Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1994 Jun;14(6):4116–4125. doi: 10.1128/mcb.14.6.4116

Participation of Ets transcription factors in the glucocorticoid response of the rat tyrosine aminotransferase gene.

M L Espinás 1, J Roux 1, J Ghysdael 1, R Pictet 1, T Grange 1
PMCID: PMC358777  PMID: 7910945

Abstract

We have previously shown that two remote glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase (TAT) gene contain multiple binding sites for several transcription factor families, including the glucocorticoid receptor (GR). We report here the identification of two novel binding sites for members of the Ets family of transcription factors in one of these GRUs. One of these binding sites overlaps the major GR-binding site (GRBS), whereas the other is located in its vicinity. Inactivation of the latter binding site leads to a twofold reduction of the glucocorticoid response, whereas inactivation of the site overlapping the GRBS has no detectable effect. In vivo footprinting analysis reveals that the active site is occupied in a glucocorticoid-independent manner, in a TAT-expressing cell line, even though it is located at a position where there is a glucocorticoid-dependent alteration of the nucleosomal structure. This same site is not occupied in a cell line that does not express TAT but expresses Ets-related DNA-binding activities, suggesting the existence of an inhibitory effect of chromatin structure at a hierarchical level above the nucleosome. The inactive Ets-binding site that overlaps the GRBS is not occupied even in TAT-expressing cells. However, this same overlapping site can confer Ets-dependent stimulation of both basal and glucocorticoid-induced levels when it is isolated from the GRU and duplicated. Ets-1 expression in COS cells mimics the activity of the Ets-related activities present in hepatoma cells. These Ets-binding sites could participate in the integration of the glucocorticoid response of the TAT gene with signal transduction pathways triggered by other nonsteroidal extracellular stimuli.

Full text

PDF
4116

Images in this article

Selected References

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

  1. Ahearn J. M., Jr, Bartolomei M. S., West M. L., Cisek L. J., Corden J. L. Cloning and sequence analysis of the mouse genomic locus encoding the largest subunit of RNA polymerase II. J Biol Chem. 1987 Aug 5;262(22):10695–10705. [PubMed] [Google Scholar]
  2. Archer T. K., Lefebvre P., Wolford R. G., Hager G. L. Transcription factor loading on the MMTV promoter: a bimodal mechanism for promoter activation. Science. 1992 Mar 20;255(5051):1573–1576. doi: 10.1126/science.1347958. [DOI] [PubMed] [Google Scholar]
  3. Beato M. Gene regulation by steroid hormones. Cell. 1989 Feb 10;56(3):335–344. doi: 10.1016/0092-8674(89)90237-7. [DOI] [PubMed] [Google Scholar]
  4. Becker P. B., Gloss B., Schmid W., Strähle U., Schütz G. In vivo protein-DNA interactions in a glucocorticoid response element require the presence of the hormone. Nature. 1986 Dec 18;324(6098):686–688. doi: 10.1038/324686a0. [DOI] [PubMed] [Google Scholar]
  5. Becker P., Renkawitz R., Schütz G. Tissue-specific DNaseI hypersensitive sites in the 5'-flanking sequences of the tryptophan oxygenase and the tyrosine aminotransferase genes. EMBO J. 1984 Sep;3(9):2015–2020. doi: 10.1002/j.1460-2075.1984.tb02084.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ben-David Y., Giddens E. B., Letwin K., Bernstein A. Erythroleukemia induction by Friend murine leukemia virus: insertional activation of a new member of the ets gene family, Fli-1, closely linked to c-ets-1. Genes Dev. 1991 Jun;5(6):908–918. doi: 10.1101/gad.5.6.908. [DOI] [PubMed] [Google Scholar]
  7. Bhat N. K., Fisher R. J., Fujiwara S., Ascione R., Papas T. S. Temporal and tissue-specific expression of mouse ets genes. Proc Natl Acad Sci U S A. 1987 May;84(10):3161–3165. doi: 10.1073/pnas.84.10.3161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bonifer C., Hecht A., Saueressig H., Winter D. M., Sippel A. E. Dynamic chromatin: the regulatory domain organization of eukaryotic gene loci. J Cell Biochem. 1991 Oct;47(2):99–108. doi: 10.1002/jcb.240470203. [DOI] [PubMed] [Google Scholar]
  9. Bosselut R., Duvall J. F., Gégonne A., Bailly M., Hémar A., Brady J., Ghysdael J. The product of the c-ets-1 proto-oncogene and the related Ets2 protein act as transcriptional activators of the long terminal repeat of human T cell leukemia virus HTLV-1. EMBO J. 1990 Oct;9(10):3137–3144. doi: 10.1002/j.1460-2075.1990.tb07511.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Boulukos K. E., Pognonec P., Begue A., Galibert F., Gesquière J. C., Stéhelin D., Ghysdael J. Identification in chickens of an evolutionarily conserved cellular ets-2 gene (c-ets-2) encoding nuclear proteins related to the products of the c-ets proto-oncogene. EMBO J. 1988 Mar;7(3):697–705. doi: 10.1002/j.1460-2075.1988.tb02865.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Boulukos K. E., Pognonec P., Sariban E., Bailly M., Lagrou C., Ghysdael J. Rapid and transient expression of Ets2 in mature macrophages following stimulation with cMGF, LPS, and PKC activators. Genes Dev. 1990 Mar;4(3):401–409. doi: 10.1101/gad.4.3.401. [DOI] [PubMed] [Google Scholar]
  12. Brüggemeier U., Rogge L., Winnacker E. L., Beato M. Nuclear factor I acts as a transcription factor on the MMTV promoter but competes with steroid hormone receptors for DNA binding. EMBO J. 1990 Jul;9(7):2233–2239. doi: 10.1002/j.1460-2075.1990.tb07393.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Carr K. D., Richard-Foy H. Glucocorticoids locally disrupt an array of positioned nucleosomes on the rat tyrosine aminotransferase promoter in hepatoma cells. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9300–9304. doi: 10.1073/pnas.87.23.9300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Carson-Jurica M. A., Schrader W. T., O'Malley B. W. Steroid receptor family: structure and functions. Endocr Rev. 1990 May;11(2):201–220. doi: 10.1210/edrv-11-2-201. [DOI] [PubMed] [Google Scholar]
  15. Cordingley M. G., Hager G. L. Binding of multiple factors to the MMTV promoter in crude and fractionated nuclear extracts. Nucleic Acids Res. 1988 Jan 25;16(2):609–628. doi: 10.1093/nar/16.2.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Danesch U., Gloss B., Schmid W., Schütz G., Schüle R., Renkawitz R. Glucocorticoid induction of the rat tryptophan oxygenase gene is mediated by two widely separated glucocorticoid-responsive elements. EMBO J. 1987 Mar;6(3):625–630. doi: 10.1002/j.1460-2075.1987.tb04800.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fujiwara S., Fisher R. J., Bhat N. K., Diaz de la Espina S. M., Papas T. S. A short-lived nuclear phosphoprotein encoded by the human ets-2 proto-oncogene is stabilized by activation of protein kinase C. Mol Cell Biol. 1988 Nov;8(11):4700–4706. doi: 10.1128/mcb.8.11.4700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fujiwara S., Koizumi S., Fisher R. J., Bhat N. K., Papas T. S. Phosphorylation of the ETS-2 protein: regulation by the T-cell antigen receptor-CD3 complex. Mol Cell Biol. 1990 Mar;10(3):1249–1253. doi: 10.1128/mcb.10.3.1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Garrity P. A., Wold B. J. Effects of different DNA polymerases in ligation-mediated PCR: enhanced genomic sequencing and in vivo footprinting. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):1021–1025. doi: 10.1073/pnas.89.3.1021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Grange T., Roux J., Rigaud G., Pictet R. Cell-type specific activity of two glucocorticoid responsive units of rat tyrosine aminotransferase gene is associated with multiple binding sites for C/EBP and a novel liver-specific nuclear factor. Nucleic Acids Res. 1991 Jan 11;19(1):131–139. doi: 10.1093/nar/19.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Grange T., Roux J., Rigaud G., Pictet R. Two remote glucocorticoid responsive units interact cooperatively to promote glucocorticoid induction of rat tyrosine aminotransferase gene expression. Nucleic Acids Res. 1989 Nov 11;17(21):8695–8709. doi: 10.1093/nar/17.21.8695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Granner D. K., Hargrove J. L. Regulation of the synthesis of tyrosine aminotransferase: the relationship to mRNATAT. Mol Cell Biochem. 1983;53-54(1-2):113–128. doi: 10.1007/BF00225249. [DOI] [PubMed] [Google Scholar]
  23. Green S., Chambon P. Nuclear receptors enhance our understanding of transcription regulation. Trends Genet. 1988 Nov;4(11):309–314. doi: 10.1016/0168-9525(88)90108-4. [DOI] [PubMed] [Google Scholar]
  24. Gutman A., Wasylyk B. Nuclear targets for transcription regulation by oncogenes. Trends Genet. 1991 Feb;7(2):49–54. doi: 10.1016/0168-9525(91)90231-E. [DOI] [PubMed] [Google Scholar]
  25. Hagman J., Grosschedl R. An inhibitory carboxyl-terminal domain in Ets-1 and Ets-2 mediates differential binding of ETS family factors to promoter sequences of the mb-1 gene. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8889–8893. doi: 10.1073/pnas.89.19.8889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Herbst R. S., Nielsch U., Sladek F., Lai E., Babiss L. E., Darnell J. E., Jr Differential regulation of hepatocyte-enriched transcription factors explains changes in albumin and transthyretin gene expression among hepatoma cells. New Biol. 1991 Mar;3(3):289–296. [PubMed] [Google Scholar]
  27. Imai E., Stromstedt P. E., Quinn P. G., Carlstedt-Duke J., Gustafsson J. A., Granner D. K. Characterization of a complex glucocorticoid response unit in the phosphoenolpyruvate carboxykinase gene. Mol Cell Biol. 1990 Sep;10(9):4712–4719. doi: 10.1128/mcb.10.9.4712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jantzen H. M., Strähle U., Gloss B., Stewart F., Schmid W., Boshart M., Miksicek R., Schütz G. Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransferase gene. Cell. 1987 Apr 10;49(1):29–38. doi: 10.1016/0092-8674(87)90752-5. [DOI] [PubMed] [Google Scholar]
  29. Kumar V., Chambon P. The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell. 1988 Oct 7;55(1):145–156. doi: 10.1016/0092-8674(88)90017-7. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. LaMarco K., Thompson C. C., Byers B. P., Walton E. M., McKnight S. L. Identification of Ets- and notch-related subunits in GA binding protein. Science. 1991 Aug 16;253(5021):789–792. doi: 10.1126/science.1876836. [DOI] [PubMed] [Google Scholar]
  32. Lemaigre F. P., Lafontaine D. A., Courtois S. J., Durviaux S. M., Rousseau G. G. Sp1 can displace GHF-1 from its distal binding site and stimulate transcription from the growth hormone gene promoter. Mol Cell Biol. 1990 Apr;10(4):1811–1814. doi: 10.1128/mcb.10.4.1811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lim F., Kraut N., Framptom J., Graf T. DNA binding by c-Ets-1, but not v-Ets, is repressed by an intramolecular mechanism. EMBO J. 1992 Feb;11(2):643–652. doi: 10.1002/j.1460-2075.1992.tb05096.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Luisi B. F., Xu W. X., Otwinowski Z., Freedman L. P., Yamamoto K. R., Sigler P. B. Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature. 1991 Aug 8;352(6335):497–505. doi: 10.1038/352497a0. [DOI] [PubMed] [Google Scholar]
  35. Macleod K., Leprince D., Stehelin D. The ets gene family. Trends Biochem Sci. 1992 Jul;17(7):251–256. doi: 10.1016/0968-0004(92)90404-w. [DOI] [PubMed] [Google Scholar]
  36. Marais R., Wynne J., Treisman R. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell. 1993 Apr 23;73(2):381–393. doi: 10.1016/0092-8674(93)90237-k. [DOI] [PubMed] [Google Scholar]
  37. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  38. Miner J. N., Yamamoto K. R. Regulatory crosstalk at composite response elements. Trends Biochem Sci. 1991 Nov;16(11):423–426. doi: 10.1016/0968-0004(91)90168-u. [DOI] [PubMed] [Google Scholar]
  39. Mueller P. R., Wold B. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science. 1989 Nov 10;246(4931):780–786. doi: 10.1126/science.2814500. [DOI] [PubMed] [Google Scholar]
  40. Nye J. A., Petersen J. M., Gunther C. V., Jonsen M. D., Graves B. J. Interaction of murine ets-1 with GGA-binding sites establishes the ETS domain as a new DNA-binding motif. Genes Dev. 1992 Jun;6(6):975–990. doi: 10.1101/gad.6.6.975. [DOI] [PubMed] [Google Scholar]
  41. Oddos J., Grange T., Carr K. D., Matthews B., Roux J., Richard-Foy H., Pictet R. Nucleotide sequence of 10 kilobases of rat tyrosine aminotransferase gene 5' flanking region. Nucleic Acids Res. 1989 Nov 11;17(21):8877–8878. doi: 10.1093/nar/17.21.8877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. PITOT H. C., PERAINO C., MORSE P. A., Jr, POTTER V. R. HEPATOMAS IN TISSUE CULTURE COMPARED WITH ADAPTING LIVER IN VIVO. Natl Cancer Inst Monogr. 1964 Apr;13:229–245. [PubMed] [Google Scholar]
  43. Pearce D., Yamamoto K. R. Mineralocorticoid and glucocorticoid receptor activities distinguished by nonreceptor factors at a composite response element. Science. 1993 Feb 19;259(5098):1161–1165. doi: 10.1126/science.8382376. [DOI] [PubMed] [Google Scholar]
  44. Pognonec P., Boulukos K. E., Gesquière J. C., Stéhelin D., Ghysdael J. Mitogenic stimulation of thymocytes results in the calcium-dependent phosphorylation of c-ets-1 proteins. EMBO J. 1988 Apr;7(4):977–983. doi: 10.1002/j.1460-2075.1988.tb02904.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rao V. N., Huebner K., Isobe M., ar-Rushdi A., Croce C. M., Reddy E. S. elk, tissue-specific ets-related genes on chromosomes X and 14 near translocation breakpoints. Science. 1989 Apr 7;244(4900):66–70. doi: 10.1126/science.2539641. [DOI] [PubMed] [Google Scholar]
  46. Reik A., Schütz G., Stewart A. F. Glucocorticoids are required for establishment and maintenance of an alteration in chromatin structure: induction leads to a reversible disruption of nucleosomes over an enhancer. EMBO J. 1991 Sep;10(9):2569–2576. doi: 10.1002/j.1460-2075.1991.tb07797.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Renkawitz R. Transcriptional repression in eukaryotes. Trends Genet. 1990 Jun;6(6):192–197. doi: 10.1016/0168-9525(90)90176-7. [DOI] [PubMed] [Google Scholar]
  48. Rigaud G., Roux J., Pictet R., Grange T. In vivo footprinting of rat TAT gene: dynamic interplay between the glucocorticoid receptor and a liver-specific factor. Cell. 1991 Nov 29;67(5):977–986. doi: 10.1016/0092-8674(91)90370-e. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Schmid W., Strähle U., Schütz G., Schmitt J., Stunnenberg H. Glucocorticoid receptor binds cooperatively to adjacent recognition sites. EMBO J. 1989 Aug;8(8):2257–2263. doi: 10.1002/j.1460-2075.1989.tb08350.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schüle R., Muller M., Kaltschmidt C., Renkawitz R. Many transcription factors interact synergistically with steroid receptors. Science. 1988 Dec 9;242(4884):1418–1420. doi: 10.1126/science.3201230. [DOI] [PubMed] [Google Scholar]
  52. Schüle R., Muller M., Otsuka-Murakami H., Renkawitz R. Cooperativity of the glucocorticoid receptor and the CACCC-box binding factor. Nature. 1988 Mar 3;332(6159):87–90. doi: 10.1038/332087a0. [DOI] [PubMed] [Google Scholar]
  53. Seif R., Cuzin F. Temperature-sensitive growth regulation in one type of transformed rat cells induced by the tsa mutant of polyoma virus. J Virol. 1977 Dec;24(3):721–728. doi: 10.1128/jvi.24.3.721-728.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Simons S. S., Jr, Schleenbaker R. E., Eisen H. J. Activation of covalent affinity labeled glucocorticoid receptor-steroid complexes. J Biol Chem. 1983 Feb 25;258(4):2229–2238. [PubMed] [Google Scholar]
  55. Strähle U., Schmid W., Schütz G. Synergistic action of the glucocorticoid receptor with transcription factors. EMBO J. 1988 Nov;7(11):3389–3395. doi: 10.1002/j.1460-2075.1988.tb03212.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Urness L. D., Thummel C. S. Molecular interactions within the ecdysone regulatory hierarchy: DNA binding properties of the Drosophila ecdysone-inducible E74A protein. Cell. 1990 Oct 5;63(1):47–61. doi: 10.1016/0092-8674(90)90287-o. [DOI] [PubMed] [Google Scholar]
  57. Wasylyk B., Hahn S. L., Giovane A. The Ets family of transcription factors. Eur J Biochem. 1993 Jan 15;211(1-2):7–18. doi: 10.1007/978-3-642-78757-7_2. [DOI] [PubMed] [Google Scholar]
  58. Wasylyk B., Wasylyk C., Flores P., Begue A., Leprince D., Stehelin D. The c-ets proto-oncogenes encode transcription factors that cooperate with c-Fos and c-Jun for transcriptional activation. Nature. 1990 Jul 12;346(6280):191–193. doi: 10.1038/346191a0. [DOI] [PubMed] [Google Scholar]
  59. Wasylyk C., Flores P., Gutman A., Wasylyk B. PEA3 is a nuclear target for transcription activation by non-nuclear oncogenes. EMBO J. 1989 Nov;8(11):3371–3378. doi: 10.1002/j.1460-2075.1989.tb08500.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wasylyk C., Gutman A., Nicholson R., Wasylyk B. The c-Ets oncoprotein activates the stromelysin promoter through the same elements as several non-nuclear oncoproteins. EMBO J. 1991 May;10(5):1127–1134. doi: 10.1002/j.1460-2075.1991.tb08053.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wasylyk C., Kerckaert J. P., Wasylyk B. A novel modulator domain of Ets transcription factors. Genes Dev. 1992 Jun;6(6):965–974. doi: 10.1101/gad.6.6.965. [DOI] [PubMed] [Google Scholar]
  62. Weih F., Nitsch D., Reik A., Schütz G., Becker P. B. Analysis of CpG methylation and genomic footprinting at the tyrosine aminotransferase gene: DNA methylation alone is not sufficient to prevent protein binding in vivo. EMBO J. 1991 Sep;10(9):2559–2567. doi: 10.1002/j.1460-2075.1991.tb07796.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Woods D. B., Ghysdael J., Owen M. J. Identification of nucleotide preferences in DNA sequences recognised specifically by c-Ets-1 protein. Nucleic Acids Res. 1992 Feb 25;20(4):699–704. doi: 10.1093/nar/20.4.699. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES