Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1988 Sep;8(9):3872–3881. doi: 10.1128/mcb.8.9.3872

Negative regulation of transcription in vitro by a glucocorticoid response element is mediated by a trans-acting factor.

S J Langer 1, M C Ostrowski 1
PMCID: PMC365446  PMID: 2851730

Abstract

In vitro experiments with cell extracts prepared from a mouse mammary epithelial cell line demonstrated that a cis-acting glucocorticoid response element (GRE) of the mouse mammary tumor virus represses transcription from its homologous promoter. Competition transcription experiments, in which a molar excess of a restriction fragment that contains the GRE is added to the cell-free assay, revealed that a nuclear factor mediates in trans the negative regulation of mammary tumor virus transcription in vitro. Gel retention assays indicated that a factor in the extracts specifically recognizes the GRE. One unusual result of the gel retention studies was that heating the GRE probe to 65 degrees C before addition to a binding assay increases the formation of the specific protein-DNA complex 20-fold. Exonuclease III footprinting demonstrated that the sequences recognized by the factor are identical for either untreated or heat-treated probe. The footprinting also demonstrated that this factor recognizes sequences that are distinct from those recognized by the glucocorticoid receptor. A synthetic oligonucleotide based on the sequences identified by the footprinting experiments repressed the activity of a heterologous enhancer-promoter in vivo, as assayed by transient expression assays. We propose that this negative transcription element may control the basal level of expression of some glucocorticoid-modulated genes and may explain the insensitivity of certain tumor cells to steroid hormone action.

Full text

PDF
3872

Images in this article

3875Image
on p.3875
3876Image
on p.3876
3876Image
on p.3876
3877Image
on p.3877
3878Image
on p.3878

Selected References

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

  1. Berger J., Hauber J., Hauber R., Geiger R., Cullen B. R. Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene. 1988 Jun 15;66(1):1–10. doi: 10.1016/0378-1119(88)90219-3. [DOI] [PubMed] [Google Scholar]
  2. Borrelli E., Hen R., Chambon P. Adenovirus-2 E1A products repress enhancer-induced stimulation of transcription. Nature. 1984 Dec 13;312(5995):608–612. doi: 10.1038/312608a0. [DOI] [PubMed] [Google Scholar]
  3. Buetti E., Kühnel B. Distinct sequence elements involved in the glucocorticoid regulation of the mouse mammary tumor virus promoter identified by linker scanning mutagenesis. J Mol Biol. 1986 Aug 5;190(3):379–389. doi: 10.1016/0022-2836(86)90009-4. [DOI] [PubMed] [Google Scholar]
  4. Chandler V. L., Maler B. A., Yamamoto K. R. DNA sequences bound specifically by glucocorticoid receptor in vitro render a heterologous promoter hormone responsive in vivo. Cell. 1983 Jun;33(2):489–499. doi: 10.1016/0092-8674(83)90430-0. [DOI] [PubMed] [Google Scholar]
  5. Charnay P., Mellon P., Maniatis T. Linker scanning mutagenesis of the 5'-flanking region of the mouse beta-major-globin gene: sequence requirements for transcription in erythroid and nonerythroid cells. Mol Cell Biol. 1985 Jun;5(6):1498–1511. doi: 10.1128/mcb.5.6.1498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen E. Y., Howley P. M., Levinson A. D., Seeburg P. H. The primary structure and genetic organization of the bovine papillomavirus type 1 genome. Nature. 1982 Oct 7;299(5883):529–534. doi: 10.1038/299529a0. [DOI] [PubMed] [Google Scholar]
  7. Cordingley M. G., Riegel A. T., Hager G. L. Steroid-dependent interaction of transcription factors with the inducible promoter of mouse mammary tumor virus in vivo. Cell. 1987 Jan 30;48(2):261–270. doi: 10.1016/0092-8674(87)90429-6. [DOI] [PubMed] [Google Scholar]
  8. Darbre P. D., King R. J. Progression to steroid insensitivity can occur irrespective of the presence of functional steroid receptors. Cell. 1987 Nov 20;51(4):521–528. doi: 10.1016/0092-8674(87)90121-8. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goodbourn S., Burstein H., Maniatis T. The human beta-interferon gene enhancer is under negative control. Cell. 1986 May 23;45(4):601–610. doi: 10.1016/0092-8674(86)90292-8. [DOI] [PubMed] [Google Scholar]
  12. Greenberg M. E., Ziff E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. doi: 10.1038/311433a0. [DOI] [PubMed] [Google Scholar]
  13. Hager G. L., Richard-Foy H., Kessel M., Wheeler D., Lichtler A. C., Ostrowski M. C. The mouse mammary tumor virus model in studies of glucocorticoid regulation. Recent Prog Horm Res. 1984;40:121–142. doi: 10.1016/b978-0-12-571140-1.50008-6. [DOI] [PubMed] [Google Scholar]
  14. Hager L. J., Palmiter R. D. Transcriptional regulation of mouse liver metallothionein-I gene by glucocorticoids. Nature. 1981 May 28;291(5813):340–342. doi: 10.1038/291340a0. [DOI] [PubMed] [Google Scholar]
  15. Huang A. L., Ostrowski M. C., Berard D., Hager G. L. Glucocorticoid regulation of the Ha-MuSV p21 gene conferred by sequences from mouse mammary tumor virus. Cell. 1981 Dec;27(2 Pt 1):245–255. doi: 10.1016/0092-8674(81)90408-6. [DOI] [PubMed] [Google Scholar]
  16. Imler J. L., Lemaire C., Wasylyk C., Wasylyk B. Negative regulation contributes to tissue specificity of the immunoglobulin heavy-chain enhancer. Mol Cell Biol. 1987 Jul;7(7):2558–2567. doi: 10.1128/mcb.7.7.2558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Jones K. A., Kadonaga J. T., Rosenfeld P. J., Kelly T. J., Tjian R. A cellular DNA-binding protein that activates eukaryotic transcription and DNA replication. Cell. 1987 Jan 16;48(1):79–89. doi: 10.1016/0092-8674(87)90358-8. [DOI] [PubMed] [Google Scholar]
  19. Luciw P. A., Bishop J. M., Varmus H. E., Capecchi M. R. Location and function of retroviral and SV40 sequences that enhance biochemical transformation after microinjection of DNA. Cell. 1983 Jul;33(3):705–716. doi: 10.1016/0092-8674(83)90013-2. [DOI] [PubMed] [Google Scholar]
  20. Majors J., Varmus H. E. A small region of the mouse mammary tumor virus long terminal repeat confers glucocorticoid hormone regulation on a linked heterologous gene. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5866–5870. doi: 10.1073/pnas.80.19.5866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McKnight S. L., Gavis E. R., Kingsbury R., Axel R. Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: identification of an upstream control region. Cell. 1981 Aug;25(2):385–398. doi: 10.1016/0092-8674(81)90057-x. [DOI] [PubMed] [Google Scholar]
  22. Mita S., Yasuda H., Marunouchi T., Ishiko S., Yamada M. A temperature-sensitive mutant of cultured mouse cells defective in chromosome condensation. Exp Cell Res. 1980 Apr;126(2):407–416. doi: 10.1016/0014-4827(80)90280-3. [DOI] [PubMed] [Google Scholar]
  23. Nir U., Walker M. D., Rutter W. J. Regulation of rat insulin 1 gene expression: evidence for negative regulation in nonpancreatic cells. Proc Natl Acad Sci U S A. 1986 May;83(10):3180–3184. doi: 10.1073/pnas.83.10.3180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Okret S., Poellinger L., Dong Y., Gustafsson J. A. Down-regulation of glucocorticoid receptor mRNA by glucocorticoid hormones and recognition by the receptor of a specific binding sequence within a receptor cDNA clone. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5899–5903. doi: 10.1073/pnas.83.16.5899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ostrowski M. C., Huang A. L., Kessel M., Wolford R. G., Hager G. L. Modulation of enhancer activity by the hormone responsive regulatory element from mouse mammary tumor virus. EMBO J. 1984 Aug;3(8):1891–1899. doi: 10.1002/j.1460-2075.1984.tb02064.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Overhauser J., Fan H. Generation of glucocorticoid-responsive Moloney murine leukemia virus by insertion of regulatory sequences from murine mammary tumor virus into the long terminal repeat. J Virol. 1985 Apr;54(1):133–144. doi: 10.1128/jvi.54.1.133-144.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Owen R. D., Ostrowski M. C. Rapid and selective alterations in the expression of cellular genes accompany conditional transcription of Ha-v-ras in NIH 3T3 cells. Mol Cell Biol. 1987 Jul;7(7):2512–2520. doi: 10.1128/mcb.7.7.2512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Payvar F., DeFranco D., Firestone G. L., Edgar B., Wrange O., Okret S., Gustafsson J. A., Yamamoto K. R. Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region. Cell. 1983 Dec;35(2 Pt 1):381–392. doi: 10.1016/0092-8674(83)90171-x. [DOI] [PubMed] [Google Scholar]
  29. Sassone-Corsi P., Verma I. M. Modulation of c-fos gene transcription by negative and positive cellular factors. Nature. 1987 Apr 2;326(6112):507–510. doi: 10.1038/326507a0. [DOI] [PubMed] [Google Scholar]
  30. Scheidereit C., Geisse S., Westphal H. M., Beato M. The glucocorticoid receptor binds to defined nucleotide sequences near the promoter of mouse mammary tumour virus. Nature. 1983 Aug 25;304(5928):749–752. doi: 10.1038/304749a0. [DOI] [PubMed] [Google Scholar]
  31. Schöler H. R., Gruss P. Cell type-specific transcriptional enhancement in vitro requires the presence of trans-acting factors. EMBO J. 1985 Nov;4(11):3005–3013. doi: 10.1002/j.1460-2075.1985.tb04036.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Spalholz B. A., Yang Y. C., Howley P. M. Transactivation of a bovine papilloma virus transcriptional regulatory element by the E2 gene product. Cell. 1985 Aug;42(1):183–191. doi: 10.1016/s0092-8674(85)80114-8. [DOI] [PubMed] [Google Scholar]
  33. Velcich A., Ziff E. Adenovirus E1a proteins repress transcription from the SV40 early promoter. Cell. 1985 Mar;40(3):705–716. doi: 10.1016/0092-8674(85)90219-3. [DOI] [PubMed] [Google Scholar]
  34. Wigler M., Silverstein S., Lee L. S., Pellicer A., Cheng Y. c., Axel R. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell. 1977 May;11(1):223–232. doi: 10.1016/0092-8674(77)90333-6. [DOI] [PubMed] [Google Scholar]
  35. Wu C. An exonuclease protection assay reveals heat-shock element and TATA box DNA-binding proteins in crude nuclear extracts. Nature. 1985 Sep 5;317(6032):84–87. doi: 10.1038/317084a0. [DOI] [PubMed] [Google Scholar]
  36. Yamamoto K. R. Steroid receptor regulated transcription of specific genes and gene networks. Annu Rev Genet. 1985;19:209–252. doi: 10.1146/annurev.ge.19.120185.001233. [DOI] [PubMed] [Google Scholar]
  37. Zaret K. S., Yamamoto K. R. Reversible and persistent changes in chromatin structure accompany activation of a glucocorticoid-dependent enhancer element. Cell. 1984 Aug;38(1):29–38. doi: 10.1016/0092-8674(84)90523-3. [DOI] [PubMed] [Google Scholar]
  38. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES