Summary
We have undertaken studies to examine the ligand requirements for the DNA binding and functional activity of human estrogen receptor (ER) within intact cells. By employing an assay using competitive binding of ER with basal transcription factors on a constitutive promoter (CMV-(HRE)n-CAT, containing hormone response element(s) between the TATA-box and the start site of transcription of the Cytomegalovirus promoter upstream of the chloramphenicol acetyltransferase gene) we were able to examine the DNA binding ability of the human ER in whole cells. We used this promoter interference assay to examine the DNA binding of ER in cell lines containing high and low levels of endogenous ER, as well as in Chinese Hamster Ovary (CHO) cells expressing wild-type and mutant ERs from cotransfected expression vectors. We have found that the ER is capable of binding to reporter templates within intact cells in the absence of ligand; however, ligand enhances this interaction. We provide some evidence that this DNA binding may result in ligand-independent transactivation of transgenes by the receptor, and that DNA binding alone is not sufficient for full receptor activity. Likewise, receptors occupied with estradiol or two classes of antiestrogens were also capable of DNA binding. Receptors occupied with the pure antiestrogen ICI 164,384 bound to DNA in whole cells and were in sufficient quantity to occupy receptor binding sites on reporter templates, indicating that neither a loss of DNA binding of ICI-receptor complexes or reduction of ER protein in target cells could fully explain the antagonistic nature of this compound. Through the analysis of a temperature-sensitive ER mutant (C447A), we provide evidence that the ligand-independent DNA binding observed for the ER is the basis of functional differences observed between the ER and other members of the steroid receptor family.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Aronica SM and Katzenellenbogen BS (1991): Progesterone receptor regulation in uterine cells: Stimulation by estrogen, cyclic adenosine 3′,5′-monophosphate, and insulin-like growth factor I and suppression by antiestrogens and protein kinase inhibitors. Endocrinol 128: 2045–2052.
Bagchi MK, Elliston JF, Tsai SY, Edwards DP, Tsai M-J and O’Malley BW (1988): Steroid hormone-dependent interaction of human progesterone receptor with its target enhancer element. Mol Endo 2: 1221–1229.
Bagchi MK, Tsai, SY, Tsai M-J and O’Malley BW (1990): Identification of a functional intermediate in receptor activation in progesterone-dependent cell-free extracts. Nature 345: 547–550.
Berry M, Metzger, M and Chambon P (1990): Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen. EMBO J 9: 2811–2818.
Brown M and Sharp, P (1990): Human estrogen receptor forms multiple-DNA complexes. J Biol Chem 265: 11238–11243.
Cairns W, Cairns C, Pongratz I, Poellinger L and Okret S (1991): Assembly of a glucocorticoid receptor complex prior to DNA binding enhances its specific interaction with a glucocorticoid response element. J Biol Chem 266: 11221–11226.
Carson-Jurica MA, Schrader WT and O’Malley BW (1990): Steroid Receptor Family: Structure and Function. Endocrine Reviews 11: 201–220.
Clarke R, Brunner N, Katzenellenbogen BS, Thompson BW, Norman MJ, Koppi C., Paik S, Lippman ME and Dickson RB (1989): Progression of human breast cancer cells from hormone-dependent to hormone-independent growth both in vitro and in vivo. Proc Natl Acad Sci USA 86: 3649–3653.
Dauvois S, Danielian PS, White R and Parker MG (1992): Antiestrogen ICI 164,384 reduces cellular estrogen receptor content by increasing its turnover. Proc Natl Acad Sci USA 89: 4037–4041.
Denis M, Poellinger L, Wikström AC and Gustafsson, JA (1988): Requirement of hormone for thermal conversion of the glucocorticoid receptor to a DNA binding state. Nature 333: 686–688.
Denis M and Gustafsson JA (1989): Translation of glucocorticoid receptor mRNA in vitro yields a nonactivated protein. J Biol Chem 264: 6005–6008.
DeMarzo AM, Beck CA, Oanate, SA and Edwards DP (1991): Dimerization of mammalian progesterone receptors occurs in the absence of DNA and is related to the release of the 90-kDa heat shock protein. Proc Natl Acad Sci USA 88: 72–76.
Edwards DP, Kuhnel B, Estes PA and Nordeen SK (1989): Human progesterone receptor binding to mouse mammary tumor virus deoxyribonucleic acid: dependence on hormone and nonreceptor nuclear factor(s). Mol Endo 3: 381–391.
El-Ashry D, Onate SA, Nordeen SK and Edwards DP (1989): Human progesterone receptor complexed with the antagonist RU 486 binds to hormone response elements in a structurally altered form. Mol Endo 3: 1545–1558.
Elliston JF, Tsai SY, O’Malley BW and Tsai MJ (1990): Superactive estrogen receptors. J Biol Chem 265: 11517–11521.
Fawell SE, White R, Hoare S, Sydenham M, Page M and Parker MG (1990a): Inhibition of estrogen receptor-DNA binding by the “pure” antiestrogen ICI 164,384 appears to be mediated by impaired receptor dimerization. Proc Natl Acad Sci USA 87: 6883–6887.
Fawell SE, Lees JA, White JA and Parker MG (1990b): Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor. Cell 60: 953–962.
Gibson MK, Nemmers LA, Beckman Jr WC, Davis VL, Curtis SW and Korach KS (1991): The mechanism of ICI 164,384 antiestrogenicity involves rapid loss of estrogen receptor in uterine tissue. Endocrinol 129: 2000–2010.
Giguere V, Hollenberg SM, Rosenfeld MG and Evans RM (1986): Functional domains of the human glucocorticoid receptor. Cell 46: 645–652.
Howard KJ, Holley SJ, Yamamoto KR and Distelhorst CW (1990): Mapping the hsp90 binding region of the glucocorticoid receptor. J Biol Chem 265: 11928–11935.
King WJ and Greene GL (1984): Monoclonal antibodies localize estrogen receptor in the nuclei of target cells. Nature (London) 307: 745–749.
Lees JA, Fawell SE and Parker MG (1989): Identification of two transactivation domains in the mouse oestrogen receptor. Nucleic Acids Res 17: 5477–5487.
Martinez E and Wahli W (1989): Cooperative binding of estrogen receptor to imperfect estrogen-responsive DNA elements correlates with their synergistic hormone-dependent enhancer activity. EMBO J 8: 3781–3791.
McDonnell DP, Nawaz Z and O’Malley BW (1991): In situ distinction between steroid receptor binding and transactivation at a target gene. Mol Cell Biol 11: 4350–4355.
Meyer ME, Gronemeyer H, Turcotte B, Bocquel MT, Tasset D and Chambon P (1989): Steroid hormone receptors compete for factors that mediate their enhancer function. Cell 57: 433–442.
Pham TA, Elliston J, Nawaz Z, McDonnell DP, Tsai MJ and O’Malley BW (1991): Antiestrogen can establish nonproductive receptor complexes and alter chromatin structure at target elements. Proc Natl Acad Sci USA 88: 3125–3129.
Picard D, Kumar V, Chambon P and Yamamoto KR (1990a): Signal transduction by steroid hormones: nuclear localization is differentially regulated in estrogen and glucocorticoid receptors. Cell Reg 1: 291–299.
Picard D, Khursheed B, Garabedian MJ, Fortin MG, Lindquist S and Yamamoto KR (1990b): Reduced levels of hsp90 compromise steroid receptor action in vivo. Nature 348: 166–168.
Power RF, Mani SK, Codina J, Conneeley OM and O’Malley BW (1991): Dopaminergic and ligand-independent activation of steroid hormone receptors. Science 254: 1636–1639.
Reese JC and Katzenellenbogen BS (1991a): Mutagenesis of cysteines in the hormone binding domain of the human estrogen receptor: alterations in binding and transcriptional activation by covalently and reversibly attaching ligands. J Biol Chem 266: 10880–10887.
Reese JC and Katzenellenbogen BS (1991b): Differential DNA-binding abilities of receptor occupied with two classes of antiestrogens: studies using human estrogen receptor overexpressed in mammalian cells. Nucleic Acids Res 19: 6595–6602.
Reese JC and Katzenellenbogen BS (1992a): Characterization of a temperature-sensitive mutation in the hormone binding domain of the human estrogen receptor: studies utilizing cell-free extracts and intact cells and implications for hormone-dependent transcriptional activation. J Biol Chem 267: 9868–9873.
Reese JC and Katzenellenbogen BS (1992b): Examination of the DNA-Binding ability of estrogen receptor in whole cells: implications for hormone-independent transactivation and the actions of antiestrogens. Mol Cell Biol 12: 4531–4538.
Schena M and Yamamoto KR (1988): Mammalian glucocorticoid receptor derivatives enhance transcription in yeast. Science 241: 965–967.
Tora L, Mullick A, Metzger D, Ponglikitmongkol M, Park I and Chambon P (1989a): The cloned human oestrogen receptor contains a mutation which alters its hormone binding properties. EMBO J 8: 1981–1986.
Tora L, White J, Brou C, Tasset D, Webster N, Scheer E and Chambon P (1989b): The human estrogen receptor has two nonacidic transcriptional activation functions. Cell 59: 477–487.
Tzukerman M, Zhang X-K, Hermann T, Wills, KN, Graupner, G and Pfahl M (1991): The human estrogen receptor has transcriptional activator and repressor functions in the absence of ligand. The New Biologist 2: 613–620.
Wrenn CK and Katzenellenbogen BS (1990): Cross-linking of the estrogen receptor to chromatin in intact MCF-7 human breast cancer cells: optimization and effect of ligand. Mol Endo 4: 1647–1654.
Note added in proof: The manuscript by Pakdel, Reese, and Katzenellenbogen is now in press. MOLEC ENDO.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1994 Birkhäuser Boston
About this chapter
Cite this chapter
Reese, J.C., Katzenellenbogen, B.S. (1994). Ligand Requirements for Estrogen Receptor Function and the Actions of Antiestrogens. In: Moudgil, V.K. (eds) Steroid Hormone Receptors: Basic and Clinical Aspects. Hormones in Health and Disease. Birkhäuser Boston. https://doi.org/10.1007/978-1-4615-9849-7_15
Download citation
DOI: https://doi.org/10.1007/978-1-4615-9849-7_15
Publisher Name: Birkhäuser Boston
Print ISBN: 978-1-4615-9851-0
Online ISBN: 978-1-4615-9849-7
eBook Packages: Springer Book Archive