Human Estrogen Receptor β Binds DNA in a Manner Similar to and Dimerizes with Estrogen Receptor α

Abstract

The cloning of a novel estrogen receptor β (denoted ERβ) has recently been described (Kuiper, G. G. J. M., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J-A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5925–5930 and Mosselman, S., Polman, J., and Dijkema, R. (1996)FEBS Lett. 392, 49–53). ERβ is highly homologous to the “classical” estrogen receptor α (here referred to as ERα), has been shown to bind estrogens with an affinity similar to that of ERα, and activates expression of reporter genes containing estrogen response elements in an estrogen-dependent manner. Here we describe functional studies comparing the DNA binding abilities of human ERα and β in gel shift assays. We show that DNA binding by ERα and β are similarly affected by elevated temperature in the absence of ligand or in the presence of 17β-estradiol and the partial estrogen agonist 4-hydroxy-tamoxifen. In the absence of ligand, DNA binding by ERα and β is rapidly lost at 37 °C, while in the presence of 17β-estradiol and 4-hydroxy-tamoxifen, the loss in DNA binding at elevated temperature is much more gradual. We show that the loss in DNA binding is not due to degradation of the receptor proteins. However, while the complete antagonist ICI 182,780 does not “protect” human ERα (hERα) from loss of DNA binding at elevated temperaturein vitro, it does appear to protect human ERβ (hERβ), suggestive of differences in the way ICI 182,780 acts on hERα and β. We further report that ERα and β can dimerize with each other, the DNA binding domain of hERα being sufficient for dimerization with hERβ. Cell and promoter-specific transcription activation by ERα has been shown to be dependent on the differential action of the N- and C-terminal transcription activation functions AF-1 and AF-2, respectively. The existence of a second estrogen receptor gene and the dimerization of ERα and β add greater levels of complexity to transcription activation in response to estrogens. The cloning of a novel estrogen receptor β (denoted ERβ) has recently been described (Kuiper, G. G. J. M., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J-A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5925–5930 and Mosselman, S., Polman, J., and Dijkema, R. (1996)FEBS Lett. 392, 49–53). ERβ is highly homologous to the “classical” estrogen receptor α (here referred to as ERα), has been shown to bind estrogens with an affinity similar to that of ERα, and activates expression of reporter genes containing estrogen response elements in an estrogen-dependent manner. Here we describe functional studies comparing the DNA binding abilities of human ERα and β in gel shift assays. We show that DNA binding by ERα and β are similarly affected by elevated temperature in the absence of ligand or in the presence of 17β-estradiol and the partial estrogen agonist 4-hydroxy-tamoxifen. In the absence of ligand, DNA binding by ERα and β is rapidly lost at 37 °C, while in the presence of 17β-estradiol and 4-hydroxy-tamoxifen, the loss in DNA binding at elevated temperature is much more gradual. We show that the loss in DNA binding is not due to degradation of the receptor proteins. However, while the complete antagonist ICI 182,780 does not “protect” human ERα (hERα) from loss of DNA binding at elevated temperaturein vitro, it does appear to protect human ERβ (hERβ), suggestive of differences in the way ICI 182,780 acts on hERα and β. We further report that ERα and β can dimerize with each other, the DNA binding domain of hERα being sufficient for dimerization with hERβ. Cell and promoter-specific transcription activation by ERα has been shown to be dependent on the differential action of the N- and C-terminal transcription activation functions AF-1 and AF-2, respectively. The existence of a second estrogen receptor gene and the dimerization of ERα and β add greater levels of complexity to transcription activation in response to estrogens. The estrogen receptor α (ERα) 1The abbreviations used are: ERα, estrogen receptor α; hERα, human estrogen receptor α; ERβ, estrogen receptor β; hERβ, human estrogen receptor β; ERE, estrogen response element; EREM, mutant ERE; E2, 17β-estradiol; OHT, 4-hydroxy-tamoxifen; ICI, ICI182780; AF, activation function; DBD, DNA binding domain; LBD, ligand binding domain; PCR, polymerase chain reaction; kDa, kilo-dalton; FCS, fetal calf serum, DMEM, Dulbecco's modified Eagle's Medium; WCE, whole cell extract; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); PBS, phosphate-buffered saline. 1The abbreviations used are: ERα, estrogen receptor α; hERα, human estrogen receptor α; ERβ, estrogen receptor β; hERβ, human estrogen receptor β; ERE, estrogen response element; EREM, mutant ERE; E2, 17β-estradiol; OHT, 4-hydroxy-tamoxifen; ICI, ICI182780; AF, activation function; DBD, DNA binding domain; LBD, ligand binding domain; PCR, polymerase chain reaction; kDa, kilo-dalton; FCS, fetal calf serum, DMEM, Dulbecco's modified Eagle's Medium; WCE, whole cell extract; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); PBS, phosphate-buffered saline. is a member of a superfamily of transcription factors that induce transcription of target genes by binding to cis-acting enhancer elements in promoters of responsive genes (for reviews, see Refs. 3Evans R.M. 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AF-1 and AF-2 can activate transcription independently and synergistically act in a promoter- and cell-specific manner (12Kumar V. Green S. Staub A. Chambon P. EMBO J. 1986; 5: 2231-2236Crossref PubMed Scopus (407) Google Scholar, 14Kumar V. Green S. Stack G. Berry M. Jin J.R. Chambon P. Cell. 1987; 51: 941-951Abstract Full Text PDF PubMed Scopus (1054) Google Scholar, 15Webster N.J.G. Green S. Jin J.R. Chambon P. Cell. 1988; 54: 199-207Abstract Full Text PDF PubMed Scopus (441) Google Scholar, 16Lees J.A. Fawell S.E. Parker M.G. Nucleic Acids Res. 1989; 17: 5477-5488Crossref PubMed Scopus (257) Google Scholar, 17Tora L. White J. Brou C. Tasset D. Webster N. Scheer E. Chambon P. Cell. 1989; 59: 477-487Abstract Full Text PDF PubMed Scopus (881) Google Scholar, 18Berry M. Metzger D. Chambon P. EMBO J. 1990; 9: 2811-2818Crossref PubMed Scopus (659) Google Scholar, 19Metzger D. Ali S. Bornert J.-M. Chambon P. J. Biol. Chem. 1995; 270: 9535-9542Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Region D is involved in binding to hsp90 (20Chambraud B. Berry M. Redeuilh G. Chambon P. Baulieu E.-E. J. Biol. Chem. 1990; 265: 20686-20691Abstract Full Text PDF PubMed Google Scholar), as well as containing nuclear localization signals (21Ylikomi T. Bocquel M.-T. Berry M. Gronemeyer H. Chambon P. EMBO J. 1992; 11: 3681-3694Crossref PubMed Scopus (248) Google Scholar), and plays a part in stabilizing DNA binding by the DBD (22Mader S. Chambon P. White J.H. Nucleic Acids Res. 1993; 21: 1125-1132Crossref PubMed Scopus (101) Google Scholar). Region F appears to play a role in modulating transcriptional activation by ERα (23Montano M.M. Muller V. Trobaugh A. Katzenellenbogen B.S. Mol. Endocrinol. 1995; 9: 814-825Crossref PubMed Google Scholar). The mechanisms of anti-estrogen action have been extensively studied. In particular, anti-estrogens such as tamoxifen and ICI 164,384 (or its derivative ICI 182,780) antagonize the effects of estrogens by competing with estrogen for binding to the estrogen receptor. Tamoxifen, and its derivative 4-hydroxy-tamoxifen, show partial agonistic activity by inhibiting trans-activation by AF-2 but enabling transcription through AF-1 (18Berry M. Metzger D. Chambon P. EMBO J. 1990; 9: 2811-2818Crossref PubMed Scopus (659) Google Scholar, 19Metzger D. Ali S. Bornert J.-M. Chambon P. J. Biol. Chem. 1995; 270: 9535-9542Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 24McInerney E.M. Katzenellenbogen B.S. J. Biol. Chem. 1996; 271: 24172-24178Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). ICI 164,384, on the other hand, is a complete antagonist that does not prevent DNA binding by ER but inhibits trans-activation by both AF-1 and AF-2 (25Metzger D. Berry M. Ali S. Chambon P. Mol. Endocrinol. 1995; 9: 579-591Crossref PubMed Google Scholar). Thus, anti-estrogens do not appear to act by preventing DNA binding by ERα and may act by inactivating (or preventing the activation of) the transcription activation functions (see also Ref.26McDonnell D.P. Clemm D.L. Hermann T. Goldman M.E. Pike J.W. Mol. Endocrinol. 1995; 9: 659-669Crossref PubMed Google Scholar). Metzger et al. (25Metzger D. Berry M. Ali S. Chambon P. Mol. Endocrinol. 1995; 9: 579-591Crossref PubMed Google Scholar) showed, however, that at elevated temperature (37 °C), hERα undergoes a reduction in its capacity to bind to an ERE in vitro. The reduction in DNA binding is much faster in the absence of ligand than in the presence of E2 or the partial agonist OHT. Interestingly, ICI 164,384 does not “protect” hERα from losing DNA binding capacity at 37 °C. In vivoICI 164,384 binding reduces the half-life of ERα (27Gibson M.K. Nemmers L.A. Beckman W.C. Davis V.L. Curtis S.W. Korach K.S. 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Cell. 1993; Full Text PDF PubMed Scopus Google Scholar, and in the DBD are for receptor the LBD are for dimerization of the ERα S.E. J.A. White R. Parker M.G. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar). have been to play in the and in the of the being involved in and in and The to in the ERα gene has also been to that its is studies from Korach and Korach K.S. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar) ERα have shown while absence of the ERα gene is not and and to are with and estrogen in mutant of the gene expression in and expression of the receptor gene not in response to estrogens. The of studies have for the of ERα in the and of the but also of the as well as for and has been from however, for the of ERα in of the and estrogen binding activity with in the of mutant Korach K.S. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar, K.S. Science. 1994; PubMed Scopus Google Scholar, Curtis S.W. J. Korach K.S. Mol. Endocrinol. 1995; 9: PubMed Google Scholar, K.S. Curtis S.W. J. S. E.M. S. Res. 1996; 51: Google Scholar). the cloning of a second estrogen receptor gene has recently been in the E. M. S. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar) and in S. J. R. Lett. 1996; PubMed Scopus Google Scholar). ERβ is highly homologous to ERα, and homology hERα and β are in the DNA and binding respectively. The ERβ is highly in the and E. M. S. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), while the levels of expression of human ERβ in the S. J. R. Lett. 1996; PubMed Scopus Google Scholar). ERβ 17β-estradiol with an affinity similar to that of ERα and has been shown to activate reporter gene expression in response to E2 in Furthermore, estrogen-dependent transcriptional activation by ERβ is by tamoxifen E. M. S. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), while human ERβ has been shown to be by ICI 164,384 S. J. R. 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The at °C. of the the shift of WCE, of and of ERE or mutant ERE in and of and gel shift at for of the ERE by of ERE and at for and and also as described in the and on polyacrylamide containing in at and The to the of and to ERE a gel and region for of hERα S. N. Metzger D. 1993; PubMed Scopus Google Scholar) that by with and with of with at for at °C, by The with for at °C, by further and the DNA binding of hERβ, to and of the the sequence S. J. R. Lett. 1996; PubMed Scopus Google Scholar). The to contain to and the into the expression also contains the enabling in transcription S. Scheer E. Nucleic Acids Res. 1988; Scopus Google Scholar), as and of S. J. R. Lett. 1996; PubMed Scopus Google Scholar) into to encoding amino the N-terminal to the to for a used to the of and In of and to the of the proteins. In of and containing the hERα L. A. Metzger D. M. Chambon P. EMBO J. 1989; PubMed Scopus Google used to and respectively. the of in in a and on by In of a at the of while in of in a of kDa, in with the from of the sequence S. J. R. Lett. 1996; PubMed Scopus Google Scholar). A of for than as be from the presence of an amino N-terminal to the of in containing FCS, with or and as described and in gel shift to a of at for in the presence of of ERE or a mutant ERE to the for at on a polyacrylamide gel for both and with the but not the with binding of hERα and β to we in the presence or absence of in and containing FCS, with or or ICI 182,780 to to shift as in each of in the presence of E2 or and and in the absence of ligand or in the presence of ICI of for as has been described (see the in the presence of ICI, levels of for In it that the and the faster in the presence of E2 than in the absence of ligand or the presence of or The described that DNA binding by hERα and affected by the different al. E.M. Katzenellenbogen B.S. J. Biol. Chem. 1996; 271: 24172-24178Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) have shown that at 37 ERE binding by the of temperature on DNA binding by hERβ, we gel shift in and at or 37 in the presence or absence of used to to differences in DNA binding due to degradation of the receptor in the gel shift of with or at or 37 for in the presence or absence of by of ERE and a further at 37 for In the presence of E2 or OHT, at or 37 on DNA binding by and and and in the absence of ligand or in the presence of ICI, at 37 in DNA binding and and and are in with described (25Metzger D. Berry M. Ali S. Chambon P. Mol. Endocrinol. 1995; 9: 579-591Crossref PubMed Google Scholar). of ligand similarly affected DNA binding by while DNA binding in the presence of E2 or reduction in DNA binding also ICI the with and for of and that the DNA binding not due to degradation of or The that hERα and β are in way by at 37 in the absence of ligand or in the presence of ICI and further to that DNA in a manner similar to hERα in the absence of ligand and in the presence of E2 or the anti-estrogens but that DNA binding by hERα and β is affected by more the of temperature on DNA binding by and the DNA binding of hERα and we further gel shift in or and cell for of at or 37 from by the of ERE and a further at 37 for at on DNA binding in the absence or presence of not at 37 differential DNA binding A and In the presence of E2, its to bind to the ERE, and in the presence of OHT, a loss of in DNA binding In the absence of ligand or the presence of ICI, however, a reduction in the of of at 37 of at 37 in the presence of E2 in reduction in DNA binding and a reduction of in DNA binding in the presence of OHT, while loss in the absence of ligand or in the presence of ICI we the of temperature on by the in the presence or absence of ligand at for by in the presence of the ERE for of at 37 °C. A similar in DNA binding for and in the presence of E2 or and DNA binding for of in the absence of ligand or the presence of ICI in the presence of ICI, DNA binding by similar to that in the presence of E2 and OHT. In the absence of ligand, DNA binding by or than that in the presence of (25Metzger D. Berry M. Ali S. Chambon P. Mol. Endocrinol. 1995; 9: 579-591Crossref PubMed Google Scholar), the and at °C, DNA binding by in the absence of ligand or the presence of E2, OHT, and ICI binding for a similar the similar hERα and β are highly with amino acid sequence in the DNA binding We to whole cell from with and in to on gel the and at different in the gel (see the presence of a at a the be of the presence of of or in an at an and in further of the A/B region or the ligand binding domain of and at a the for or of and also in an and more on not of amino of also the presence of that the DNA binding domain is sufficient for of hERα and β for whole cell of with and or to shift as described for and of the presence or absence of and the of the and similar for each The mechanisms of ERα function have been extensively in the its and a of has also been on the mechanisms of action of estrogen tamoxifen and ICI studies have that ERα activates gene expression by binding to in responsive genes through the action of transcription activation functions AF-1 and The partial agonist tamoxifen (or inhibits AF-2 but not The of action of ICI 164,384 its derivative ICI 182,780) is more while it does not prevent DNA binding by ERα, it does not “protect” ERα from losing DNA binding in vitro, reduces its half-life due in to its of of ERα, as well as inhibiting transcription activation by both AF-1 and AF-2 (see the and comparison with other nuclear as well as have that the N-terminal domain of ERα in particular, amino the C of are for target gene (see the The of amino acid sequence hERα and β in region C and the of the that ERβ to the DNA elements as et al. E. M. S. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar) and et al. S. J. R. Lett. 1996; PubMed Scopus Google Scholar) have shown that ERβ can activate expression of reporter genes containing In we have the DNA binding of in the absence of and the presence of E2 or the anti-estrogens and ICI and in DNA binding by with DNA binding by We show that to the gene vitro. of of the ERE DNA binding by hERβ. is to bind to an ERE in the absence of ligand, in the presence of E2, or the estrogen and We that in DNA binding by is affected by at 37 in the absence of ligand or in the presence of ICI, with DNA binding in the presence of also from loss in DNA binding to a greater than in the absence of ligand or the presence of as described for hERα (25Metzger D. Berry M. Ali S. Chambon P. Mol. Endocrinol. 1995; 9: 579-591Crossref PubMed Google Scholar), differences in DNA binding are for the different the and are at DNA binding is a of at appears to be sufficient to protect from loss in DNA binding at 37 in the presence of ICI, a in DNA binding is for hERα in the presence of show that while E2 and OHT, but not ICI, prevent of hERα, DNA binding by is affected by temperature that ICI can “protect” from to In hERα and β bind to the DNA and in DNA binding are similar for both in the presence of E2 or the partial antagonist 4-hydroxy-tamoxifen. In the absence of ligand or in the presence of the antagonist ICI in DNA binding by hERα is affected at temperature (37 The mechanisms of loss in DNA binding are but are not due to degradation and may from loss of for DNA a similar for and are at 37 °C, at sufficient for in DNA binding at 37 °C, that greater in the absence of estrogens. acid sequence also that ERα and β may gel shift and show that hERα and β and that amino of hERα are sufficient for A dimerization function is in the ligand binding domain of hERα, and and hERα or more than with has been described for by hERα (25Metzger D. Berry M. Ali S. Chambon P. Mol. Endocrinol. 1995; 9: 579-591Crossref PubMed Google Scholar), hERα and β similar of to being in the absence of ligand or in the presence of E2, OHT, or hERα and β that in may in gene expression in cell in are The of ERβ in is as ERα and are that ERα is sufficient for the effects of estrogens. is however, that ERα and β act and ERα is sufficient to the (or in it is that ERβ is more than ERα in ERα and β can bind to similar DNA elements with by the existence of a second ER gene the of differences in trans-activation of genes due to the presence of ERα β. to as by also differential transcriptional activation by ERα and β. We and Chambon for of and and for ICI We are to of the and to for