Estrogen Receptor Binding to DNA Is Not Required for Its Activity through the Nonclassical AP1 Pathway

In the classical signaling pathway, the estrogen receptor (ER) binds directly to estrogen response elements (EREs) to regulate gene transcription. To test the hypothesis that the nonclassical pathway involves ER interactions with other proteins rather than direct binding to DNA, mutations were introduced into the DNA binding domain (DBD) of the mouse ERα. The effects of these DBD mutations were examined in DNA binding assays using reporter constructs containing either EREs (classical) or AP1 (nonclassical) response elements. Using the AP1 reporter, there was a reversal of ER action relative to that seen with the ERE reporter. Estradiol induced suppression, and the antiestrogen ICI 182,780 stimulated transcription of the AP1 reporter. DBD mutations in the proximal (P-box) of the first zinc finger of the ER (E207A/G208A and E207G/G208S) eliminated ERE binding. These mutants were inactive using the ERE reporter but retained partial or full activity with the AP1 reporter. The DBD mutant ERs interacted with Jun when tested in mammalian cell two-hybrid assays. Two mutations (K366D and I362R) in the ER ligand binding domain known to alter coactivator interactions impaired transcriptional responses using either the ERE or AP1 reporters. We concluded that ER action through the AP1 response element involves interactions with other promoter-bound proteins instead of, or in addition to, direct binding to DNA. Interactions with coactivators were required for both pathways. These data supported a model in which ER-mediated transcriptional activation or repression is dependent on the ligand and the nature of the response element in the target gene. In the classical signaling pathway, the estrogen receptor (ER) binds directly to estrogen response elements (EREs) to regulate gene transcription. To test the hypothesis that the nonclassical pathway involves ER interactions with other proteins rather than direct binding to DNA, mutations were introduced into the DNA binding domain (DBD) of the mouse ERα. The effects of these DBD mutations were examined in DNA binding assays using reporter constructs containing either EREs (classical) or AP1 (nonclassical) response elements. Using the AP1 reporter, there was a reversal of ER action relative to that seen with the ERE reporter. Estradiol induced suppression, and the antiestrogen ICI 182,780 stimulated transcription of the AP1 reporter. DBD mutations in the proximal (P-box) of the first zinc finger of the ER (E207A/G208A and E207G/G208S) eliminated ERE binding. These mutants were inactive using the ERE reporter but retained partial or full activity with the AP1 reporter. The DBD mutant ERs interacted with Jun when tested in mammalian cell two-hybrid assays. Two mutations (K366D and I362R) in the ER ligand binding domain known to alter coactivator interactions impaired transcriptional responses using either the ERE or AP1 reporters. We concluded that ER action through the AP1 response element involves interactions with other promoter-bound proteins instead of, or in addition to, direct binding to DNA. Interactions with coactivators were required for both pathways. These data supported a model in which ER-mediated transcriptional activation or repression is dependent on the ligand and the nature of the response element in the target gene. Estrogen has a wide range of physiologic activities, including the control of development, reproduction, and metabolism as well as effects on cell growth and differentiation. Most, if not all, actions of estrogen occur through its receptors, ERα1 and ERβ. The functional domains of the ER are relatively well defined. These domains include the N-terminal domain (A/B regions), DNA binding domain (C), hinge (D), ligand binding domain (E), and the C-terminal domain (F). A ligand-dependent activation function 2 in the C-terminal region of the ligand binding domain (LBD) and a ligand-independent activation function 1 in the N-terminal domain have also been characterized (1Tsai M.J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2689) Google Scholar, 2Ribeiro R.C. Kushner P.J. Baxter J.D. Annu. Rev. Med. 1995; 46: 443-453Crossref PubMed Scopus (216) Google Scholar). In the traditional model of ER action, the receptor binds as homodimers (3Kumar V. Chambon P. Cell. 1988; 55: 145-156Abstract Full Text PDF PubMed Scopus (957) Google Scholar) or heterodimers (4Tremblay G.B. Tremblay A. Labrie F. Giguere V. Mol. Cell. Biol. 1999; 19: 1919-1927Crossref PubMed Google Scholar, 5Ogawa S. Inoue S. Watanabe T. Hiroi H. Orimo A. Hosoi T. Ouchi Y. Muramatsu M. Biochem. Biophys. Res. Commun. 1998; 243: 122-126Crossref PubMed Scopus (449) Google Scholar, 6Pace P. Taylor J. Suntharalingam S. Coombes R.C. Ali S. J. Biol. Chem. 1997; 272: 25832-25838Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 7Cowley S.M. Hoare S. Mosselman S. Parker M.G. J. Biol. Chem. 1997; 272: 19858-19862Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar) to estrogen response elements (EREs) in the promoters of many, though not all, estrogen-responsive genes. Similar to other nuclear receptors, the ER recruits an array of transcriptional cofactors (coactivators and corepressors) that bind to the receptor and also interact with other transcription factors, including components of the general transcription factor apparatus. Some of the cofactors also possess chromatin-remodeling activities or recruit additional proteins to the complex to mediate transcription (reviewed in Ref. 8McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1648) Google Scholar). It is now recognized that the type of ligand bound to the ER influences its interaction with cofactors. The crystal structures of the ER LBD when bound to an agonist (estradiol) or an antagonist (raloxifene) have been solved. Comparison of these structures suggests a molecular basis for the differential ligand-dependent cofactor binding (9Brzozowski A.M. Pike A.C. Dauter Z. Hubbard R.E. Bonn T. Engstrom O. Ohman L. Greene G.L. Gustafsson J.A. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2929) Google Scholar). The binding of 17β-estradiol induces a major shift in the position of helix 12, one of several helices that form the coactivator interaction surface. Substitution of raloxifene or 4-hydroxytamoxifen (10Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2240) Google Scholar) for estradiol changes the orientation of helix 12 in a manner that partially obscures the residues involved in the coactivator interaction. Antagonist-bound ER binds to corepressors in vitro (11Smith C.L. Nawaz Z. O'Malley B.W. Mol. Endocrinol. 1997; 11: 657-666Crossref PubMed Scopus (556) Google Scholar, 12Lavinsky R.M. Jepsen K. Heinzel T. Torchia J. Mullen T.M. Schiff R. Del-Rio A.L. Ricote M. Ngo S. Gemsch J. Hilsenbeck S.G. Osborne C.K. Glass C.K. Rosenfeld M.G. Rose D.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2920-2925Crossref PubMed Scopus (581) Google Scholar), but these interactions are not as strong as those seen with certain other nuclear receptors such as the thyroid hormone or retinoic acid receptors. The region of cofactor binding has been localized to a hydrophobic surface of the LBD. Not all genes that are regulated by the ER contain an ERE. The mechanism for estrogen action through this “nonclassical” pathway (or pathways) is not clear. However, several lines of evidence suggest that the ER interacts with other transcription factors bound to their response elements (e.g. NF-κB, SP1, electrophile response element, AP1) in these target genes. Repression exerted through the NF-κB site has been examined in the context of the human interleukin 6 promoter (13Stein B. Yang M.X. Mol. Cell. Biol. 1995; 15: 4971-4979Crossref PubMed Google Scholar, 14Ray P. Ghosh S.K. Zhang D.H. Ray A. FEBS Lett. 1997; 409: 79-85Crossref PubMed Scopus (226) Google Scholar). In this case, repression is dependent on two transcription factors, NF-κB and CAAT enhancer-binding protein β. A direct interaction of NF-κB and ER has been demonstrated and requires the DBD and the D region of ER (13Stein B. Yang M.X. Mol. Cell. Biol. 1995; 15: 4971-4979Crossref PubMed Google Scholar). This direct protein binding contributes to interleukin 6 promoter repression by estrogen (14Ray P. Ghosh S.K. Zhang D.H. Ray A. FEBS Lett. 1997; 409: 79-85Crossref PubMed Scopus (226) Google Scholar). The ER has also been shown to affect gene expression from promoters containing an AP1 site. In some cases, such as the collagenase (15Paech K. Webb P. Kuiper G.G. Nilsson S. Gustafsson J. Kushner P.J. Scanlan T.S. Science. 1997; 277: 1508-1510Crossref PubMed Scopus (2064) Google Scholar, 16Webb P. Lopez G.N. Uht R.M. Kushner P.J. Mol. Endocrinol. 1995; 9: 443-456Crossref PubMed Google Scholar, 17Webb P. Nguyen P. Valentine C. Lopez G.N. Kwok G.R. McInerney E. Katzenellenbogen B.S. Enmark E. Gustafsson J.A. Nilsson S. Kushner P.J. Mol. Endocrinol. 1999; 13: 1672-1685Crossref PubMed Google Scholar), human insulin growth factor 1 (18Umayahara Y. Kawamori R. Watada H. Imano E. Iwama N. Morishima T. Yamasaki Y. Kajimoto Y. Kamada T. J. Biol. Chem. 1994; 269: 16433-16442Abstract Full Text PDF PubMed Google Scholar), or chicken ovalbumin (19Gaub M.P. Bellard M. Scheuer I. Chambon P. Sassone-Corsi P. Cell. 1990; 63: 1267-1276Abstract Full Text PDF PubMed Scopus (419) Google Scholar) promoters, estrogen activates expression. Of interest, in the context of the collagenase promoter ER antagonists also stimulate expression (15Paech K. Webb P. Kuiper G.G. Nilsson S. Gustafsson J. Kushner P.J. Scanlan T.S. Science. 1997; 277: 1508-1510Crossref PubMed Scopus (2064) Google Scholar, 17Webb P. Nguyen P. Valentine C. Lopez G.N. Kwok G.R. McInerney E. Katzenellenbogen B.S. Enmark E. Gustafsson J.A. Nilsson S. Kushner P.J. Mol. Endocrinol. 1999; 13: 1672-1685Crossref PubMed Google Scholar). Other genes containing an AP1 site in their promoters are negatively regulated by estrogen, including the ovine follicle-stimulating hormone β (20Miller C.D. Miller W.L. Endocrinology. 1996; 137: 3437-3446Crossref PubMed Scopus (46) Google Scholar) and human choline acetyltransferase gene (21Schmitt M. Bausero P. Simoni P. Queuche D. Geoffroy V. Marschal C. Kempf J. Quirin-Stricker C. J. Neurosci. Res. 1995; 40: 152-164Crossref PubMed Scopus (37) Google Scholar). In this report, we further examine the mechanism by which the ER acts through the nonclassical pathway, using the AP1 response element as a model. We demonstrate, using selective DBD mutations, that DNA binding by the ER is not necessary for its activity through this nonclassical pathway. The mouse ERα expression vector was provided by Malcolm Parker (Imperial Cancer Research Fund, London, United Kingdom) and subcloned into pcDNA3.1(−) (Invitrogen). Point mutations were introduced using overlapping polymerase chain reactions, and the sequence of the mutated cDNA was by DNA The expression vector the DNA binding domain to a of human its DNA binding domain acid to the in I. M. Res. PubMed Scopus Google Scholar). The DNA binding domain in was as a The reporter has been Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). The reporter AP1 to a promoter the reporter a of collagenase promoter to containing one AP1 site P. M. R. B. C. P. M. Cell. Full Text PDF PubMed Scopus Google Scholar). The reporter of the sequence of in the vector T. R. Biochem. Biophys. Res. Commun. 1998; PubMed Scopus Google Scholar). from estrogen receptor human B. R. 15: Google Scholar), were in with receptor from human provided by V. were in with and were using and in and with with were to or in 1 to were with as T. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and were with as Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). and reporter were with receptor expression vector or vector as a cell two-hybrid the reporter to activity and its interaction with ER mutants was from and ICI 182,780 was provided by Estradiol and ICI 182,780 were to as in was to control in the activity was or by using an and as relative The and of or are shown for were or with The ERE and the for shift assays were M. Y. T. Mol. Endocrinol. 1999; 13: PubMed Scopus Google Scholar). were by in in vitro with a binding containing DNA, and 1 17β-estradiol for and with ERE for in a of The were and to through and was by were with the expression and in with were as C. S. T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The were using and was using mouse ER and were using an to the The response to ER and antagonists was examined using classical and nonclassical pathway reporter the classical pathway, which two of the gene ERE of the of was the nonclassical pathway, two AP1 were which a of the collagenase promoter to and a AP1 site P. M. R. B. C. P. M. Cell. Full Text PDF PubMed Scopus Google Scholar), and which AP1 of a promoter were in In the of hormone not alter the activity of of these reporter the of ER or other estrogen-responsive not ER was with the ERE reporter, estradiol and the antiestrogen ICI 182,780 transcription The responses to and antagonists were when the reporter was Estradiol and the antiestrogen ICI 182,780 stimulated promoter activity AP1 These effects were for the of AP1 response was using a reporter the AP1 but the of the promoter and the vector not A reporter containing a of the collagenase promoter to which a AP1 a response to the reporter and all were with with the effects of estradiol and ICI and 4-hydroxytamoxifen stimulated transcription not constructs were also into in the of the ER expression those in Estrogen the ERE reporter and the and ICI 182,780 the ERE reporter and the and The ligand effects on the nonclassical pathway were when ER was into with the reporter not To the nonclassical pathway requires ER binding to DNA, mutations were introduced into the ER The mutant receptors were examined for DNA binding and functional activity using the ERE and AP1 reporters. These mutations were to ER action through the classical pathway but to the to interact with other transcription factors, actions through the nonclassical pathway. The of the zinc and the of introduced mutations are in The first mutants are all the of the first zinc a region known to mediate interaction with DNA V. S. M. Chambon P. Cell. Full Text PDF PubMed Scopus Google Scholar). The mutant has been demonstrated to binding to the ERE S. V. H. Chambon P. Nature. PubMed Scopus Google Scholar). The mutant was with the of the protein by with In the was to the direct interaction of the with DNA L. D. Cell. Full Text PDF PubMed Scopus Google Scholar). The is in the of the zinc a region that has been in and DNA binding L. D. Cell. Full Text PDF PubMed Scopus Google Scholar). The DNA binding of these mutants were tested in shift assays binding and binding relative to the type The in the domain retained a of ERE binding The was than the other mutants DNA binding The of these mutants to through the classical and nonclassical was examined in assays A and of the mutants or estrogen through the classical pathway The mutant retained estrogen through the ERE reporter. The of activity with the and mutants was using containing EREs from other estrogen genes such as human and human not of nuclear demonstrated expression of the and mutant proteins In the AP1 reporter the and mutants retained the nonclassical of The mutant was the with responses to those of the selective of ERE binding and transcriptional control by the classical pathway but full by the nonclassical pathway. The mutant the to through the nonclassical pathway. A mammalian two-hybrid was to test the hypothesis that ER interacts with AP1 proteins in the nonclassical pathway. or mutant ER was with a using as a reporter In control the DBD not the reporter, either in the or of expression but not to estradiol or ICI ER was estradiol ICI 182,780 a that the activity seen in the AP1 reporter assays. The activities of the DBD mutants were also tested in this a mutant that the nonclassical response for AP1 transcription in the of ICI 182,780 The and which activity in the AP1 reporter to a than or ER all activity in both the AP1 reporter and in the interaction the transcriptional of the ER DBD mutants are in the and AP1 reporter assays. The LBD of ER an in cofactor which are to ER activity through the ERE. Two mutations and were introduced into ER helix which with helices and 12 a hydrophobic that is involved in cofactor The were on their of when with nuclear receptors and mutations have been to interactions with including receptor coactivator 1 P.M. E. Parker M.G. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar, Hoare S. P.M. Parker M.G. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). Using a mammalian two-hybrid these mutant ERs were to or all of their interaction with and in the of estradiol not The eliminated the effects of estradiol and ICI 182,780 and the ERE reporter, activity was as were estradiol activation and ICI the AP1 reporter, estradiol was eliminated and ICI was The of the was dependent on the ligand Estradiol of the ERE reporter and of the AP1 reporter was ICI 182,780 stimulated the ERE reporter and a with the AP1 reporter. These that ER mutations that alter binding to transcriptional cofactors ER action through both the classical and nonclassical pathways. The effects of the helix mutants in the mammalian two-hybrid assays with were to those seen with the AP1 reporter, with the that ER actions on the AP1 reporter are through interactions with In the classical pathway, ER action is by direct receptor binding to binding induces an ER that interactions with coactivators and general transcription factors, in transcription. bound to an the ER not interact with coactivators in not transcription. In the mechanism by which ER nonclassical is well We that this mechanism involves ER interactions with other rather than direct binding to DNA. genes were as a model for the nonclassical pathway. these reporter and antagonists transcription in the of We a DBD mutant of ER that through the classical pathway but retained of the nonclassical pathway. These data into by which nonclassical ER signaling estrogen receptor or antagonists have an on the nonclassical AP1 pathway, and antagonists transcription. a reversal of activity suggests that a mechanism ER activity through the nonclassical AP1 pathway. is to the of ER to bind to an ERE and activity through the nonclassical pathway. However, as the ER DBD interacts with other such as and this interaction requires a the interaction ER and cofactors has functional both in the classical and the nonclassical pathways. In the nonclassical pathway, we that estradiol and ICI 182,780 activates transcription the AP1 reporter, a that is the of the effects on the ERE reporter. the activation of transcription by an antagonist is with other (15Paech K. Webb P. Kuiper G.G. Nilsson S. Gustafsson J. Kushner P.J. Scanlan T.S. Science. 1997; 277: 1508-1510Crossref PubMed Scopus (2064) Google Scholar, 16Webb P. Lopez G.N. Uht R.M. Kushner P.J. Mol. Endocrinol. 1995; 9: 443-456Crossref PubMed Google Scholar, 17Webb P. Nguyen P. Valentine C. Lopez G.N. Kwok G.R. McInerney E. Katzenellenbogen B.S. Enmark E. Gustafsson J.A. Nilsson S. Kushner P.J. Mol. Endocrinol. 1999; 13: 1672-1685Crossref PubMed Google Scholar), the of estradiol on the collagenase promoter has not been of in However, of gene expression is a physiologic and genes such as interleukin 6 (13Stein B. Yang M.X. Mol. Cell. Biol. 1995; 15: 4971-4979Crossref PubMed Google Scholar, 14Ray P. Ghosh S.K. Zhang D.H. Ray A. FEBS Lett. 1997; 409: 79-85Crossref PubMed Scopus (226) Google Scholar), factor S. S. S. R. J. 1999; PubMed Scopus Google Scholar), follicle-stimulating hormone β (20Miller C.D. Miller W.L. Endocrinology. 1996; 137: 3437-3446Crossref PubMed Scopus (46) Google Scholar), choline acetyltransferase (21Schmitt M. Bausero P. Simoni P. Queuche D. Geoffroy V. Marschal C. Kempf J. Quirin-Stricker C. J. Neurosci. Res. 1995; 40: 152-164Crossref PubMed Scopus (37) Google Scholar), Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar), and H. H. Y. T. T. K. K. M. Y. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In cases, involves the AP1 site (20Miller C.D. Miller W.L. Endocrinology. 1996; 137: 3437-3446Crossref PubMed Scopus (46) Google Scholar, M. Bausero P. Simoni P. Queuche D. Geoffroy V. Marschal C. Kempf J. Quirin-Stricker C. J. Neurosci. Res. 1995; 40: 152-164Crossref PubMed Scopus (37) Google H. H. Y. T. T. K. K. M. Y. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). ER action through the nonclassical pathway not ER binding to DNA and to by the in the first zinc finger DNA binding and ERE activation but activity through the nonclassical pathway. A for the interaction in the nonclassical pathway is a of the AP1 protein This is supported by evidence for ER interaction with in mammalian two-hybrid assays and by the that ER mutants have effects when tested in interaction assays or in AP1 reporter assays. The interaction Jun and ER in a two-hybrid not as other proteins (e.g. in the interaction. to Jun with ER in shift assays or to a not direct interactions not It is that these interactions are not strong to the cofactors such as receptor coactivator 1 or other proteins or the have demonstrated a direct interaction ER and using assays P. Lopez G.N. Uht R.M. Kushner P.J. Mol. Endocrinol. 1995; 9: 443-456Crossref PubMed Google Scholar, M. D. J. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). It is that in the mammalian two-hybrid the interaction was induced by ICI 182,780 but not by that changes the protein The mechanism of repression but by the promoter context of the activation of interleukin 6 promoter requires the activity of two transcription factors, CAAT enhancer-binding protein β and In this case, repression to interactions with of these factors (13Stein B. Yang M.X. Mol. Cell. Biol. 1995; 15: 4971-4979Crossref PubMed Google Scholar, 14Ray P. Ghosh S.K. Zhang D.H. Ray A. FEBS Lett. 1997; 409: 79-85Crossref PubMed Scopus (226) Google Scholar). repression mechanism of the Jun N-terminal pathway C. A. 1997; 11: PubMed Scopus Google Scholar). repression of the receptor of NF-κB ligand to of expression and a in Jun by the N-terminal A.C. Pike Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). with the AP1 reporter, ER and the DBD mutants dependent transcription in the of ICI We that a containing the and domains of ER not affect transcription from the AP1 reporter the the domain but containing the DBD transcription in the manner as ER not These are with several P. Lopez G.N. Uht R.M. Kushner P.J. Mol. Endocrinol. 1995; 9: 443-456Crossref PubMed Google Scholar, Y. Kawamori R. Watada H. Imano E. Iwama N. Morishima T. Yamasaki Y. Kajimoto Y. Kamada T. J. Biol. Chem. 1994; 269: 16433-16442Abstract Full Text PDF PubMed Google M. D. J. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar) and the that ER interactions with Jun a region the It is also that the activity through the nonclassical pathway, as well as interaction with though this mutant partial activity through the ERE. ER zinc finger is the protein the zinc in It is well that coactivators are involved in the classical pathway. Using selective ER mutants and that alter interactions with we that these mutants not affect the classical pathway but also ER action through the nonclassical AP1 pathway. The hydrophobic these mutations were introduced interacts with both coactivators and the of residues to and in thyroid hormone receptor activation but also repression and the interaction with corepressors nuclear receptor and for retinoic acid and thyroid hormone receptors Nature. 1999; PubMed Scopus Google Scholar). It is that the and mutations affect interactions with corepressors we not interactions ER and corepressors in two-hybrid assays not these interactions are than the that the reversal of the effects on the nonclassical pathway is of the reversal of cofactors bound to It is that the antagonist ICI 182,780 an interaction the ER and coactivators and that estradiol an interaction the ER and However, is that when the ER is involved in a interaction instead of binding to an the hydrophobic cofactor binding a the interactions of the region of the LBD both coactivators and corepressors Nature. 1999; PubMed Scopus Google Scholar), the that changes in the protein a of proteins recognized by the The mutant an additional in of the hypothesis that changes in protein have a on the of This not the interaction with coactivators and transcription through the classical pathway in the of However, this mutant also activates transcription in the of an In this the some mutations of helix 12 that an antagonist to an include the mutant of human ERα K. A.L. Katzenellenbogen B.S. Mol. Endocrinol. 1996; PubMed Scopus Google Scholar) and the mutants and of mouse estrogen receptor A. E. S. Parker M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), which are by ICI and We that for the AP1 response element, ER interacts with other proteins instead of, or in addition to, DNA to its transcriptional The nonclassical pathway the for cofactors. However, the of cofactor binding as demonstrated by the reversal of activities through the AP1 reporter. These data the of ER activity to changes in its by mutations or by an of We Malcolm Parker for for ICI and for and the human and human ERE reporter estrogen receptor ligand binding domain estrogen response element DNA binding domain sequence type mouse ERα