Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

The EMBO Journal logoLink to The EMBO Journal
. 1988 Jun;7(6):1653–1660. doi: 10.1002/j.1460-2075.1988.tb02992.x

Expression of human estrogen receptor mutants in Xenopus oocytes: correlation between transcriptional activity and ability to form protein-DNA complexes.

I Theulaz 1, R Hipskind 1, B ten Heggeler-Bordier 1, S Green 1, V Kumar 1, P Chambon 1, W Wahli 1
PMCID: PMC457149  PMID: 3169000

Abstract

The human estrogen receptor (hER) is a trans-acting regulatory protein composed of a series of discrete functional domains. We have microinjected an hER expression vector (HEO) into Xenopus oocyte nuclei and demonstrate, using Western blot assay, that the hER is synthesized. When nuclear extracts from oocytes were prepared and incubated in the presence of a 2.7 kb DNA fragment comprising the 5' end of the vitellogenin gene B2, formation of estrogen-dependent complexes could be visualized by electron microscopy over the estrogen responsive element (ERE). Of crucial importance is the observation that the complex formation is inhibited by the estrogen antagonist tamoxifen, is restored by the addition of the hormone and does not take place with extracts from control oocytes injected with the expression vector lacking the sequences encoding the receptor. The presence of the biologically active hER is confirmed in co-injection experiments, in which HEO is co-introduced with a CAT reporter gene under the control of a vitellogenin promoter containing or lacking the ERE. CAT assays and primer extensions analyses reveal that both the receptor and the ERE are essential for estrogen induced stimulation of transcription. The same approach was used to analyze selective hER mutants. We find that the DNA binding domain (region C) is essential for protein--DNA complex formation at the ERE but is not sufficient by itself to activate transcription from the reporter gene. In addition to region C, both the hormone binding (region E) and amino terminal (region A/B) domains are needed for an efficient transcription activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Full text

PDF
1653

Images in this article

Selected References

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

  1. Berg J. M. Potential metal-binding domains in nucleic acid binding proteins. Science. 1986 Apr 25;232(4749):485–487. doi: 10.1126/science.2421409. [DOI] [PubMed] [Google Scholar]
  2. Danielsen M., Northrop J. P., Ringold G. M. The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing and expression of wild-type and mutant receptor proteins. EMBO J. 1986 Oct;5(10):2513–2522. doi: 10.1002/j.1460-2075.1986.tb04529.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Druege P. M., Klein-Hitpass L., Green S., Stack G., Chambon P., Ryffel G. U. Introduction of estrogen-responsiveness into mammalian cell lines. Nucleic Acids Res. 1986 Dec 9;14(23):9329–9337. doi: 10.1093/nar/14.23.9329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dynan W. S., Tjian R. Control of eukaryotic messenger RNA synthesis by sequence-specific DNA-binding proteins. 1985 Aug 29-Sep 4Nature. 316(6031):774–778. doi: 10.1038/316774a0. [DOI] [PubMed] [Google Scholar]
  5. Fauque J., Borgna J. L., Rochefort H. A monoclonal antibody to the estrogen receptor inhibits in vitro criteria of receptor activation by an estrogen and an anti-estrogen. J Biol Chem. 1985 Dec 15;260(29):15547–15553. [PubMed] [Google Scholar]
  6. Fortune J. E. Steroid production by Xenopus ovarian follicles at different developmental stages. Dev Biol. 1983 Oct;99(2):502–509. doi: 10.1016/0012-1606(83)90299-3. [DOI] [PubMed] [Google Scholar]
  7. Galli G., Hofstetter H., Stunnenberg H. G., Birnstiel M. L. Biochemical complementation with RNA in the Xenopus oocyte: a small RNA is required for the generation of 3' histone mRNA termini. Cell. 1983 Oct;34(3):823–828. doi: 10.1016/0092-8674(83)90539-1. [DOI] [PubMed] [Google Scholar]
  8. Germond J. E., ten Heggeler B., Schubiger J. L., Walker P., Westley B., Wahli W. Vitellogenin B2 gene in Xenopus laevis: isolation, in vitro transcription and relation to other vitellogenin genes. Nucleic Acids Res. 1983 May 25;11(10):2979–2997. doi: 10.1093/nar/11.10.2979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gordon M. S., Notides A. C. Computer modeling of estradiol interactions with the estrogen receptor. J Steroid Biochem. 1986 Aug;25(2):177–181. doi: 10.1016/0022-4731(86)90414-0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Green S., Chambon P. Oestradiol induction of a glucocorticoid-responsive gene by a chimaeric receptor. Nature. 1987 Jan 1;325(6099):75–78. doi: 10.1038/325075a0. [DOI] [PubMed] [Google Scholar]
  12. Green S., Walter P., Kumar V., Krust A., Bornert J. M., Argos P., Chambon P. Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature. 1986 Mar 13;320(6058):134–139. doi: 10.1038/320134a0. [DOI] [PubMed] [Google Scholar]
  13. Greene G. L., Gilna P., Waterfield M., Baker A., Hort Y., Shine J. Sequence and expression of human estrogen receptor complementary DNA. Science. 1986 Mar 7;231(4742):1150–1154. doi: 10.1126/science.3753802. [DOI] [PubMed] [Google Scholar]
  14. Hipskind R. A., Reeder R. H. Initiation of ribosomal RNA chains in homogenates of oocyte nuclei. J Biol Chem. 1980 Aug 25;255(16):7896–7906. [PubMed] [Google Scholar]
  15. Hollenberg S. M., Giguere V., Segui P., Evans R. M. Colocalization of DNA-binding and transcriptional activation functions in the human glucocorticoid receptor. Cell. 1987 Apr 10;49(1):39–46. doi: 10.1016/0092-8674(87)90753-7. [DOI] [PubMed] [Google Scholar]
  16. Jost J. P., Seldran M., Geiser M. Preferential binding of estrogen-receptor complex to a region containing the estrogen-dependent hypomethylation site preceding the chicken vitellogenin II gene. Proc Natl Acad Sci U S A. 1984 Jan;81(2):429–433. doi: 10.1073/pnas.81.2.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Keegan L., Gill G., Ptashne M. Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. Science. 1986 Feb 14;231(4739):699–704. doi: 10.1126/science.3080805. [DOI] [PubMed] [Google Scholar]
  18. King W. J., Greene G. L. Monoclonal antibodies localize oestrogen receptor in the nuclei of target cells. Nature. 1984 Feb 23;307(5953):745–747. doi: 10.1038/307745a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Klein-Hitpass L., Schorpp M., Wagner U., Ryffel G. U. An estrogen-responsive element derived from the 5' flanking region of the Xenopus vitellogenin A2 gene functions in transfected human cells. Cell. 1986 Sep 26;46(7):1053–1061. doi: 10.1016/0092-8674(86)90705-1. [DOI] [PubMed] [Google Scholar]
  21. Klein-Hitpass L., Schorpp M., Wagner U., Ryffel G. U. An estrogen-responsive element derived from the 5' flanking region of the Xenopus vitellogenin A2 gene functions in transfected human cells. Cell. 1986 Sep 26;46(7):1053–1061. doi: 10.1016/0092-8674(86)90705-1. [DOI] [PubMed] [Google Scholar]
  22. Knowland J., Theulaz I., Wright C. V., Wahli W. Injection of partially purified estrogen receptor protein from Xenopus liver nuclei into oocytes activates the silent vitellogenin locus. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5777–5781. doi: 10.1073/pnas.81.18.5777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Koike S., Sakai M., Muramatsu M. Molecular cloning and characterization of rat estrogen receptor cDNA. Nucleic Acids Res. 1987 Mar 25;15(6):2499–2513. doi: 10.1093/nar/15.6.2499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Krust A., Green S., Argos P., Kumar V., Walter P., Bornert J. M., Chambon P. The chicken oestrogen receptor sequence: homology with v-erbA and the human oestrogen and glucocorticoid receptors. EMBO J. 1986 May;5(5):891–897. doi: 10.1002/j.1460-2075.1986.tb04300.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kumar V., Green S., Stack G., Berry M., Jin J. R., Chambon P. Functional domains of the human estrogen receptor. Cell. 1987 Dec 24;51(6):941–951. doi: 10.1016/0092-8674(87)90581-2. [DOI] [PubMed] [Google Scholar]
  26. Kumar V., Green S., Staub A., Chambon P. Localisation of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor. EMBO J. 1986 Sep;5(9):2231–2236. doi: 10.1002/j.1460-2075.1986.tb04489.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ladiges W. C., Raff R. F., Brown S., Deeg H. J., Storb R. The canine major histocompatibility complex. Supertypic specificities defined by the primed lymphocyte test (PLT). Immunogenetics. 1984;19(4):359–365. doi: 10.1007/BF00345410. [DOI] [PubMed] [Google Scholar]
  28. Liebhaber S. A., Goossens M. J., Kan Y. W. Cloning and complete nucleotide sequence of human 5'-alpha-globin gene. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7054–7058. doi: 10.1073/pnas.77.12.7054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Martinez E., Givel F., Wahli W. The estrogen-responsive element as an inducible enhancer: DNA sequence requirements and conversion to a glucocorticoid-responsive element. EMBO J. 1987 Dec 1;6(12):3719–3727. doi: 10.1002/j.1460-2075.1987.tb02706.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Miesfeld R., Godowski P. J., Maler B. A., Yamamoto K. R. Glucocorticoid receptor mutants that define a small region sufficient for enhancer activation. Science. 1987 Apr 24;236(4800):423–427. doi: 10.1126/science.3563519. [DOI] [PubMed] [Google Scholar]
  31. Probst E., Kressmann A., Birnstiel M. L. Expression of sea urchin histone genes in the oocyte of Xenopus laevis. J Mol Biol. 1979 Dec 15;135(3):709–732. doi: 10.1016/0022-2836(79)90173-6. [DOI] [PubMed] [Google Scholar]
  32. Ptashne M. Gene regulation by proteins acting nearby and at a distance. Nature. 1986 Aug 21;322(6081):697–701. doi: 10.1038/322697a0. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Scheidereit C., Westphal H. M., Carlson C., Bosshard H., Beato M. Molecular model of the interaction between the glucocorticoid receptor and the regulatory elements of inducible genes. DNA. 1986 Oct;5(5):383–391. doi: 10.1089/dna.1986.5.383. [DOI] [PubMed] [Google Scholar]
  35. Seiler-Tuyns A., Walker P., Martinez E., Mérillat A. M., Givel F., Wahli W. Identification of estrogen-responsive DNA sequences by transient expression experiments in a human breast cancer cell line. Nucleic Acids Res. 1986 Nov 25;14(22):8755–8770. doi: 10.1093/nar/14.22.8755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Walker P., Germond J. E., Brown-Luedi M., Givel F., Wahli W. Sequence homologies in the region preceding the transcription initiation site of the liver estrogen-responsive vitellogenin and apo-VLDLII genes. Nucleic Acids Res. 1984 Nov 26;12(22):8611–8626. doi: 10.1093/nar/12.22.8611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Weiler I. J., Lew D., Shapiro D. J. The Xenopus laevis estrogen receptor: sequence homology with human and avian receptors and identification of multiple estrogen receptor messenger ribonucleic acids. Mol Endocrinol. 1987 May;1(5):355–362. doi: 10.1210/mend-1-5-355. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. ten Heggeler-Bordier B., Hipskind R., Seiler-Tuyns A., Martinez E., Corthésy B., Wahli W. Electron microscopic visualization of protein-DNA interactions at the estrogen responsive element and in the first intron of the Xenopus laevis vitellogenin gene. EMBO J. 1987 Jun;6(6):1715–1720. doi: 10.1002/j.1460-2075.1987.tb02422.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. ten Heggeler B., Wahli W. Visualization of RNA polymerase II ternary transcription complexes formed in vitro on a Xenopus laevis vitellogenin gene. EMBO J. 1985 Sep;4(9):2269–2273. doi: 10.1002/j.1460-2075.1985.tb03925.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES