Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Sep;11(9):4350–4355. doi: 10.1128/mcb.11.9.4350

In situ distinction between steroid receptor binding and transactivation at a target gene.

D P McDonnell 1, Z Nawaz 1, B W O'Malley 1
PMCID: PMC361296  PMID: 1875926

Abstract

We have developed a DNA interference assay in the yeast Saccharomyces cerevisiae that is designed to indicate the intracellular DNA-binding status of the estrogen receptor. The assay utilizes a promoter containing multiple copies of a GAL4-estrogen receptor binding sequence. This element is designed so that either an estrogen receptor or a GAL4 molecule, but not both, can occupy it simultaneously. The assay is extremely sensitive, and at concentrations of estrogen receptor below that required for maximal transcriptional activation of its target estrogen response element, a quantitative inhibition of GAL4-mediated transcription is seen. Inhibition occurs thought the disruption of complex cooperative interactions among the GAL4 molecules in this reporter. The data obtained from our experiments show that at low concentrations of receptor, hormone is required to promote DNA binding. Overexpression of receptor leads to occupation of the estrogen receptor element in the absence of ligand. In contrast, this latter receptor form will not activate transcription. Our results are consistent with a two-step process for receptor activation. Ligand first causes dissociation of receptor from an inhibitory complex within the cell and produces a DNA-binding form. Second, it converts receptor to a transcriptionally competent form. With use of this yeast model system, these two steps can be distinguished in situ.

Full text

PDF
4350

Selected References

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

  1. Akerblom I. E., Slater E. P., Beato M., Baxter J. D., Mellon P. L. Negative regulation by glucocorticoids through interference with a cAMP responsive enhancer. Science. 1988 Jul 15;241(4863):350–353. doi: 10.1126/science.2838908. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Brown M., Sharp P. A. Human estrogen receptor forms multiple protein-DNA complexes. J Biol Chem. 1990 Jul 5;265(19):11238–11243. [PubMed] [Google Scholar]
  4. Carson-Jurica M. A., Lee A. T., Dobson A. W., Conneely O. M., Schrader W. T., O'Malley B. W. Interaction of the chicken progesterone receptor with heat shock protein (HSP) 90. J Steroid Biochem. 1989;34(1-6):1–9. doi: 10.1016/0022-4731(89)90060-5. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Denis M., Gustafsson J. A. Translation of glucocorticoid receptor mRNA in vitro yields a nonactivated protein. J Biol Chem. 1989 Apr 15;264(11):6005–6008. [PubMed] [Google Scholar]
  7. Denis M., Gustafsson J. A., Wikström A. C. Interaction of the Mr = 90,000 heat shock protein with the steroid-binding domain of the glucocorticoid receptor. J Biol Chem. 1988 Dec 5;263(34):18520–18523. [PubMed] [Google Scholar]
  8. Denis M., Poellinger L., Wikstöm A. C., Gustafsson J. A. Requirement of hormone for thermal conversion of the glucocorticoid receptor to a DNA-binding state. Nature. 1988 Jun 16;333(6174):686–688. doi: 10.1038/333686a0. [DOI] [PubMed] [Google Scholar]
  9. Denis M., Wikström A. C., Cuthill S., Jörnvall H., Gustafsson J. A. Interaction of the glucocorticoid receptor with the Mr approximately 90,000 heat shock protein. Biochem Soc Trans. 1988 Oct;16(5):688–690. doi: 10.1042/bst0160688. [DOI] [PubMed] [Google Scholar]
  10. Denner L. A., Bingman W. E., 3rd, Greene G. L., Weigel N. L. Phosphorylation of the chicken progesterone receptor. J Steroid Biochem. 1987;27(1-3):235–243. doi: 10.1016/0022-4731(87)90315-3. [DOI] [PubMed] [Google Scholar]
  11. Dobson A. D., Conneely O. M., Beattie W., Maxwell B. L., Mak P., Tsai M. J., Schrader W. T., O'Malley B. W. Mutational analysis of the chicken progesterone receptor. J Biol Chem. 1989 Mar 5;264(7):4207–4211. [PubMed] [Google Scholar]
  12. Drouin J., Trifiro M. A., Plante R. K., Nemer M., Eriksson P., Wrange O. Glucocorticoid receptor binding to a specific DNA sequence is required for hormone-dependent repression of pro-opiomelanocortin gene transcription. Mol Cell Biol. 1989 Dec;9(12):5305–5314. doi: 10.1128/mcb.9.12.5305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Elliston J. F., Tsai S. Y., O'Malley B. W., Tsai M. J. Superactive estrogen receptors. Potent activators of gene expression. J Biol Chem. 1990 Jul 15;265(20):11517–11521. [PubMed] [Google Scholar]
  14. Evans R. M. The steroid and thyroid hormone receptor superfamily. Science. 1988 May 13;240(4854):889–895. doi: 10.1126/science.3283939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fawell S. E., Lees J. A., White R., Parker M. G. Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor. Cell. 1990 Mar 23;60(6):953–962. doi: 10.1016/0092-8674(90)90343-d. [DOI] [PubMed] [Google Scholar]
  16. Giniger E., Ptashne M. Cooperative DNA binding of the yeast transcriptional activator GAL4. Proc Natl Acad Sci U S A. 1988 Jan;85(2):382–386. doi: 10.1073/pnas.85.2.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Glass C. K., Holloway J. M., Devary O. V., Rosenfeld M. G. The thyroid hormone receptor binds with opposite transcriptional effects to a common sequence motif in thyroid hormone and estrogen response elements. Cell. 1988 Jul 29;54(3):313–323. doi: 10.1016/0092-8674(88)90194-8. [DOI] [PubMed] [Google Scholar]
  18. Graupner G., Wills K. N., Tzukerman M., Zhang X. K., Pfahl M. Dual regulatory role for thyroid-hormone receptors allows control of retinoic-acid receptor activity. Nature. 1989 Aug 24;340(6235):653–656. doi: 10.1038/340653a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Jakobsen B. K., Pelham H. R. Constitutive binding of yeast heat shock factor to DNA in vivo. Mol Cell Biol. 1988 Nov;8(11):5040–5042. doi: 10.1128/mcb.8.11.5040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Johnston M. A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae. Microbiol Rev. 1987 Dec;51(4):458–476. doi: 10.1128/mr.51.4.458-476.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Klein-Hitpass L., Ryffel G. U., Heitlinger E., Cato A. C. A 13 bp palindrome is a functional estrogen responsive element and interacts specifically with estrogen receptor. Nucleic Acids Res. 1988 Jan 25;16(2):647–663. doi: 10.1093/nar/16.2.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Klein-Hitpass L., Tsai S. Y., Weigel N. L., Allan G. F., Riley D., Rodriguez R., Schrader W. T., Tsai M. J., O'Malley B. W. The progesterone receptor stimulates cell-free transcription by enhancing the formation of a stable preinitiation complex. Cell. 1990 Jan 26;60(2):247–257. doi: 10.1016/0092-8674(90)90740-6. [DOI] [PubMed] [Google Scholar]
  24. Lees J. A., Fawell S. E., Parker M. G. Identification of two transactivation domains in the mouse oestrogen receptor. Nucleic Acids Res. 1989 Jul 25;17(14):5477–5488. doi: 10.1093/nar/17.14.5477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mak P., McDonnell D. P., Weigel N. L., Schrader W. T., O'Malley B. W. Expression of functional chicken oviduct progesterone receptors in yeast (Saccharomyces cerevisiae). J Biol Chem. 1989 Dec 25;264(36):21613–21618. [PubMed] [Google Scholar]
  26. McDonnell D. P., Pike J. W., Drutz D. J., Butt T. R., O'Malley B. W. Reconstitution of the vitamin D-responsive osteocalcin transcription unit in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Aug;9(8):3517–3523. doi: 10.1128/mcb.9.8.3517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Metzger D., White J. H., Chambon P. The human oestrogen receptor functions in yeast. Nature. 1988 Jul 7;334(6177):31–36. doi: 10.1038/334031a0. [DOI] [PubMed] [Google Scholar]
  28. O'Malley B. The steroid receptor superfamily: more excitement predicted for the future. Mol Endocrinol. 1990 Mar;4(3):363–369. doi: 10.1210/mend-4-3-363. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Quarmby V. E., Fox-Davies C., Newbold R. R., Korach K. S. Response of the mouse uterus to nafoxidine stimulation: agonism and antagonism. Biol Reprod. 1988 May;38(4):945–954. doi: 10.1095/biolreprod38.4.945. [DOI] [PubMed] [Google Scholar]
  31. Scheidereit C., von der Ahe D., Cato A. C., Wenz M., Suske G., Carlson C., Bosshard H., Westphal H. M., Beato M. Protein-DNA interactions at steroid hormone regulated genes. Endocr Res. 1989;15(4):417–440. doi: 10.3109/07435808909036347. [DOI] [PubMed] [Google Scholar]
  32. Schena M., Yamamoto K. R. Mammalian glucocorticoid receptor derivatives enhance transcription in yeast. Science. 1988 Aug 19;241(4868):965–967. doi: 10.1126/science.3043665. [DOI] [PubMed] [Google Scholar]
  33. Selleck S. B., Majors J. E. In vivo DNA-binding properties of a yeast transcription activator protein. Mol Cell Biol. 1987 Sep;7(9):3260–3267. doi: 10.1128/mcb.7.9.3260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Smith D. F., Faber L. E., Toft D. O. Purification of unactivated progesterone receptor and identification of novel receptor-associated proteins. J Biol Chem. 1990 Mar 5;265(7):3996–4003. [PubMed] [Google Scholar]
  35. Tasset D., Tora L., Fromental C., Scheer E., Chambon P. Distinct classes of transcriptional activating domains function by different mechanisms. Cell. 1990 Sep 21;62(6):1177–1187. doi: 10.1016/0092-8674(90)90394-t. [DOI] [PubMed] [Google Scholar]
  36. Tora L., Mullick A., Metzger D., Ponglikitmongkol M., Park I., Chambon P. The cloned human oestrogen receptor contains a mutation which alters its hormone binding properties. EMBO J. 1989 Jul;8(7):1981–1986. doi: 10.1002/j.1460-2075.1989.tb03604.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tora L., White J., Brou C., Tasset D., Webster N., Scheer E., Chambon P. The human estrogen receptor has two independent nonacidic transcriptional activation functions. Cell. 1989 Nov 3;59(3):477–487. doi: 10.1016/0092-8674(89)90031-7. [DOI] [PubMed] [Google Scholar]
  38. Tzukerman M., Zhang X. K., Hermann T., Wills K. N., Graupner G., Pfahl M. The human estrogen receptor has transcriptional activator and repressor functions in the absence of ligand. New Biol. 1990 Jul;2(7):613–620. [PubMed] [Google Scholar]
  39. White R., Lees J. A., Needham M., Ham J., Parker M. Structural organization and expression of the mouse estrogen receptor. Mol Endocrinol. 1987 Oct;1(10):735–744. doi: 10.1210/mend-1-10-735. [DOI] [PubMed] [Google Scholar]
  40. Willmann T., Beato M. Steroid-free glucocorticoid receptor binds specifically to mouse mammary tumour virus DNA. Nature. 1986 Dec 18;324(6098):688–691. doi: 10.1038/324688a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES