Proteolytic enzymes initiating cell division and escape from contact inhibition of growth. Nature. 1970 Jul 11;227(5254):170–171. doi: 10.1038/227170a0. [DOI] [PubMed] [Google Scholar]
  4. Burn P., Kupfer A., Singer S. J. Dynamic membrane-cytoskeletal interactions: specific association of integrin and talin arises in vivo after phorbol ester treatment of peripheral blood lymphocytes. Proc Natl Acad Sci U S A. 1988 Jan;85(2):497–501. doi: 10.1073/pnas.85.2.497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Darrow A. L., Rickles R. J., Pecorino L. T., Strickland S. Transcription factor Sp1 is important for retinoic acid-induced expression of the tissue plasminogen activator gene during F9 teratocarcinoma cell differentiation. Mol Cell Biol. 1990 Nov;10(11):5883–5893. doi: 10.1128/mcb.10.11.5883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DeCaprio J. A., Ludlow J. W., Figge J., Shew J. Y., Huang C. M., Lee W. H., Marsilio E., Paucha E., Livingston D. M. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell. 1988 Jul 15;54(2):275–283. doi: 10.1016/0092-8674(88)90559-4. [DOI] [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. Dynan W. S., Tjian R. Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II. Cell. 1983 Mar;32(3):669–680. doi: 10.1016/0092-8674(83)90053-3. [DOI] [PubMed] [Google Scholar]
  13. Fagan J. B., Sobel M. E., Yamada K. M., de Crombrugghe B., Pastan I. Effects of transformation on fibronectin gene expression using cloned fibronectin cDNA. J Biol Chem. 1981 Jan 10;256(1):520–525. [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Hamann L., Bayer K. U., Jensen K., Harbers K. Interaction of several related GC-box- and GT-box-binding proteins with the intronic enhancer is required for differential expression of the gb110 gene in embryonal carcinoma cells. Mol Cell Biol. 1994 Sep;14(9):5786–5793. doi: 10.1128/mcb.14.9.5786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hara E., Nakada S., Takehana K., Nakajima T., Iino T., Oda K. Molecular cloning and characterization of cellular genes whose expression is repressed by the adenovirus E1a gene products and growth factors in quiescent rat cells. Gene. 1988 Oct 15;70(1):97–106. doi: 10.1016/0378-1119(88)90108-4. [DOI] [PubMed] [Google Scholar]
  19. Herman B., Pledger W. J. Platelet-derived growth factor-induced alterations in vinculin and actin distribution in BALB/c-3T3 cells. J Cell Biol. 1985 Apr;100(4):1031–1040. doi: 10.1083/jcb.100.4.1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hirst R., Horwitz A., Buck C., Rohrschneider L. Phosphorylation of the fibronectin receptor complex in cells transformed by oncogenes that encode tyrosine kinases. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6470–6474. doi: 10.1073/pnas.83.17.6470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Horwitz A., Duggan K., Buck C., Beckerle M. C., Burridge K. Interaction of plasma membrane fibronectin receptor with talin--a transmembrane linkage. Nature. 1986 Apr 10;320(6062):531–533. doi: 10.1038/320531a0. [DOI] [PubMed] [Google Scholar]
  22. Hynes R. O. Integrins: a family of cell surface receptors. Cell. 1987 Feb 27;48(4):549–554. doi: 10.1016/0092-8674(87)90233-9. [DOI] [PubMed] [Google Scholar]
  23. Hynes R. O., Yamada K. M. Fibronectins: multifunctional modular glycoproteins. J Cell Biol. 1982 Nov;95(2 Pt 1):369–377. doi: 10.1083/jcb.95.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hynes R. Molecular biology of fibronectin. Annu Rev Cell Biol. 1985;1:67–90. doi: 10.1146/annurev.cb.01.110185.000435. [DOI] [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. Ishii T., Shimizu M., Kanayama Y., Nakada S., Nojima H., Oda K. Differential activation of cyclin and cyclin-dependent kinase genes by adenovirus E1A12S cDNA product. Exp Cell Res. 1993 Oct;208(2):407–414. doi: 10.1006/excr.1993.1262. [DOI] [PubMed] [Google Scholar]
  27. Jackson S. P., Tjian R. Purification and analysis of RNA polymerase II transcription factors by using wheat germ agglutinin affinity chromatography. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1781–1785. doi: 10.1073/pnas.86.6.1781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jochemsen A. G., Bernards R., van Kranen H. J., Houweling A., Bos J. L., van der Eb A. J. Different activities of the adenovirus types 5 and 12 E1A regions in transformation with the EJ Ha-ras oncogene. J Virol. 1986 Sep;59(3):684–691. doi: 10.1128/jvi.59.3.684-691.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. Kimura G., Itagaki A., Summers J. Rat cell line 3y1 and its virogenic polyoma- and sv40- transformed derivatives. Int J Cancer. 1975 Apr 15;15(4):694–706. doi: 10.1002/ijc.2910150419. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Kornblihtt A. R., Umezawa K., Vibe-Pedersen K., Baralle F. E. Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene. EMBO J. 1985 Jul;4(7):1755–1759. doi: 10.1002/j.1460-2075.1985.tb03847.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  36. Manley J. L., Fire A., Cano A., Sharp P. A., Gefter M. L. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855–3859. doi: 10.1073/pnas.77.7.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Matsuno K., Hui C. C., Takiya S., Suzuki T., Ueno K., Suzuki Y. Transcription signals and protein binding sites for sericin gene transcription in vitro. J Biol Chem. 1989 Nov 5;264(31):18707–18713. [PubMed] [Google Scholar]
  38. Nakajima T., Nakamura T., Tsunoda S., Nakada S., Oda K. E1A-responsive elements for repression of rat fibronectin gene transcription. Mol Cell Biol. 1992 Jun;12(6):2837–2846. doi: 10.1128/mcb.12.6.2837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nakamura T., Nakajima T., Tsunoda S., Nakada S., Oda K., Tsurui H., Wada A. Induction of E1A-responsive negative factors for transcription of the fibronectin gene in adenovirus E1-transformed rat cells. J Virol. 1992 Nov;66(11):6436–6450. doi: 10.1128/jvi.66.11.6436-6450.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Patel R. S., Odermatt E., Schwarzbauer J. E., Hynes R. O. Organization of the fibronectin gene provides evidence for exon shuffling during evolution. EMBO J. 1987 Sep;6(9):2565–2572. doi: 10.1002/j.1460-2075.1987.tb02545.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pietenpol J. A., Stein R. W., Moran E., Yaciuk P., Schlegel R., Lyons R. M., Pittelkow M. R., Münger K., Howley P. M., Moses H. L. TGF-beta 1 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains. Cell. 1990 Jun 1;61(5):777–785. doi: 10.1016/0092-8674(90)90188-k. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Ruoslahti E. Fibronectin and its receptors. Annu Rev Biochem. 1988;57:375–413. doi: 10.1146/annurev.bi.57.070188.002111. [DOI] [PubMed] [Google Scholar]
  45. Staudt L. M., Singh H., Sen R., Wirth T., Sharp P. A., Baltimore D. A lymphoid-specific protein binding to the octamer motif of immunoglobulin genes. Nature. 1986 Oct 16;323(6089):640–643. doi: 10.1038/323640a0. [DOI] [PubMed] [Google Scholar]
  46. Tamura T., Miura M., Ikenaka K., Mikoshiba K. Analysis of transcription control elements of the mouse myelin basic protein gene in HeLa cell extracts: demonstration of a strong NFI-binding motif in the upstream region. Nucleic Acids Res. 1988 Dec 23;16(24):11441–11459. doi: 10.1093/nar/16.24.11441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tokunaga K., Taniguchi H., Yoda K., Shimizu M., Sakiyama S. Nucleotide sequence of a full-length cDNA for mouse cytoskeletal beta-actin mRNA. Nucleic Acids Res. 1986 Mar 25;14(6):2829–2829. doi: 10.1093/nar/14.6.2829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Whyte P., Buchkovich K. J., Horowitz J. M., Friend S. H., Raybuck M., Weinberg R. A., Harlow E. Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature. 1988 Jul 14;334(6178):124–129. doi: 10.1038/334124a0. [DOI] [PubMed] [Google Scholar]
  49. Yamada K. M. Cell surface interactions with extracellular materials. Annu Rev Biochem. 1983;52:761–799. doi: 10.1146/annurev.bi.52.070183.003553. [DOI] [PubMed] [Google Scholar]
  50. Zerler B., Moran B., Maruyama K., Moomaw J., Grodzicker T., Ruley H. E. Adenovirus E1A coding sequences that enable ras and pmt oncogenes to transform cultured primary cells. Mol Cell Biol. 1986 Mar;6(3):887–899. doi: 10.1128/mcb.6.3.887. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES