CA2301143A1 - Differential ligand activation of estrogen receptors er.alpha. and er.beta. at ap1 sites - Google Patents

Abstract

This invention provides methods of screening test compounds for the ability to activate or inhibit estrogen receptor .beta. (ER.beta.) mediated gene activation at an AP1 site. In particular, the methods involve providing a cell comprising an estrogen receptor .beta. (ER.beta.), AP1 proteins, and a construct comprising a promoter comprising an AP1 site which regulates expression of a first reporter gene. The cell is contacted with the test compound and changes in expression levels of the reporter gene are detected indicating whether the test compounds activate transcription, inactivate transcription or have no effect at the AP1 site.

Description

DIFFERENTIAL LIGAND ACTIVATION OF ESTROGEN
RECEPTORS ERa AND ER(i AT AP1 STTES
CROSS-REFERENCE TO RELATED INVENTIONS
[ Not Applicable ]
This invention was made with the Government support under Grant No.
GM 50872, awarded by the National Institutes of Health. The Government of the United States of America may have certain rights in this invention.

Estrogens, antiestrogens, and other nuclear transcription factor ligands are used in a wide variety of therapeutic contexts. Thus, for example, estrogens are used in the treatment of osteoporosis and other aspects {e.g., vasomotor instability) of menopause, in the treatment of hypoestrogenism, and in the regulation of fertility.
Antiestrogens are used in the treatment of cancer. Tamoxifen, for example, is an antiestrogen that is used in breast cancer chemotherapy and is believed to function as an antitumor agent by inhibiting the action of the estrogen receptor (ER) in breast tissue (see, e.g., {Sutherland et al. {1987) Cancer Treat. Revs, 15: 183-194).
Glucocorticoids are used in the treatment of pure red cell anemia, acute renal failure due to acute glomerulonephritis or ~ lymphocytic leukemias, lymphomas, and other conditions.
Progestins or progestational agents such as medroxyprogesterone or megestrol acetate are used in the treatment of endometrial carcinoma and breast carcinoma, and are used in the regulation of fertility.
It has long been known that nuclear transcription factor ligands may have profound and contradictory effects upon patients depending on physiological context. For example, estrogen and estrogen agonists may have beneficial effects, such as preveming osteoporosis and reducing serum cholesterol (hove, et al. ( 1992) New Eng. J.
Med. 326:
852-856; Love, etal. (1990)J. Natl. Cancerlnst. 82:1327-1332). Conversely, agonistic activity may also be harmful. Tamoxifen for example sometimes increases endometrial tumor incidence (Tmo et al. (1991) Cancer Tread &Res. 53: 228-237) or switches from 0 inhibition to stimulation of estrogen dependent growth in breast tumor progression (Parker ( 1992), Cancer Surveys 14: Growth Regulation byNuclear Hormo~re Receptors.
Cold Spring Harbor Laboratory Press).
The related benzothiophene analog raloxifene (Figure 1 A) has been reported to retain the amiestrogen properties of tamoxifen in breast tissue and to show minimal estrogen effects in the uterus; in addition, it has potentially beneficial estrogen-like effects (in nonreproductive tissue such as bone and cardiovascular tissue (Jones et al.
(1984) J. Med Chem., 27: 1057-1066; Black et al. (1994) J. Clip. Irrvest., 93:
63-69;
Sato et al. (1996) FASEB J., 10: 905-912; Yang et al. (1996) Endocrinol., 137:

2084; Yang et al., (1996) Science, 273: 1222-1225).
One explanation for these tissue-specific actions of antiestrogens is that the ligand-bound ER has different transactivation properties when bound to different types of DNA enhancer elements. The estrogen receptor (ER) has been shown to mediate gene transcription both from the classical estrogen response element (ERE) and from an AP 1 enhancer element that requires ligand and the AP1 transcription factors Fos and Jun for transcriptional activation (Fig.1B). In transactivation experiments, tamoxifen inhibits the transcription of genes that are regulated by a classical ERE, but like the natural estrogen hormone 17/3-estradiol [F.,~ (Fig. 1 A)], tamoxifen activates the transcription of genes that are under the control of an AP 1 element (Webb, et al ( 1995) Mol. Endo., 9:
443-456).
At the end of 1995, a second ER (ER~i) was cloned from a rat prostate cDNA library (Kuiper et al. (1996) Proc. Natl. Acac~ Sci., USA, 93: 5925-5930). The human (Mosselman et al. (1996) FEES Lett., 392: 49-53) and mouse (Tremblay et al.
(1997) Mol. E~rdocrinol., 11: 353-365) homologs have also been cloned. The first identified ER has been renamed ERa (Kuiper et al. ( 1996) supra. ). The existence of two ERs was postulated to present a potential new mechanism tissue-specific estrogen regulation.
From the foregoing, it is clear that the activity and regulation of nuclear transcription fiictor ligands, especially estrogens, is complex and the use of various transcription factor ligands can lead to contradictory and often adverse consequences.
Thus, when electing to use a nuclear transcription factor ligand in a therapeutic context, it is desirable to eluadate as precisely as possible the various modes of action (biological wo 99W~6o PGT/US98J18030 0 activities) of the agents) under consideration. Similarly, it has long been known that various environmental compounds have estrogenic and possl'bly antiestrogenic activity.
When evaluating the impact of such environmernal estrogens and/or antiestrogens, it is desirable to evaluate their effect on all metabolic pathways in which they might prove active.
SUMMARY OF THE INVENTION
The present invention provides methods to rapidly and effectively screen compounds for their ability to activate or inactivate gene transcription in a previously unknown regulatory pathway: an estrogen receptor beta (ER~i)-mediated AP 1 pathway.
This invemion is premised, in part, on the surprising discovery that ER~i is capable of intersecting with AP 1 to induce transcription of a gene under AP 1 control.
Even more surprising was the discovery that ER~i-mediated AP 1 interactions can produce results significantly different than ERac-mediated AP 1 interactions. For example, estradiol, which activates gene expression through an ERai-mediated AP1 interaction, actually inhibits gene activation through an ERa-mediated AP 1 interaction.
In one embodiment, this invention provides methods of screening test compounds for differential ERac-mediated and ERA-mediated activation at an APl site.
The methods typically involve providing a first cell comprising an estrogen receptor (3 (ER~i), an AP 1 protein, and a conshuct comprising a promoter comprising an AP
1 site which regulates expression of a first reporter gene. The first cell is contacted with the test compound and the expression of the first reporter gene is compared with ERa-mediated expression of a gene at an AP 1 site in response to the same test compound.
The cell can contain a heterologous estrogen receptor beta (ER~i) and preferred ER(3s comprise an amino acid seqeunce of SEQ ID NO: 3 or SEQ ID NO: 5. The cell can also contain a heterologous AP1 protein. Preferred reporter genes used in this assay include chloramphenicol acetyl transferees (CAT), luciferase, (3 -galactosidase (~i-gal), alkaline phosphatase, horse radish peroxidase (HIZP), growth hormone (GH), and green fluorescent protein (GFP) with a luciferase gene or a green fluorescent protein (gene) being preferred. The test compound can be a compound known or suspected to have anti-estrogenic activity. The method can be one in which the ERa-mediated expression of a gene at an AP1 site is determined by providing a second cell comprising an estrogen 0 receptor a (ERac), AP1 proteins, and a construct comprising a promoter comprising an AP1 site which regulates expression of a second reporter gene. The second cell is contacted with the test compound; and expression of the second reporter gene is detected.
One preferred standard estrogen response element is from the Xenopus vitellogenin AZ
gene. The second reporter gene and the first reporter gene can be the same species of reporter gene. The cell and the second cell are the same cell.
In one embodiment, this invention provides methods screening a test compound for the ability to activate or inhibit estrogen receptor beta (ER(3) mediated gene activation at an AP 1 site. The methods typically involve providing a first cell comprising an estrogen receptor ji {ERA), AP 1 proteins, and a construct comprising a promoter comprising an AP1 site which regulates expression of a first reporter gene.
The cell is contacted with a test compound and expression ofthe first reporter gene is detected. The cell can contain a native or heterologous estrogen receptor beta (ER~i). In a preferred embodiment, the ER j3 the amino acid sequence of Sequence ID No: 3 or Sequence ID No:

5. The first cell can also contain a heterologous AP1 protein (e.g,. jun and/or fos).
Virtually any reporter gene may be used. Preferred reporter genes include, but are not limited to chloramphenicol acetyl transferase (CAT), luciferase, (3 -galactosidase (~i-gal), alkaline phosphatase, horse radish peroxidase (HItP), or Been fluorescent protein (GFP) with a luciferase or a green fluorescent protein (GFP) being most preferred.
Virtually any compound can be screened according to the methods of this invention. However, preferred test compounds are compounds known to have anti-estrogenic activity.
In another embodiment, the above method can fiuther involve providing a second cell comprising an estrogen receptor a (ERa), AP 1 proteins, and a construct comprising a promoter comprising an AP1 site which regulates expression of a second reporter gene. The second cell is contacted with the test compound and the expression of the second reporter gene is then detected. In addition, or alternatively, the above method can involve providing a third cell comprising an estrogen receptor a (ERa), and a construct comprising a promoter comprising a standard estrogen response element (ERE) which regulates expression of a third reporter gene. The third cell is contacted with the test compound; and expression of the third reporter gene is then detected. One standard estrogen response element can be from the Xenopus vitellogenin A2 gene.

wo ~nm6o rcrnrsmso3o 0 Additionally or alternatively, the above method can also imrolve providing a fourth cell comprising an estrogen receptor ~ (ER(i), and a construct comprising a promoter comprising a standard estrogen response element (ERE) which regulates expression of a fourth reporter gene. The fourth cell is contacted with the test compound and expression of the fourth reporter gene is detected. Again the standard estrogen response element can 5 be from the Xenopus vitellogenin A2 gene. In one embodiment, the first cell and said third cell are the same cell, while in another embodiment, the first cell and said fourth cell are the same cell.
Any of the above-described assays can be nm to detect or identify inhibitors that block compounds that activate ER/i-mediated AP 1 gene transcription. This typically involves performing the assays as described above, but, in addition, contacting the first cell with a second compound, in addition to the test compound, wherein said second compound is known to activate transcription through estrogen receptor (3 (ER(3) mediated gene activation at an AP 1 site. Detecting then comprises detecting test compound mediated decrease in said estrogen receptor [i (ERA) mediated gene activation at an AP1 site. In a particularly preferred embodiment, the detecting can involve comparing the expression of the first reporter gene in the presence of the test compound and the second compound with the expression of the reporter gene in the presence of the second compound without the test compound.
In one embodiment, the second compound known to activate transcription through estrogen receptor (3 (ER(i) mediated gene activation at an AP1 site is identified by a method involving providing a second cell comprising an estrogen receptor ~ (ER~i), and AP 1 protein, and a construct comprising a promoter comprising an AP 1 site that regulates expression of a second reporter gene. The second cell is contacted with the s~;ond compound and the expression of the second reporter gene is detected where an increase in expression of the second reporter gene produced by the compound indicates that said second compound activates transcription through ERA at an AP1 site.
The assays ofthis invention can also be used to detect or identify inhibitors that block compounds that inhibit ER/3-mediated AP 1 gene transcription. These methods involve performing the assays as described above, while additionally contacting the first cell with a second compound, in addition to the test compound, where the second w0 99/11760 PCT/US98/18030 0 compound is known to inhibit transcription through estrogen receptor ~ (ERA) mediated activity at an AP1 site. Expression of the reporter gene is detected where the detection comprises detecting test compound mediated increase in estrogen receptor (3 (ERA) mediated gene activation at an AP 1 site. The detecting can involve comparing expression of the first reporter gene in the presence of both the second compound and the test compound with expression of the first reporter gene in the presence of the second compound without the test compound.
The second compound known to inhibit transcription through estrogen receptor ø (ERA) mediated gene activation at an AP 1 site can be identified by providing a second cell comprising an estrogen receptor (3 (ER~i), and AP 1 protein, and a construct comprising a promoter comprising an AP1 site that regulates expression of a second reporter gene. The second cell is contacted with the second compound; and expression of the second reporter gene is detected. A decrease in expression of said second reporter gene produced by the second compound indicates that the second compound inhibits transcription through ERA at the AP 1 site.
This invention also provides for any ofthe cells described above or herein.
In one embodiment the cell comprises an estrogen receptor (3 (ER~i), an AP 1 protein (e.g., jun or fos), and a construct comprising a promoter comprising an AP 1 site which regulates expression of a first reporter gene. The cell can additionally include a receptor for a nuclear transcription factor ligand preferably for a nuclear transcription factor ligand other than estrogen. The cell preferably contains a heterologous ER(i, more preferably an ER(i comprising an amino acid sequence of Sequence 1D No: 3 or Sequence m No:
5. The AP 1 protein can be a native AP 1 protein or a heterologous AP 1 protein. The reporter gene can be one selected from the group consisting of chloramphenicol acetyl transferees (CAT), luciferase, (i -galactosidase ([i-gal), alkaline phosphatase, horse radish peroxidase (HRP), and green fluorescent protein (GFP), but in particulary preferred embodiment, the reporter gene encodes a luciferase or a green fluorescent protein (GFP).
The cell can additionaly include a standard estrogen response element (FRE) which regulates expression of a second reporter gene. One preferred standard estrogen response element is from the Xenopus viteUogenin A2 gene. Preferred cells of this invention are mammalian cells and particularly preferred cells are derived from breast tissue or from wo ~ni~6o rc~r~smso3o 0 uterine tissue. The cells may be nooplastic cells. Air of the above-described assays can be run to detect or identify inhibitors that block compounds that activate ERø-mediated AP 1 gene transcription.
In still another embodiment, this invention provides methods of screening a nuclear tr~aascription factor ligand for the ability to modulate estrogen receptor (3 S mediated activation or inactivation of transcription at an AP 1 site. The methods involve providing a first cell containing an estrogen receptor ~ (ER~i), an AP 1 protein, a receptor for the nuclear transcription factor ligand, and a construct comprising a promoter comprising an AP1 site which regulates expression of a first reporter gene.
The cell is contacted with the transcription fi~ctor ligand and with a compound having ERA
mediated activity at the AP 1 site. Expression of the first reporter gene is then detected.
The method can fiuther involve providing a second cell containing an estrogen receptor (3 (ER~i), a receptor for the nuclear transcription fi~ctor ligand, and a construct comprising a promoter comprising an estrogen response element (ERE) that regulates expression of a second reporter gene. The second cell is contacted with the transcription factor ligand and with the compound having AP-1 mediated estrogenic activity and expression of the second reporter gene is detected. The first and second cells can be the same or different.
Alternatively, or in addition, the method can further involve providing a second cell comaining a cognate receptor of the transcription factor ligand, and a promoter comprising a response element for the cognate receptor that regulates expression of a second reporter gene. The second cell is contacted with the transcription factor ligand and with the compound having compound having ER(i mediated activity at said AP1 site expression of the second reporter gene is detected. Again, the first and second cells can be the same or different cells.
In any of the above-described methods the nuclear transcription factor ligand can be selected from the group consisting of a glucocorticoid, a progestin, vitamin D, retinoic acid, a an androgen, a mineralcorticoid, and a prostaglandin.
Similarly, the cognate receptor can be selected from the group consisting of an estrogen receptor, a glucocorticoid receptor, a progestin PR A receptor, and progestin PR B
receptor, androgen receptor, a mineralcorticoid receptor, and a prostaglandin receptor.
In a 0 particularly preferred ~nbodiment, the ERS comprises an amino acid sequence of Figure or Figure 6A. The ERA can be a heterologous ER~i. Similarly, the receptor for the nuclear transcription factor ligand can be heterologous to the cell. The cell can express an AP 1 protein (e.g., jun or fos) from a heterologous DNA. In one particularly preferred embodiment, the nuclear transcription factor is a progestin; and said receptor for the 5 nuclear transcription factor ligand is a progestin receptor. In another preferred embodiment, the nuclear transcription factor is a glucocorticoid and said receptor for said nuclear transcription factor ligand is a GR receptor.
This invention also provides methods of screening an agent for the ability to alter modulation of estrogen receptor [3 (ER~i) activation or inactivation of transcription at an AP 1 site by a nuclear transcription factor ligand. The methods involve providing a first cell containing an estrogen receptor ji (ER/3), an AP 1 protein, a receptor for the nuclear transcription factor ligand, and a promoter comprising an API
site which regulates expression of a first reporter gene. The first cell is contacted with the transcription factor ligand, with a compound having ERA mediated activity at an AP 1 site, and with the agent and expression of the first reporter gene is detected.
This method can further involve providing a second cell containing an estrogen receptor (3 (ER~i), a receptor for the nuclear transcription factor ligand, and a promoter comprising an estrogen response element (ERE) that regulates expression of a second reporter gene. The second cell is contacted with the transcription factor ligand and with the compound having AP-1 mediated estrogenic activity and expression of the reporter gene is detected. The first and second cell can be the same cell or different cells.
The nuclear transcription factor can be one selected from the group consisting of a glucocorticoid, a progestin, vitamin D, retinoic acid, an androgen, a mineralcorticoid, a 2S prostaglandin. Similarly, the nuclear transcription factor ligand is selected from the group consisting of an estrogen receptor, a glucocorticoid receptor, a progestin PR
A receptor, progestin PR-B receptor, an androgen receptor, a mineralcorticoid receptor, and a prostaglandin receptor. Again, in any of the assays described herein, the ERj3 can be a heterologous ER(i and in a preferred embodiment, the ER~i comprises an amino acid sequence of Sequence m No: 3 or S~uence ID No: 5 or is encoded by a nucleic acid WO 99/11760 PCTlUS98/18030 0 sequence of Sequence ID No: 3 or Sequence ID No: 6. The AP 1 proteins) and/or the receptor for the nuclear transcription factor ligand can also be native to the cell or heterologous. In one particularly preferred embodimem, the nuclear transcription factor is a progestin; and the receptor for said nuclear transcription factor ligand is a progestin receptor, while in another preferred embodiment, the nuclear transcription factor is a glucocorticoid and the receptor for said nuclear transcription factor ligand is a GR
receptor.
This invention also provides kits for screening a compound for the ability to activate or inhibit estrogen receptor (3 (ER(3) mediated gene activation at an AP1 site.
The kits can include a container containing a cell comprising an estrogen receptor ~3 (ER~i), an AP 1 protein (e.g., jun and/or fos), and a construct comprising a promoter comprising an AP 1 site which regulates expression of a first reporter gene.
The cell of the kits can further a receptor for a nuclear transcription factor ligand, preferably a nuclear transcription factor ligand other than estrogen. The kits can also further include instructional materials containing protocols for the practice of any of the assay methods described herein.
DEFINITIONS
The terms "activate transcription" or "inhibit transcription" as used herein refer to the upregulation of transcription of a gene or the downregulation of transcription of a gene. It will be appreciation that either complete, or partial, "turning on" or "turning off' are is regaxded herein as activation or inhibition, respectively.
Activation and inhibition of transcription are typically measured with respect to a control or controls where the comrol or controls involve a similar treatment lacking the compound or agent in question and/or contain a standard agent (e.g., E2 or tamoxifen). It will also be appreciated that there may exist a baseline level of transcription (e.g, of a particular reporter gene) even where an assay cell of this imrention is '~nstimulated"
(e.g. the receptor in question is unliganded), i. e., without exogenously supplied ligand). In this case, it may be possible to see inhibition without necessarily applying exogenous activator see, e.g., Example 1).
As used herein an antiestrogen is a compound that substantially inhibits estrogen activity as measured in an assay for estrogenic activity, for example, cellular 0 assays as din Webb et al. Mol. Ei~OCrir~ol., 6:157-167 (1993). More generally, a "transcription factor antagonist" is a compound that substamially inhibits transcription factor activity as meas~ued in a standard assay for that transcription factor activity.
A "nuclear transcription factor" as used herein refers to members of the nuclear transcription factor supetfiunily. This is a family of receptors that are capable of 5 entering the nucleus of a cell and once there, effecting the up-regulation or down-regulation of one or more genes. A "nuclear transcription factor ligand" is a compound that binds to a nuclear transcription factor. Preferred nuclear transcription factors are typically steroid receptors, however, the group is not so limited. Nuclear transcription factor ligands include, but are not limited to estrogen, progestins, androgens, 10 mineralcorticoids, glucocorticoids, retinoic acid, vitamin D, and prostaglandins.
Transcription factor ligands also include analogues of naturally occurring factors~and blocking agents (antagonists) of such factors. Transcription factors also include, as they are identified, the ligands that bind orphan receptors (those nuclear transcription factors which have been identified by sequence homology, but whose ligand is yet unidentified).
It will be appreciated that when used in the context of a modulator of estrogen activity, the nuclear transcription factor ligand is typically one other than estrogen (or other than the estrogen or estrogen agonist whose activity is being modulated). Nuclear transcription factors typically mediate their activity through binding of a cognate receptor in the cell nucleus. The term cognate receptor" refers to a receptor of the type that is typically bound by the transcription ligand in question. Thus; the cognate receptor for an estrogen is an estrogen receptor, the cognate receptor for a glucocorticoid is a glucocorticoid receptor, the receptor for a progestin is a progestin receptor, and so forth.
The cognate receptor includes the native (naturally occurring) form as well as modified receptors.
The phrase estrogen receptor beta (ER~irmediated activation or inactivation of gene transcription at an AP 1 site refers to the activation or inactivation of a gene (e.g., a reporter gene) under control of an AP 1 site by the interaction of that AP 1 site with a liganded ER(i receptor. Similarly ERa-mediated activation or inactivation refers to gene regulation mediated by the interaction of ERa. Inactivation or inactivation at an ERE refers to activation or inactivation of a gene under control of an ERE.

WO 99/11'160 PCT/US98/18030 0 The phrase "digsl ERa-mediated and ERA-mediatod activation at an AP 1 site" refers to differences between ERa- and ER(3-mediated gene activation at an AP1 site in response to the same ligand. Differential activation can be reflected in significant differences in levels of gene activation or inactivation by the same ligand depending on whether it interacts with ERa or ERA. Differential activation can also reflect differences in the "sign" of gene activation. Thus differential activation can refer to ER[3-mediated activation oftranscription at an AP 1 site and ERa-mediated inactivation of gene transcription at an AP1 site in response to the same ligand.
Conversely, differential activation can refer to ERj3-mediated inactivation oftranscription at an AP1 site and ERa-mediated activation of gene transcription at an AP 1 site in response to the same ligand.
API-mediated estrogenic/agonist activity, as used herein, refers to activation of a gene under the control of an AP 1 site (also referred to as an AP 1 response element) mediated by the interaction of a nuclear transcription factor with the AP1 site.
When used in reference to ER mediated activation of a gene controlled by the AP1 site, the pathway is referred to as the indirect estrogen response (in contrast to the classical estrogen response which is mediated through an ERE). A general description of the AP 1 site is found in Angel & Kann, Biochem. Bioplrys Acta:, 1072: 129-157 (1991) and Angel, et al., Cell, 49: 729-739 (1987).
A "compound having AP 1 mediated estrogenic activity" refers to a compound that, when present in a cell containing a gene under control of an AP
1 site and AP 1 proteins, activates transcription of the gene under comrol of the AP 1 site.
A "compound having the ability to inactivate or inhibit estrogen receptor beta (ER~i) mediated gene activation at an AP1 site refers to a compound that is capable of upregulating or downregulating transcription of a gene under the control of an AP 1 site through its imeraetion (e.g., binding) of an ERA.
The phrases "modulate estrogen activation" or "modulation of estrogen activation" refer to alteration of the estrogen induced expression of a particular gene.
Where the phrase additionally recites "at an AP 1 site or at an ERE" the phrase refers to alteration of the level of estrogen induced expression of one or more genes under control of the AP1 site or ERE site respectively. The phrase "detecting expression"
when used wo ~nm6o rc~rnrs9snso3o 0 with reference to a reports gene refers to detection of presence or absence of expression of the reporter gene or to quantification of expression level of the reporter gene. The quantification can be either an absolute measurement or a relative measurement (e.g., in comparison to another expressed gale).
The term "operably linked" refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or transcription factor binding site) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence.
The term "recombinant" when used with reference to a cell indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid. Recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also express genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means. Recombinant expression refers to the expression of the heterologous nucleic acid by such a recombinant cell.
A "heterologous nucleic acid", as used herein, is one that originates from a foreign source (or species) or, if from the same source, is modified from its original form. Thus, a heterologous nucleic acid operably linked to a promoter is from a source different from that from which the promoter was derived, or, if from the same source, is modified from its original form. Modification of the heterologous sequence may occur, e.g., by treating the DNA with a restriction enzyme to generate a DNA fragment that is capable of being operably linked to the promoter. Techniques such as sit~directed mutagenesis are also useful for modifying a heterologous sequence. Similarly, a "heterologous protein" refers to a protein 'that originates from a foreign source (e.g., different cell or species) or, if from the same source, is modified from its original form, or is expressed fibm a heterologous nucleic acid.
A "recombinant expression cassette" or simply an "expression cassette"
is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of effecting expression of a structural gene in hosts compatible with such sequences. Expression cassettes include at least promoters and optionally, wo ~nm6o Pc~rnrsmso3o 0 transcription termination signals. Typically, the recombinant expression cassette includes a nucleic acid to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a promoter. Additional factors necessary or helpful in effecting expression may also be used as described herein. For example, an expression cassette can also include nucleotide sequences that encode a signal sequence that directs secretion of an expressed protein from the host cell.
Xenogens are defined here to include any compound having estrogenic activity in the assays described herein, which is derived from a source outside the human body. Environmental compounds as used herein can be derived from a wide variety of sources including plants, soil, water, foods. They also include synthetic compounds such as chlorinated organics, polycyclic aromatic hydrocarbons, herbicides, pesticides, pharmaceuticals and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 A illustrates the structure of five estrogen receptor (ER) ligands:
Estradiol (E~, diethylastilbestrol (DES), ICI 184,384, raloxifene (Ral), and tamoxifen (Tam).
Figure iB illu~rates two estrogen receptor (ER) response elements: a simple (classical) estrogen response element (ERE) and an ER dependent AP 1 element described also in USSN 08/410,807, in USSN 60/051,309, and by Webb etal ( 1995)Mol.
Endo., 9: 443-456.
Figure 2 illustrates ER~i action at an estrogen response element (ERE).
HeLa cells were transfected with an ERE-regulated luciferase reporter plasmid and an expression vector for rat ER(3 as described herein. Transfected cells were treated with the ligands (E2, 0.1 pM; DES, 1 pM, Ral, l pM, tamoxifen 5 pM; and ICI,1 ~ or an ethyl alcohol (EtOH) vehicle control. All assays were done with at least triplicate transfections.
Error bars show deviations between wells from a single representative transfection.
Figure 3 illustrates ERa action at an AP 1 element. HeLa cells were traasfected with an AP 1 reporter plasmid and an ERa expression plasmid and treated with the five Iigands (see, e.g., Figure 2). Ligand concentrations were E2, 0.1 pM;
DES, 1 ~M,; Ral, 1 pM; Tam, 5 p,M, and ICI, 1 pM. Error bars are as in Figure 2.

wo ~nm6o rcrnJS9snso3o 0 Figure 4 illustrates ER~3 activation and inhibition at AP1. (A) ERA action at an AP 1 response element. HeLa cells were fed with an AP 1 reporter plasmid and a rat ER[3 expression plasmid as described herein. Transfected cells were treated with the following ligand concentrations: E2, 0.1 pM; DES,1 pM; Ral, l pM, Tam, 5 pM; and ICI, 1 E,~M. (B) Dose response of raloxifene induction with ERS at an AP1 element.
HeLa cells transfected as described for A were treated with the indicated range of raloxifene concentrations. (D) Comparative inhibition of raloxifene induction by E= and DES. HeLa cells were transfected as described for (A) and treated with ligands. The left panel shows transactivation induction by raloxifene ( 1 pM), the lack of induction by E2 (0.1 uM) and induction to the amount observed with the control (no ligand added). The right panel shows the dose dependence of inhibition of raloxifene (1 ~
induction by DES (solid line) and EZ (Dashed line). (D) Raloxifene overriding EZ
inhibition. HeLa cells were transfected as described for (A) and treated with Iigands. The left panel shows the transcription induction resulting from the vehicle control (EtOH), Ral ( 1 pM) plus EZ ( 10 nM), and EZ (10 nM) alone. The right panel shows the dose dependence of raloxifene induction in the presence of E~ (10 nMJ.
Figure 5 illustrates ligand-dependent ER~i activity in three cell types;
Ishikawa cells, MCF7 cells and MDA453 cells. (A) Ligand-dependent ER~3 action at an AP 1 element in Ishikawa cells. Ishikawa cells were transfected with an AP 1-regulated luciferase reporter plasmid and an ER(3 expression plasmid. Transfected cells were treated with one or two ligands as indicated (Ez, 0.1 pM; DES, 1 ~M; Ral, 1 pM, Tam, 5 pM;
and ICI, 1 pM; or an EtOH vehicle (co~rol)). (B) Ligand dependent ERø action at an AP 1 eleme~ in MCF7 cells. MCF7 cells were treated and analyzed as described for (A).
Ligand dependent ERA action at an AP1 element in MDA453 cells. MDA453 cells were treated and analyzed as described for (A).
DETAILED DESCRIPTION
Antiestrogens are therapeutic agents for the treatment and possible prevention of breast cancer. Tamoxifen (Figure 1 A), for example, is an antiestrogen that is used in breast cancer chemotherapy and is believed to function as an antitumor agent by inhibiting the action of the estrogen receptor (ER) in breast tissue (Grainger et al.
(1996) Nature Med, 2: 381-385). Paradoxically, tamoxifen appears to function as an WO 99111760 ~ PCT/US98/18030 0 estrogen-like ligand in uterine tissue, and this tissue-specific iatrogenic effect may explain the increased risk of uterine cancer that is observed with prolonged tamoxifen therapy (Kedar et al. (1994) Lancet, 343: 1318-1321).
The related benzothiophene analog raloxifene (Fig. l A) has been reported to retain the antiestrogen properties of tamoxifen in breast tissue and to show minimal 5 estrogen effects in the uterus; in addition, it has potentially beneficial estrogen-like effects (in nonreproductive tissue such as bone and cardiovascular tissue (Jones et al. (1984) J.
Med Chem., 27: 1057-1066; Black et al. (1994) J. Clip. Imest., 93: 63-69; Sato et al.
(1996) FASEB J., 10: 905-912; Yang et al. (1996) F.~docrinol., 137: 2075-2084;
Yang et al., ( 1996) Science, 273 :1222-1225)). One explanation for these tissue-specific actions 10 of antiestrogens is that the Ggand-bound ER may have different transactivation properties when bound to different types of DNA enhancer elements.
The classical estrogen response element (ERE) is composed of two inverted hexanucleotide repeats, and ligand-bound ER binds to the ERE as a homodimer (Fig. 1B). The ER also mediates gene transcription from an AP1 ecihancer element that 15 requires ligand and the AP 1 transcription factors Fos and Jun for t<ansaiptional activation (Fig. l B) (Umayahara et al. ( 1994) J. Biol. Chem., 269:16433-16442). In transactivation experiments, tamoxifen inhibits the transcription of genes that are regulated by a classical ERE, but like the natural estrogen hormone 17b-estradiol jE2 (Fig. 1 A)], tamoxifen activates the transcription of genes that are under the control of an AP 1 element (Webb et al. ( 1995) Mol. Endocrinol., 9: 443-456).
At the end of 1995, a second ER (ERA) was cloned firm a rat prostate cDNA library (Kuiper et al. (1996) Proc. Natl. Acad Sci.USA, 93: 5925-5930).
The human (Mosselman et al. (1996) FEES Lett., 392: 49=53) and mouse (Tremblay et al.
{1997)Mol. E»dOCrinol., 11: 353-365) homologs were also cloned. The first idemified ER has been renamed ERa (Kuiper et al. (1996) supra). It was a discovery of this invention that ERA presents another source of tissue-specific estrogen regulation, particularly as mediaxed through the AP 1 site. In particular, it was a discovery of this invention that ERa and ER[i respond differently to certain Ligands at an AP 1 element.
The results described herein suggest different regulatory fixnctions for the two ER
subtypes. This invention thus provides materials and methods for screening for WO 99!11760 PCTNS98/18030 0 compounds that exhibit differential activity depending on whether their activity is mediated through ERa or ER~i. In addition, this invention provides materials and methods for determining whether a compound is capable of activate or inhibit estrogen receptor (3 (ER~i) mediated gene activation (transcription) at an AP1 site.
I. Screening Methods and Compositions.
It was a discovery of this ERA can interact with a AP 1 site to activate or inactivate expression (e.g.transcription) of a gene under the control of the AP1 site.
Moreover, it was a particularly surprising discovery that putative estrogens can actually demonstrate "antiestrogenic" activity in an ER~/AP 1 pathway (where antiestrogenic activity in this context is as compared to the activity of an estrogen in the classical ERaJERE pathway). Thus, where an estrogen would activate transcription in an ERa/ERE pathway the estrogen inactivates transcription in an ERaIAP l pathway.
Conversely, putative antiestrogens can demonstrate estrogenic activity in an pathway. This invention thus provides methods for detecting antiestrogenic activity of putative estrogens, or for detecting estrogenic activity of putative antiestrogens. More generally, as explained below, this invention provides methods of screening compounds for the ability to activate or inhibit estrogen receptor [3 (ER(3) mediated gene activation at an AP 1 site. This allows identification of previously unsuspected environmental estrogens or antiestrogens or for screening of compounds for those that have desirable estrogenic or antiestrogenic properties. Such compounds are expected to be useful for the treatment or the prevention of various cancers (e.g.breast cancer, ovarian cancer, endometrial cancer) and other diseases (e.g. endometriosis) mediated by estrogen.
A) Screening for ER[3 mediated AP 1 activation or inhibition.
This invention provides efficient ways to screen large numbers of test compounds for the ability to activate or inhibit estrogen receptor a (ERj3) mediated gene activation at an AP1 site. In one embodiment, the methods utilize a cell containing an estrogen receptor beta (ER~i), an AP 1 protein, and a construct comprising a promoter and reporter gene under the contro! of an AP 1 site such that ER~i irneraction with the AP 1 site, can increase or inhibit expression (e.g., transcription) ofthe reporter gene. The cell is contacted with one or more compounds whose ER(3 activity at AP1 it is desired to evaluate. In a preferred embodiment, the expression level of the reporter gene in the cell wo ~nt~6o rcrms9mso3o 0 contacted with the compound is compared to the expression level of a cell contacted by a control (e.g., identical culture conditions lacking the test compound and/or with a reference compound e.g., estradiol or tamoxifen). A decrease in expression level of the reporter gene indicates that the test compound inhibits ER(3-mediated expression (transcription) at an AP1, site, while an increase in expression level of the reporter gene indicates that the test compound activates ER[3-mediated expression (transcription) at an AP 1 site.
The criteria used to evaluate a change in expression level of the reporter gene in this assay, and the other assays described herein, are those standard in the art.
Thus, for example, a statistically significant difference in expression level between the test and control experiments are scored as a valid change. In a preferred embodiment, the expression level may change by a factor 1.5 or more, preferably by factor of 2 or more, more preferably by a factor of 4 or more, and most preferably by a factor of 5 or even 10 or more.
Screening for differential ERac and ER/i mediated activity.
it will be appreciated that using the methods of this invention, the ability of compounds to activate or inhibit ERA-mediated transcription at an AP 1 site can be compared to the ability of those compounds to activate or inhibit ER(~-mediated activity at an ERE site or to the ability of those compounds to activate or inhibit ERa-mediated activity at an APlor ERE. In this manner, compounds having a highly specific mode of activity across a wide tissue distribution, or alternatively compounds having a highly variable mode of activity can be identified.
Four preferred estrogen receptor based assays are illustrated in Table 1.
These correspond to ERa-mediated ERE activity, ERa-mediated AP1 activity, ER(3-mediated ERE activity, and ER~i-mediated AP 1 activity. It was a discovery ofthe present invention that various compounds exhibit differential activity in these various assays.

Table 1. Iuush~ation of estrogen receptor based assays.
ER ER

a ERElreporter Cla (3 exre ssical pathwaclassical S AP1/reporter Ind a ene irect ath indirect athwa This is illustrated in Table 2, where it can be seen that estrogen activates transcription in both the classical response (at an ERE) and in the indirect response (at an AP 1 ) when the interaction is mediated by ERa. In contrast, estrogen acts as an inhibitor of transcription at AP 1 when the interaction is mediated by ER~i. In contrast, the estrogen antagonist tamoxifen appears to always act as an inhibitor at an ERE, but an activator of transcription at an AP 1 site. Moreover, the activity of ERA does not appear to be tissue restricted.

0 Tsble 2. Illustration of the activity of estradiol (E~ and an estrogen antagonist (tamoxifen) in each of the ER assays.
ER ER
a ERFJreporter gene Ac Ac Estradiol tivates tivates Inh Inh Tamoxifen ibits ibits AP 1/reporter gene Ac Inh Estradiol tivates ibits Ac Ac Tamoxifen tivates tivates The assay for ERA-mediated AP 1 activity is described above. The remaining assays are performed in an analogous meaner. Thus, the ERa-mediated activity assays simply involve substituting ERa for ER(3, and the ERE activity assays simply involve substituting the EREJreporter gene construct for the AP 1 /reporter gene construct.
The ERa assays (both for ERE and AP1 activity) are described in detail in USSN
08/410,807, in USSN 60/051,309, and by Webb et al (1995) Mol. Enafo., 9: 443-456).
The assay for ERA-mediated ERE activity utilizes a cell comaining an estrogen receptor beta (ER(3), and a construct comprising a promoter and reporter gene under the control of an ERE site such that ERA imeraction with the ERE site, can increase or inhibit expression (e.g., transcription) of the reporter gene. The cell is contacted with one or more compounds whose ERj3 activity at an ERE it is desired to evaluate.
In a preferred embodiment, the expression level of the reporter gene in the cell contacted with the compound is compared to the expression level of a cell contacted by a comrol (e.g., identical culture conditions lacking the test compound and/or with a reference compound eg., estradiok or tamoxifen). A decrease in expression level of the reporter gene indicates that the test compound inhibits ERA-mediated expression (transcription) at an ERE, while wo ~nm6o pc~rivsmso3o 0 an increase in acpression level of the reporter gene indicates that the test compound activates ERA-mediated expression (transcription) at an ERE site.
While, in a preferred embodiment, each assay is performed in a separate cell, it will be appreciated that AP1 and ERE assays can be combined and performed in a single cell. In this case, the APllreporter gene construct preferably utilizes a different 5 reporter gene than the ERE/reporter gene construct so that AP 1 activation or inactivation can be distinguished from ERE activation or inactivation.
Screening for inhibitor activity.
The above-describe assays can also be used to identify (screen for) compounds that inhibit other compounds which have ERa-mediated or ERA-mediated 10 activity an ERE or at an AP-1 site. These assays are performed in the same manner as the assays described above. In this instance, however, the cell is contacted with two compounds, a test compound that is being screened for inhibitory activity and a second compound for which an inhibitor (or alternatively an agonist) is sought.
Thus, for example, where it is desired to identify a test compound having 15 ER(3-mediated estrogen inhibitory activity at an AP 1 site, the cell containing ERA, an AP 1 protein, and a reporter gene under control of an AP1 site is contacted with estrogen and the test compound. If the compound inhibits the characteristic ER/i-mediated estrogen activity at API, the compound is an inlu'bitor. It should be noted that in this case, ERJ3-mediated estrogen activity at AP 1 inhibits transcription, thus an estrogen inhibitor in this 20 context actually increases ER(i-mediated transcription at AP1. This is illustrated in Example 1, where it is shown that tamoxifen is one such inhibitor.
Inhibitors, or agonists, of ERø-mediated or ERa-mediated estrogenic or antiestrogenic activity at ERE and at AP 1 can be screened in an analogous manner.
D) Screening for environmental estrogens or antiestrogens.
As indicated above, this invention allows for screening of test compounds for estrogenic or antiestrogenic activity mediated through ERA or ERa at an ERE or at an AP1 site. The assays are particularly useful for screening environmental compounds for estrogenic or antiestrogenic activity. Environmental compounds having estrogenic activity are referred to here as xenoestrogens. Xenoestrogens include any compound derived from a source outside the human body, having estrogenic activity in the assays 0 described herein. Environmental compounds as used herein can be derive from a wide variety of sources including plants, soil, water, foods. They also include synthetic compounds such as chlorinated organics, polycyclic aromatic hydrocarbons, herbicides, pesticides, pharmaceuticals and the like.
It will be appreciated that environmental estrogens often are only weakly active. Consequently, particularly when testing an environmental compound for estrogenic or antiestrogenic activity, it is often desirably to maximize sensitivity of the assay. This may be accomplished by using cells that produce the methods typically comprise cultured cells that produce high levels of the human estrogen receptor (ERa or ER~i).
Such cells include, but are not limited to MCF-7 cells (ATCC No. I-iTB 22), MDA453 cells (ATCC
No. HTB 131), ZR-75-1 cells (ATCC No. CRL 1500) or ERC1 cells described in Kushner et al. (1990) Mol. F~idocri»ol., 4:1465-1473, and ERC2 and ERC3 cells as described by Webb et al. (1993) Mol. F.»docrirrol., 6:157-167.
It is also known that environmental estrogens may show synergistic activity in combination. Thus, in one embodiment, two or more suspected environmental estrogens are assayed according to the above methods in combination. It will be recogNZe~, however, that such combined testing is not limited simply to environmental estrogens but rather, amr combination of agents can be screened simultaneously.
Screening for transcription factor modulation of ER(3 activity at AP 1.
It has been demonstrated that various nuclear transcription factors (e.g., progesterone, gtucocorticoids, etc. ) interact with the ERa-mediated estrogenic activity at the AP1 site (see, e.g., USSN 60/051,309). It is believed that ER(3 is also capable of such interactions at AP1. Thus, in another embodiment, this invention provides assays (methods of screening) nuclear transcription factor ligands, and putative or known transcription factor ligand agonists or antagonists for the ability to modulate ER(3-mediated activation or inactivation of transcription at an AP 1 site.
These assays are performed in the same manner as the assays described above, however the assay cell additionally contains a receptor for a second nuclear transcription ligand (preferably a ligand other than estrogen). Thus, the cell contains an estrogen receptor beta (ER~i), an AP 1 protein, a receptor for a second nuclear transcription factor ligand, and a construct comprising a promoter comprising an AP 1 site 0 which regulates expression of a reporter gene. The cell is contacted with both a transcription factor ligand that is to be wed and with a compound having ER[3 mediated activity at an AP i site.
Alteration of the typical activity (level of AP1 regulated reporter gene expression) ofthe compound having ER[3-mediated activity at an AP 1 site by the presence of the compound being scxeened (the test transcription factor iigand) indicates that the screened compound is capable of modulating an ER~i-mediated AP 1 response of the compound having ER(i-mediated activity at an AP 1 site. Preferred second nuclear transcription factor ligands include, but are not limited to glucocorticoids, progestins, vitamin D, retinoic acid, androgens, mineralcorticoids, and prostaglandins.
Similarly, inhibitors, or agonists, of the test compound can be screened by running the same assay in the presence of the inhibitor that is to be screened.
II. Cell Types The assay methods of this invention provide methods for evaluating the ability of a test, or control, compound to activate or inhibit transcription through interaction with a transcription factor receptor (e.g., estrogen receptor).
Thus, in a preferred embodiment, the cells used in the assays of this invention preferably contain at least one transcription factor receptor.
For example, where it is desired to screen for activity of a compound mediated by the estrogen receptor a (ERa) cells are preferably provided that contain ERa and where it is desired to screen for activity of a compound mediated by estrogen receptor ~3 (ER[3) cells are preferably provided that contain ER~i.
Where it is desired to screen for the ability of a nuclear tt~nscxiption factor ligand modulate estrogen receptor (a or (i) mediated activation or inactivation of transcription at an AP 1 site, the cell preferably include, in addition to the particular ERa or ER[3 at least a second nuclear transcription. factor receptor (e.g., glucocorticoid receptor (GR)). Cells that naturally express one or more of the desired receptor types can be used in the assays of this imemion. Alternatively, cells can be modified (e.g., through recombinant DNA techniques) to express ERa and/or ER.(~ and/or the transcription factor receptor of choice.

WO 99/11760 ~ PGT/US98/18I130 0 Suitable cells for practicing the methods of this invention include, but are not limited to cells derived from a uterine cervical adenocarcinoma (HeZ,a) , a hypothalamic cel! line (GT1-1 (Melton et al. (1990) Neuron, 5: 1-10), MCF-7 cells (ATCC No. HTB 22), MDA453 cells (ATCC No. HTB 131), ZR 75-1 cells (ATCC No.
CRL 1500) or ERC1 cells described in Kushnex et al., Mol. Faralocrinol., 4:1465-1473 (1990). ERC2 and ERC3 calls as described by Webb, et al. Mol. F,ndocrinol., 6:157-167 (1993). It will be appreciated that the invention is not limited to practice in mammalian cells and may be practiced, for example in yeast and insect cells, transfected with the appropriate genes and recombinant constructs.
A) Cells naturally expressing two or more receptor types.
_ Many cells that express a second transcription factor receptor in addition to the estrogen receptor (ER) are well known to those of skill in the art.
Thus, for example, in the uterus there is evidence that ER and glucocorticoid receptors (GR) co-exist in the endometrium (Prodi et al. (1979) Tumor. 65: 241-253). In the brain, maps of ER and GR immunoreactivity and mRNA localization suggest co-localization in certain cerebral nuclei such as the paraventricular nucleus of the hypothalamus, the hypothalamic arcuate nucleus, and the central nucleus of the amygdala (Fuxe et al. ( 1985) Endocrinol., 118: 1803-1812; Simerly et al. (1990) J. Comp. Neurol. 294: 76-95). In bone, ER and have been found in cultured osteoblast-like cells (Liesegang et al. (1994) J.
Andrology, 14: 194-199). ER has also been demonstrated in osteoclasts {Oursler et al.
(1994) Proc.
Natl. Acad Sci., USA, 91: 5227-5231) and data suggest that the glucocorticoid dexamethasone (Dex) regulates metaboli~n in these cells (along (1979) J. Biol.
Chem., 254: 6337-b340) raising the possibility that osteoclasts contain functional GR
as well. In addition, numerous tumor cell lines have been demonstrated to have both ER and GR
(Swing et al. (1989) Int. J. Cancer., 44: 744-752.
B) Cells recombinamly modified to express two or more receptor types.
Cel~s normally lacking the ERa or ER~i or other transcription factor cognate receptors can be recombinantly modified to express one or more of the desired receptors. Typically this involves transfecting the cell with an expression cassette comprising a nucleic acid encoding the receptor of interest and culturing the cell under conditions where the receptor is expressed (e.g., in the presence of an appropriate inducer 0 if the promoter regulating exp~ion of the receptor is inducible). Typically, the cassette is selected to provide constitutive expression of the receptor.
A cell that naturally expresses one receptor need only be modified to express the second receptor. However, if the cell expresses neither receptor, it may be transfected with expression cassettes expressing both receptors. Even where a cell S naturally expresses one or both receptors, it may be recombinantly modified to express those receptors at a higher level (e.g., by introducing expression cassettes encoding the receptors) whose expression level it is desired to increase).
The cells need not contain "native" receptors, but may be modified to provide truncated or chimeric receptors to provide increased affnity and/or sensitivity of the assay. Thus, for example, Berry, et al.(1990), F.~I~BD J., 9: 2811-2818, describe the production of cells containing truncated or chimeric ER receptors.
Methods of modifying cells to express particular receptors are well known to those of skill in the art. Thus, for example, cells modified to express high levels of estrogen receptor are described by Kushner et al. ( 1990), Mol. Er~iocrinol. , 4:1465-1473.
See also ITlrst et al. (1990) Mol. Endocrinol., 4: 162-170). Transfection of cells to express ERac is described below, in the Examples, and in US SN 08/410, 807.
Transfection of cells to express ER~i is described herein, and transfection of cells to express glucocorticoid receptors (GR), progestin receptors (PR), and other receptors is described in copending USSN 60/043,059.
C) Cells Containing AP1 proteins.
In assays that involve screening for transcription factor receptor mediated activation or inactivation oftranscription at APl, the cells preferably contain one or more AP 1 proteins (the Jun or Fos proteins or other members of that protein family, see Bohmaan, et al. (1987) Science, 238: 1386-1392) in addition to the transcription factor receptor(s).
The cells can naturally express the AP 1 proteins) or they can be modified (e.g., by transfection with a suitable expression cassette) to express a heterologous AP1 protein. Methods of expressing AP 1 proteins are well known to those of skill in the art (see, e.g., Turner et al. (1989) Science, 243:1689-1694 and Cohen et al.
(1989) Genes wo ~nm6o rrrnJSmso3o 0 & Dev., 3 : 173-184, and Example 1 ). Cells that naturally express one or more AP 1 proteins may still be so modified to increase intracellular jun and/or fos levels.
IlT) Expression of Nuclear Transcription Factor Receptors.
As explained above the assays of this invention utilize cells containing one or more nuclear transcription factor receptors (e.g., ERa, ER~3, GR, PR, etc.
) an estrogen 5 receptor and a receptor for a nuclear transcription factor (typically a transcription factor other than estrogen). The factor can be one that is expressed endogenously by the cell or, alternatively, the cell can be modified (e.g., a recombinant cell) so that it expresses the receptor.
A) Estrogen Receptor Alpha (ERa) 10 An estrogen receptor, as used herein, includes an estrogen receptor alpha (ERa) in its native (naturally occurring) form as well as modified estrogen receptors.
Numerous modifications of estrogen receptors are known to those of skill in the art.
These include, but are not limited to VP 16-ER, V-ER, a chimeric receptor comprising the strong VP 16 transcriptional activation domain linked to the amino terminus of the ER, V-15 ER in which the ER DNA binding domain (DBD) is deleted, Hl 1 an ER lacking the DNA
binding domain, and the like (see e.g., Kumar et al., Cell, 51: 941-951 (1987) and Elliston et al. ( 1990) J Biol Chem 265:11517-21 ).
Means of recombinantly expressing the estrogen receptor alpha (ERa) are well known to those of skill in the art (see, e.g., USSN 08/410,807 and Webb et al ( 1995) 20 Mol. Fardocrinol., 9: 443-456).
B) Estrogen Receptor Beta (ER(3).
Estrogen receptor beta (F.R(3) is a second estrogen receptor (ER) cloned from a rat prostate cDNA library (Kuiper et al. ( 1996) Proc. Natl. Acad Sci.
USA, 93 5925-5930). Subsequently the human (Mosselman et al. (1996) FEBSLett., 392: 49-53) 25 and mouse (Tremblay et al. (1997) Mol. Errdocrinol., 11: 353-365) homologs were cloned. Accordingly, the original estrogen receptor (ER) has been renamed ERa (Kuiper et al. ( 1996) supra. ).
Using the known sequence information one of skill in the art can routinely construct vectors that express an ER~i when transfected into a suitable host cell. Detailed 0 protocols for the preparation of an ER(3 vector can be found in Kuiper et al. ( 1996) Proc.
Natl. Acad Sci. USA, 93: 5925-5930 and in WO 97/09348.
It will be appreciated that exist s number of different estrogen beta receptors comprising various splice variants, mutations, and so forth. It will be appreciated that ER~i as used herein is intended to include all FR~3 variants.
However, in a preferred embodiment, the ER(3 variants used in this invention correspond to the so called "intermediate length" ER~i variants such as those described in WO
97/09348.
Particularly preferred ERA variants are shown in sequence listings 3, 4, and 5 herein which correspond to figures 1 and 13A and 13B of WO 97/09348, C) Nuclear transcription factor ligand and cognate receptor As indicated above, the in addition to the estrogen receptor (ERa and/or FR~i), the cells can contain a cognate receptor for a nuclear transcription factor ligand whose interaction (preferably a cognate receptor other than an estrogen receptor). As used herein, the term "cognate receptor" refers to a receptor of the type that is typically bound by the transcription factor ligand in question. Thus, the cognate receptor for an estrogen is an estrogen receptor, the cognate receptor for a glucocorticoid is a glucocorticoid receptor, the receptor for a progestin is a progestin receptor, and so forth.
As with the estrogen receptor, the cognate receptor includes the native (naturally occurring ) form as well as modified receptors.
Natural and modified cognate receptors for nuclear transcription factor ligands, particularly for steroid nuclear transcription factors, are well known to those of skill in the art. These include, but are not limited to the glucocorticoid receptors, the progestin receptors (e.g., PR A, PR B (see, e.g., Law et al. ( 1987) Proc.
Natl. AcaaL Sci.
USA 84: 2877-2881; Wei et al. (1988) Mol. Fr~do. 2: 62-72; and Kushner et al.
(1990) Mol. Endocrinol, 4:1465-1473), vitamin D receptors, mineralcorticoid receptors, androgen receptors, and thyroid hormone receptors (see, Mangelsdorf (1995) Cell, 83:
835-839).
IV. ERE and AP1 Reporter Constructs The cells of this invention preferably contain (e.g., are transfected with) nucleic acid constzucts comprising one or more reporter genes under the control of a response element (either the AP1 site or estrogen response element (ER,E)).
Where two 0 different response elements are monitored in a single cell, two differ~t reporter genes are used. Thus, for example, one gene can reports transcription induced by the classical estrogen response system (ERE), while the other gene reports transcription induced by the indirect (AP 1 ) estrogen response. The two reporter genes and response elements are typically placed in separate cells, but the methods can also be used with both constructs in the same cell.
A) AP1/Reporter construct.
In one embodiment the methods of this invention imrolve providing a cell containing an estrogen receptor (ERa or ER(3), and a promoter comprising an AP1 site that regulates expression of a reporter gene (also referred to herein as the reporter gene for the indirect estrogen response pathway (see, e.g., USSN 08/410,807 and Webb et al (1995) Mol. Endocrinol., 9: 443-456).
The reporter gene for the indirect estrogen response pathway comains an AP1 site preferably upstream of the target promoter and capable of regulating (i.e., operably linked to) that promoter. AP 1 site are sites that are bound by AP 1 (the Jun and Fos proteins) or other members of that protein family. In a preferred embodiment, the consensus AP1 site (or AP1 response element) is TGA(C/G)TCA (SEQ ID NO: 1).
One of skill would recognize that the particular AP1 site used is not a critical aspect of the imrention. Any sequence capable of being bound by AP 1 or members of that family and regulating a promoter is suitable. This would include promoters which encompass a naturally occurring AP 1 site. Typical promoters include, but are not restricted to meralloprotease genes such as stromelysin, gelatinase, matrilysin, and the human collagenase gene.
Alternatively promoters may be construcxed which contain a non-naturally occurring AP1, or related, binding site. This facilitates the creation of reporter gene systems that are not typically found under the control of AP 1. In addition, promoters may be constructed which contain multiple copies of the AP1 site thereby increasing the sensitivity or possibly modulating the response the reporter gene system.
B) ERE/Reporter Construct The methods of this invention can also involve providing a cell containing a promoter comprising an estrogen response element that regulates expression of a 0 reporter gene (also referred to herein as the reporter gene for the direct or classical estrogen response pathway (see, e.g., U.S.S.N. 08/410,807 and Webb, etal.
(1995)Mol.
E~alo., 9: 443-456). This permits detection of the "direct" (classical) estrogen response and evaluation of the interaction or modulation of the classical response by the nuclear transcription factor ligand.
Typically, the estrogen response element (ERE) is upstream of the target promoter and capable of regulating that promoter. In a preferred embodiment the FRF
may be the consensus estrogen response element AGGTCACAGTGACCT (SEQ ID NO:
2) from the Xenopus vitellogenin A2 gene. The particular ERE used in the cell is not a critical aspect of the invention and the present invention is not limited to the use of any one particular ERE. Suitable EREs are well known to those of skill. For instance, other sources of naturally occx~iTing EREs include the vitellogenin B2 gene, the chicken ovalbumin gene, and the PS2 gene. Alternatively, non-naturally occurring EREs may be inserted into particular promoters. The consensus ERE from theXenopus vitellogenin A2 gene is widely used for this purpose, but other EREs may be used as well C) Reporter Genes) The present invention is not limited to a particular reporter gene. Any gene that expresses an easily assayable product will provide a suitable indicator for the present assay. Suitable reporter genes are weU known to those of skill in the art.
Examples of reporter genes include, but are not limited to CAT
(chloramphenicol acetyl transferees) (Alton and Vapnek ( 1979) Nature 282: 864-869), luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al.
( 1987) Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht et al. ( 1984) Proc.
Natl. Aced Sci., USA,1: 4154-4158; Baldwin et al. ( 1984) Biochemistry 23 :3663-3667);
alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem.182: 231-238; Hall et al. (1983) J. Mol. Appl. Gen. 2: 1 O 1 ), and green fluorescent protein.
One of skill will recognize that various recombinant constructs comprising the AP-1 site can be used in combination with any promoter and reporter gene compatible with the cell being used. The promoter will preferably be one susceptible to regulation by the AP 1 site.
D) Construction of the Promoter/Reporter Expression Cassette.

wo ~nm6o rcr~s9snso3o 0 The promoter/reporter ion cassettes and, other expression cassettes (constructs) described herein, can be constructed according to ordinary methods well known to those of skill in the art. Construction of these cassettes is variously exemplified in Example 1, in USSN 08/410,807, in Webb et al. (1995)Mol. Endo. 9: 443-456, and in other references cited herein.
The constructs can all be created using standard amplification and cloning methodologies well known to those of skill in the art. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide toMolecular Cloning Techniques: Methods in Fnzymology, 152 Academic Press, Inc., San Diego, CA; Sambrook et al. ( 1989) Molecular Cloning -A LaboratoryManual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY,; Current Protocols in Molecular Biology, Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley &
Sons, Inc., (1994 Supplement) (Ausubel); Cashionetal., U.S. Patent No:
5,017,478; and Carr, European Patent No. 0,246, 864. Examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger supra., Sambrook supra., and Ausubel supra., as well as Mullis et al., (1987) U.S. Patent No.
4,683,202;
Inr>is et al. ( 1990) PCR Protocols A Guide to Methods and Applications, Academic Press Inc. San Diego, CA; Arnheim & Levinson (October 1, 1990) C&EN 36-47; The Journal Of MH Research ( 1991 ) 3 : 81-94; Kwoh et al. ( 1989) Proc. Natl. Acac~ Sci.
USA 86:
1173; Guatelli et al. ( 1990) Proc. Natl. Acad Sci. USA 87, 1874; Lomell et al. ( 1989) J.
Clin. Chem., 35: 1826; Landegren et al., (1988) Science, 241: 1077-1080; Van Brunt (1990) Biotechnology, 8: 291-294; Wu and Wallace, (1989} Gene, 4: 560; and Barringer et al. (1990) Gene, 89: 117.
V. ER(3-mediated Activation through tethered coativactors.
In still another embodiment, ERA can mediate gene activation through virtually any response element using a tethered transcription factor coactivator strategy.
The methods involve contacting a nucleic acid that includes the gene of interest operably linked to a response element with a tethered coactivator. The tethered coactivator is composed of a polypeptide that comprises an activation fimction derived from a transcriptional coactivator, and a DNA binding moiety that is capable of specifically 0 binding to the response element. The tethered coactivator is cornacted with an activated transcription factor polypeptide (e.g., ER.~) that includes an activation function derive from a tt~ansaiption factor. The contacting of the tethered coactivator with the activated transcription factor polypeptide stimulates expression of the gene. The transcription factor can be, for example, a nuclear hormone receptor such as the estrogen receptor or 5 the estrogen receptor beta, or an AP 1 transcription factor, however, in a preferred embodiment, the transcription factor is ER~i. Detailed protocols for the tethered transcription factor activation strategy are provided in copending USSN
60/043,059.
VI. Detection of the reporter genes.
Detection of the reporter genes of this imrention is by standard methods 10 well known to those of skill in the art. Where the reporter gene is detected through its enzymatic acxivity this typically involves providing the enzyme with its appropriate substrate and detecting the reaction product (e.g., light produced by luciferase). The detection may involve simply detecting presence or absence of reporter gene produce, or alternatively, detection may involve quantification of the level of expression of reporter 15 gene products. The quantification can be absolute quantification, or alternatively, can be comparative e.g., with respect to the expression levels of one or more "housekeeping"
genes. Methods of quantifying the expression levels of particular reporter genes are well known to those of skill in the art. It will be appreciated that such detection can be performed "manually" or may be automated e.g., as in a high-throughput screening 20 system.
I~gh throughput assays for the presence, absence, or quantification ofgene expression (e.g., via the detection ofthe transcribed nucleic acid (mRNA) or the detection of gene expression (protein product)) are well known to those of skill in the art. Thus, for example, U. S. Patent 5,559,410 discloses highthroughput screening methods for proteins, 25 U.S. Patent 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays), while U.S. Patents 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.
In addition, high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH;
30 Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.).

wo 99n1760 PGT/US98118030 0 These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detectors) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization.
Compounds to be Screened.
It will be appreciated that virtually any compound can be screened by the methods of this invention. Such compounds include, but are not limited to known or suspected estrogens or antiestrogens including environmernal estrogens or environmental antiestrogens as described above.
It will be appreaated that compounds are expected to be show the most estrogenic or antiestrogenic activity if they are capable of penetrating to the nucleus of a cell and binding to a transcription factor receptor (e.g., ERa or ER(i). Such compounds are often lipophilic or capable of entering cells passively through pores or gates, through active transport, or through endocytosis. Particularly preferred compounds include, but are not limited to, steroid compounds or steroid analogs.
VIII. Assay Kits In another embodiment, this invention provides kits for the practice of the methods of this imrention. The kits preferably include one or more cornainers containing the cells described herein for the practice of the assays of this invention.
Thus, for example, the cells may include, but are not limited to, cells containing an estrogen receptor ~i (ERA), AP 1 protein(s), and a construct comprising a promoter comprising an AP 1 site which regulates expression of a first reporter gene, or such cells additionally containing a receptor for a nuclear transcription factor ligand other than estrogen. The AP1/recporter gene and the ERE/reporter gene constructs can be in separate cells or together in the same cell. The cells may additionally express high levels of AP 1 proteins such as fos and/or jun. Alternatively, or in addition, the kits can contain the AP 1 /reporter gene and/or the ERFJreporter gene constructs described herein and/or the ERa, ER[i, or other nuclear transcription factor receptor vectors. The kits may optionally contain any of the buffers, reagents, culture media, culture plates, reporter gene detection reagents, and so forth that are useful for the practice of the methods of this invernion.

WO 99/il~b0 PCT/US98/18030 0 In .addition, the kits may include instructional materials containing directions (i. e., protocols) for the pracxiice of the assay methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Arly medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to Internet sites that provide such instructional materials.
EXAMPLES
The following examples are offered to illustrate, but not to limit the present invention.
Example 1 Comparison of the Transac~ivation Properties of ERa and ERA
This example describes the investigation of the transactivation properties of ERa and ER(i with a panel of five ER ligands with the use of a reporter gene under the control of either a classical ERE or an AP 1 element. The results presented herein show that ERa and ERA respond differently to certain ligands at an AP 1 element suggesting different regulatory fimctions for the two ER subtypes.
Screeni»gMethais The transactivation properties of ERa and ER(i were compared with a panel of five estrogen receptor (ER) ligands using a reporter gene under the control of either a classical estrogen response element {ERE) or an AP 1 element. The ERE
and AP 1 driven luciferase reporter plasmids (EREII-LucG145 and Ocoll78, respectively) and the ERa expression plasmid (pSGS-HEO) were used as described in Webb et al. ( 1995) Mol.
Endocrinol., 9: 443-456, and in USSN 08/410,807 now issued as U. S. Patent The rat ER(i expression vector has been previously described (Kuiper et al. (1996) Proc. Natl. Acad Sci.USA, 93: 5925-5930). The foil-length human ER~i cDNA which was isolated from an ovarian cDNA library and found to be identical to the previously reported partial cDNA clone (Mossehnan et al. (1996) FEBSLett., 392: 49-wo ~n mho rcTnrs9srt solo 0 53) was cloned into the pCMVS eukaryotic expression vector and the resulting ERj3 expression vector was used for these experiments (see, Kuiper et al. ( 1996) Proc. Natl.
Accu~ Sct. USA, 93: 5925-5930). The ligands used to compare ERa and ER(~
transactivation properties included the estrogens ~i-estradiol (E~ and diethylstilbestrol (DES) and the antiestrogens Imperial Chemical Industries (ICI) 164384, tamoxifen, and raloxifene. Raloxifene was synthesized according to published procedure (Jones et al.
( I 984) J. Med Chem., 27: 1057). Structure and purity were verified by 'H
nuclear magnetic resonance (NMit), "C NMR, ultraviolet thin layer chromatography, and high resolution mass spectrometry. ICI 164384 was obtained from a private source and the other compounds were obtained from commercial sources.
The experiments were conducted by transfecting HeLa cells with either an ERa or ER~i expression plasmid along with a reporter plasmid that contained a luciferase gene under the transcriptional control of an estrogen response elerilent (ERE).
Cells were gown in Nunc Delta Surface tissue culture plates to a density of not more than 5 x 104 per cm2. Cells were grown in 0.1 pm sterile filtered Coon's Modified Medium (Sigma Cell Culture) with 15 mM Hepes, 0.438 g/L L-glutamine, 1.338 g/L NaHC03, 10% Seru-Max 4 (an iron supplemented , formula fed newborn calf serum, Sigma Cell culture; from a lot tested for low estrogenic activity), 0.05 mg/mL Gentamycin, 100 mg/ml Streptomycin SO,, and 100 units/ml penicillin "G".
Ishikawa cells were gown in a medium containing 100 nM tamoxifen and MCF-7 cells were grown in medium containing 10 nM estradiol.
For the transfection assays, cells were suspended 0./5 ml ofelectroporation buffer in 0.4 cm gap electroporation cuvettes (BioRad) at 106 to 2 x 106 cells per cuvette.
The electroporation buffer was prepared as a solution of 500 ml phosphate buffered saline (PBS), 5 ml of 10% glucose, and 50 pL of Biobrene. Five pg of reporter plasmid and 6 pg ofER expression plasmid were added and the cuvette was agitated to facilitate mixing of the solution and homogeneous cell distribution in the cuvette. Cells were then immediately transfected by electroporation with a BioRad GenePulser electroporation apparatus at a potential of 0.25 kV and a capacitance of 960 uF. To the electroporation cuvettes was added 1 ML growth medium (described above).

0 The transfected cells for one experiment were pooled and carefully resuspended in growth medium at a density of 8 x 10'- 1.6 x 10' cells/mL.
After a homogenous cell distribution was obtained by thorough mixing cells were plated on Nunc 6-well dishes at 2 mL per well. After 2 h of incubation hormones were added and the medium was mixed by gentle swirling. Cells were then incubated in the presence of hormone for 40-48 hours.
Growth medium was removed from the wells, and the cells were washed with Mg2+ and Ca2+ free PBS, and then they were lysed chemically with 0.2 mL
of 100 mM potassium phosphate buffer (pH 7. S) containing 0.2% Triton X-100 and 1 mM
DTT).
The plates were then frozen to -80'C, thawed and scraped with a rubber policeman to loosen and break up cell Fragments. The lysate was centrifuged in a microfuge for 2 min, 0.1 mL of the supernatant was combined with 0.3 mL luciferase assay solution, and the chemiluminescence was measured immediately for a period of 10 s.
The luciferase assay solution consisted of 25 nM gtycylglycine, 15 mM
MgS04, 4 mM EGTA, 15 mM potassium phosphate at pH 7.8, with the addition of DTT
to a f>rtal concentration of 1 mM, ATP to a final concentration of 2 mM and luciferin (Analytical Luminescence Laboratories) to a final concentration of200 uM
shortly before commencing the assay. Luminescence measurements were performed on a MonoGght 1500 (Analytical Luminescence Laboratories). The relative light units reported here were adjusted to a scale of 100 for uniformity.
The data were collected using the HEO ER variant. HEO shows reduced transactivation response from the unliganded receptor compared with the wild-type ER
resulting in clearer ligand-induced transactivation data Each experiment with ERac was also checked with the wild-type ER (HEGO), and the general ligand induction trends were found to the same as those obtained with HEO. The only difference was that the ligand-induced ttansactivation responses were lower with HEGO than with the control (no ligand added).
Transacdvation experiments were performed with both rat and human ER~i and identical trends in ligand behavior and similar induction levels were seen with both ER~is in HeLa cells. The data shown in Figure 2B and Figure 4 were obtained with the rat ERA expression plasmid.

WO 99/11760 PCT/US98/1$030 Fxperime»ts and Results The transactivation properties of ERa and ER(3 at a classical ERE in response to the estrogens EZ and diethylstilbestrol (DES) and the antiestrogens Imperial Chemical Industries (ICn 164384, tamoxifen, and raloxifene were first investigated. Both 5 ERa (18) and ER~i (Fig. 2) showed the same transactivation profiles with the panel of ligands. E2 and DES stimulated luciferase production 10-fold over ICI 164384;
raloxifene, tamoxifen, and the control (no ligand added). The antiestrogens blocked EZ
stimulation in ligand competition experiments.
Next, the ligand-induced transactivation behavior of ERa and ERA at an 10 AP1 site was examined. With ERa, all five ligands stimulated luciferase transcription, including the antiestrogens ICI 164384, tamoxifen, and raloxifene (Fig. 3).
This stimulation was dependent on h~ansfected ER, as cells transfected with only the reporter plasmid showed no induction of reporter transcription. Of the five ligands, raloxifene induced transcription the least, showing twofold induction compared with the sixfold 15 inductions typically seen with EZ and tamoxifen. The raloxifene-induced transactivation was dose dependent with a concentration value required for one-half maximal activation (ECM) of about 1 nM. In addition, raloxifene reduced the activation caused by EZ in a dose-dependent manner to the amount observed with raloxifene alone, demonstrating that raloxifene induction is weaker than induction by EZ and that raloxifene-induced 20 transactivation results from binding to ERa. If EZ is classified as a full activator of ERa at an AP 1 element (ERac-AP 1 ), then raloxifene fimctions as a partial activator and tamoxifen functions as a fill activator.
In contrast to the results seen with ERa-AP1, a difference in the ligand activation profile of ER[3 at an AP1 element (ER~i-AP1) was observed. In cells 25 transfected with ER[i, treatment with the estrogens EZ and DES did not increase luciferase transcription over the control (no ligand added), whereas treatment with the antiestrogens ICI 164384, raloxifene, and tamoxifen increased luciferase transcription (Fig.
4A). This transcription activation required transfected ER(I, as wells that were transfected with only the reporter plasmid did not show transcriptional activation by the antiestrogens. The 30 transcriptional activation caused by raloxifene was dose dependent with an ECM value of 0 about 50 nM (Fig. 4B). In ligand competition experiments, both E2 and DES
were able to block the raloxifene induction, and both estrogen ligands were able to reduce raloxifene induction to the basal level of transcription in a dose-dependent manner with concentration values required for one-half maximal inhibition of 1 to 10 nM
(Fig. 4C).
In a different ligand competition experiment, the inln'bitory effect on transcription resulting from EZ treatment could be overcome by higher concentrations of raloxifene in a dose-dependent manner (Fig. 4D). Thus, it appears that the pharmacology of ER ligands is reversed at an AP 1 element with ER(3; with F.R~i-AP 1, the antiestrogens act as transcription activators, and the estrogens act as transcription inhibitors.
It was next investigated whether the action ofER[3-AP 1 could be observed in cell lines derived from estrogen target tissues such as the uterus and breast.
Transactivation assays for ERj3-AP1 were performed in Ishikawa cells (a human uterine cell line) (Fig. SA) and in MCF7 (Fig. 5B) and IVIDA453 (Fig. Sc) human breast cancer cells. (The human ERA was used for transactivation in these cells.) In each of these cell lines, the ligands acted the same as they did in the HeLa cells; the thr~
antiestrogens activated and the estrogens inhibited ER(3-dependent transcription from an APl site (Fig.
5). No induction was seen with cells that were not transfected with the ER.~
expression plasmid, indicating that the antiestrogen induction required ER(3.
Antiestrogen induction in the breast cell lines was higher than that observed in HeLa cells.
Transfected MCF7 cells treated with raloxifene gave a 20- to 80-fold transactivation response over the control (no ligand added). In addition, raloxifene and ICI 164384 induced transcription more than tamoxifen in the breast cell lines(Fig. 5, B and C).
MCF7 cells did not appear to contain high concentrations of endogenous ER(i mRNA (Kuiper et al. (1997) Endocri»ol., 138: 553); however, the results suggest that the additional transactivation machinery required for ERA-AP 1 function is present in these cells. With two of these target tissue cell lines, E~ treatment reduced the amount of transcription to less than that seen with the control (no ligand added). In MDA453 (Fig.
SC) and Ishikawa cells (Fig. SA), E~ treatment resulted in a consistent 40 to 75%
reduction of reporter transcription levels compared with the control. This effect was also observed in ligand competition experiments (Fig. 5, A and C); EZ and DES
blocked raloxifene induction and reduced the amount of transcription to less than that seen for the 0 control. Thus, when ERA is bound by the estrogen hormone E2 or the synthetic estrogen DES, it functions as a negative regulator of genes controlled by an ER
dependent AP1 element.
The ER is the only known member of the steroidal subfamily of nuclear receptors that has different subtypes (Mangeldorf et al. (1996) Cell, 83: 835-839).
Nuclear receptors that respond to nonsteroidal hormones that have different known subtypes include the thyroid receptor {TRa and TR~i), the retinoic acid receptor (RARa, RARE, and RARY), and the retinoid X receptor (RXRa, RXR[3, and RXRy) (Mangelsdorf et al. (1996) Cell, 83: 841-850). The results presented herein demonstrate that two nuclear receptor subtypes can respond in opposite regulatory modes to the natural hormone from the same DNA response element. Moreover, the ligand-induced responses with ER(3 at an AP 1 site provide an example of negative transcriptional regulation by the natural hormone and strong positive regulation by synthetic antiestrogens.
(The genes for transforming growth factor and quinone reductase are ER regulated genes controlled by promoters containing nonclassical EREs that are activated by antiestrogens.
However, the action of ERA at either of these promoters has not been reported. The action of ERa on the quinone reductase gene shows a similar ligand profile to that of ERA at an AP1 site; antiestrogens are transcription activators, and E= is a transcription inhibitor.
If signaling from ER dependent AP 1 elements occurs in estrogen target tissues, the finding herein that ERa and ER[3 respond differently to ligands at AP1 sites reveals a potential control mechanism for transcriptional regulation ofestrogen-responsive genes and adds a layer of complexity in analyzing the pharmacology of antiestrogen therapeutics. The role of E2 completed to ER(3 would be to turn off the transcription of these genes, whereas the antiestrogens raloxifene, tamoxifen, and ICI 164384 could override this blockade and activate gene transcription. It wil! be T helpful to search for genes in estrogen target tissues that are transcriptionally regulated by ER[3 at an AP 1 site and to characterize the phenotype of cells in which these genes are activated.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and wo ~nm6o rcrnrs9i;nso3o 0 purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: THE REGENTS OF THE UN111ERSiTY OF CALIFORNIA
(ii) TITLE OF INVENTION: DIFFERENTIAL LIGAND ACTIVATION OF
ESTROGEN RECEPTORS ERalpha AND ERbeta AT AP1 SITES
(Iii) NUMBER OF SEQUENCES: 6 (iv) OORRESPOIiDENCE ADDRESS:
(A) ADDRESSEE: Fulbri9ht i ,lerarski L.L.P.
(8) STREET: 865 S. FiQueroa Street, 29'" Floor (C) CITY: Los A~pelee (D) STATE: Cslifornia (E) COWITRY: USA
(F) ZIP: 90017-2576 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC caa~pstible (C) OPERATING SYSTQI: PC-DOS/MS-DOS
(D) SOFTWRE: Patentln Release d1.0, Version 51.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION N<18:ER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: BERLINER, Robert (B) REGISTRATI011 NtlIIBER: 20,121 (C) REFERENCE/DOCKET NUMBER: 5555-497 (ix) TELECOIIIUNICATION INFORMATIO11:
(A) TELEPIIOIIE: 213-892-9200 (B) TELEFAIf: 213-680-4518 (2) INFORMATION FOR SE0 tD N0:1:
(i) SEQUEtICE CHARACTERISTICS:
(A) LENGTH: 7 base pain (B) TYPE: nucleic said (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (oenoinic) (ix) FEATURE:
(A) NAIIE/KEY: -(B) LOCATI011: 1..7 (D) OTHER INFORMATION: /note~ «AP1 response elaiaent"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

(2) INFORMATI011 F0lt SE0 ID 110:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: sin4le (D) TOPOLOGY: linear (ii) lbLECULE TYPE: DNA (~noaiie) (ix) FEATINtE:
(A) NIWE/KET: -(8) LOCUTION: 1..15 (D) OTHER INFORMATION: /note" "ERE fraa~the Xenopta vitellopenin A2 Gene"
(xi) SEqJENt~ DESCRIPTIOIIs SE0 ID 110:2:

(2) INFORMATION FOR SEG ID N0:3:
(i) 5E4VENCE CHARACTERISTICS:
(A) LENGTH: 2568 base pairs (B) TYPE: nucleic acid (C) STRAIDEDNESS: side (D) TOPOLOGY: linear (ii) MO<.ECULE TYPE: DNA (9eno~ie) (ix) FEATURE:
(A) NAI~/KEY: CDS
(B) LOGTION: 424..1878 (D) OTHER INFORMATION: /note" "Il~ino acid sequence of a rat ERbeta"
(xi) SEGIH:NCE DESCRIPTION: SE0 ID N0:3:

GGMTTTCGG GGCIIGCTCGC CCAGGGGGAG CGGCtGGTGC60 TGCGCTGGC ATCCCTAGGC

GAGCGACAJIC GGTGaCTGG

GAGTCCGGCT CTGTGfiCTGA GGMAGCACC TGTCTGGTT180 tCTGIIGAAG TMTGTCGT

TCTGAAGIIG TGGAGATCM

AMCTGCCG TCGIIGt'.CTTA GTtCCCTGCT TCCTATMCT360 GTAGCGGTC GTCCTACCC

CTGGAGCACG GCCCGTCTA GTCCCTTCC TCCTACGTAGi20 ACMCCGCG TGAGTATTG

GCT ATG AG TTC TAC AGT CCT GCT GTG ATG <68 MC TAC AGT GTt CCC GGC

Met Thr Phe Tyr Ser Pro Ala Val Net Asn tyr Ser Val Pro Ely AGC ACC AGT MC CTG GAC GGt GGG CCT GTC 516 CGA CTG AGC AG AGC CG

Ser Thr Ser Asn Leu Asp Gly Gly Pro Vat Arp Leu Ser Thr Ser Pro MT GTG CTA TGG CG ACT TCT GGG CAC CTG 5b4 TCT CCT TTA GCG ACC GT

Asn Val Leu Trp Pro Thr Ser Gly His Leu Ser Pro Leu Ala Thr His MG AGT CCt TCG TGT

Cys Gtn Ser Ser Lnu Leu Tyr Ala Glu Pro Gln Lys Ser Pro Trp Cys Glu Ala Arp Ser Leu Glu His Thr Leu Pro Yal Asn ArS Glu Thr Leu MG AGG AAG CTT AtiT GGG AGC AGT TGT GCC 708 AGC CCT GTT ACT AGT CG

Lys Arp Lys Leu Ser Gly Ser Ser Cys Ala Ser Pro Vat Thr Ser Pro 80 85 9p g5 wo ~nm6o rcrnrsmeo3o GTC TGC AGC GAT TAt 6G

Asn Als Lys Arg Asp Ala Nis Phe Cys pro Val Cys Ser Asp Tyr Als GM GGf1 TGT MG GCC tTT

Ser Gly Tyr Nis Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Als Phe ATC TGT CG GCC ACG

Phe Lys Arg Ser Ile Gln Gly Nis Asn Asp Tyr Ila Cys Pro Ala Thr AM AGC TGC GG GCC TGC

Asn Gln Cys Thr ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Als Cys GTC MG TGT GGi1 TCC AGG

Arg Leu Arg Lys Cys Tyr Gtu Vat Gly Net Val Lys Cys Gly Ser Arg AGA GM CGG TGT GCG TAC CGT ATA GtG CGG 996 AGG GG AGA AGT TCT AGC

Arg Glu Arg Cys Gly Tyr Arg Ile Val Arg Arg Gln Arg Ser Ser Ser MC GGT GGG GT GG

Glu Gln Yal His Cys Leu Ser Lys Ala Lys Arg Asn Gly Gly His Ala TTG AGT CG GAG ClU1 CTG

Pro Arg Val Lys Glu Leu Leu Lsu Ser Thr Leu Ser Pro Glu Gln Leu Mt GTG CTG GTG AGC CGT

Vel Leu Thr Leu Leu Glu Ala Glu Pro Pro Asn Val Leu Val Ser Arg 225 ~0 ~5 ATG ATG tCC CTC ACT MG

Pro Ser Net Pro Phe Thr Glu Ala Ser Met Net Net Ser Leu Thr Lys 240 245 250 ~5 GGC TGG GCC MG AM ATC

Leu Als Asp Lys Glu Lw Vat Nis Net Ile Qty Trp Als Lys Lys Ile 2~ 265 270 CM GTC CGG CTC TTA GM

Pro Gly Phe Val Glu Lau Ser Leu Leu Mp Gln Val Arg Leu Leu Glu CTG ATG TGG CGC TCC ATC

Ser Cys ~ Net Glu Val Leu ~t Val Gly Leu Net Arg Ser Ile 3~

GAC GC txC GGC MG CTC ATT TTC GCT CCC 1380 GAC CTC GTT CTG GAC AGG

Asp His Pro Gly Lys Leu lle Phe Ala Pro Asp Leu Val Leu Asp Arg 3~ 310 315 Asp Glu Gly Lys Cys Val Gtu Gly Ile Leu Glu Ile Phe Asp Net Leu AM CTC GG GC MG GAG

Leu Ala Thr Thr Ser Arg Phe Arg Glu Leu Lys Leu Gln His Lys Glu MC TCC AGT ATG TAC CCC

Tyr Leu Cys Val Lys Ala Net Ile Leu Leu Asn Ser Ser Net Tyr Pro 355 ~0 ~5 CGG MG CTG AG GC

Leu Ala Ser Ala Asn Gln Glu Ata Glu Ser Ser Arg Lys Leu Thr Nis Leu ~ Asn Ala Vat Thr A~ Ale Leu Yal Trp 3~ Ile Als Lya Ser GGT ATC TCC tCC GG CAG GG TG GTC CGA 1668 CTG GCC MC CTC CtG ATG

Gly Ile Ser Ser Gln Gln Gln Ser Yal Arg Leu Ala Asn Leu Lau !!et MG GGC ATG GM GT CTG

Leu Leu Ser Nis Yal Arg Nis Ile Ser Asn Lys Gty Met Glu His Leu GtG TAT GAC CTG CTG CTG

Leu Ser Met Lys Cys Lys Mn Vat Vat Pro Vel Tyr Asp Leu Leu Leu TAC AAG TCC TG ATC TCG

Glu lief Leu Asn Ala Nis Thr Leu Arg Gly Tyr lys Ser Ser Ile Ser MG MC AM GAG AGC TCC

Gly Ser Glu Cys Ser Ser thr Glu Asp Ser Lys Asn Lys Glu Ser Ser GAGGACTAC

Gln Asn Leu Gln Ser Gln AGAGATGGTC MMGTGGM GTGtACCCT AGGTCTGGG 1968 GGTTCCTCTT AGGGCTGCCT

GGGGTGGTT GTGTGGCGGT

AGTCTGAM GGTTCTGGM

CTAMGGTG MGTCTGATT TGGAACGlITT GTCCTTAGTC2208 TTGAGATG GCTGTTTCG

TGGGTTATTC TATGMGAC

CAGMlIGATA GTGCAAGCTT AGATGTACCT TGTTCCTCCT2448 CCGGACCCT TGGGTTAGT

TGTGTGGGT TMGATTTM

TTCTCTCTCT CCCGGMTTT

(2) INFORMATION FOR SEQ ID N0:4:
<i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 485 amino acids tB) TTPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATtNtE:
(A) (B) -(D) OTHER INFORMATION: /note= "Amino acid seyue:xe of a rat ERbeta"
(xi ) SEQUENCE DESCRIPTt011: SEQ 10 110:4:
Met Thr Phe Tyr Ser Pro Ala Val Met Asn Tyr Ser Val Pro Gly Ser WO 99!11760 PGT/US98/18030 Thr Ser Asn Lau Asp Gly Gly Pro Val Arg Leu Ser Thr Ser Pro Asn Yat teu Trp Pro Thr Ser Gly His Leu Ser Pro Leu Ala Thr His Cys Gln Ser Ser Lau Leu Tyr Ala Glu Pro Gln tys Ssr Pro Trp Cys Glu 5o s5 bo Ala Arg Ser Leu Glu His Thr Leu Pro Vat Asn Arg Gtu Thr Leu Lys 65 70 75 gp Arg Lys Lau Ser Gly Ser Ser Cya Ala Ser Pro Vsl Thr Ser Pro Asn Ala Lys Arg Asp Ata Nis Phe Cys Pro Val Cys Ser Asp Tyr Ata Ser G!y Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ata Phe Phe Lys Arg Ser Ile Gtn Gly His Asn Asp Tyr Ile Cys Pro Ala Thr Asn Gln Cys Thr ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys Arg Leu Arg Lys Cys Tyr Glu Val Gly list Val Lys Cys Gly Ssr Arg Arg Glu Arg Cys Gly Tyr Arg Ile Val Arg Arg Gln Arg Ser Ser Ser Glu Gln Val His Cys Leu Ser Lys Ala Lys Arg Asn Gly Gly His Ala Pro Arg Vat Lys Glu Leu Leu Lsu Ser Thr Leu Ser Pro Glu Gtn Leu Val Lau Thr Lsu Leu Glu Ale Glu Pro Pro Asn Vat Leu Val Ser Arg Pro Ser list Pro Phe Thr Glu Als Ser Het Met list Ser Leu Thr Lys Leu Ala Asp lys Glu Leu yal His Het Ile Gly Trp Ala Lys Lys Ile Pro Gly Phe Vsl Glu Leu Ser Leu Lau Asp Gln Val Arg Leu Leu Glu Ser Cys ~ filet Glu.Val Lsu ~t Val Gly Leu Net Trip Arg Ser Ile Asp His Pro Gly tys Leu Its Phe Ale Pro Asp Leu Val Leu Asp Arg Asp Glu Gly Lys Cys Val Glu Gly Its Leu Glu Ile Phe Asp Net Leu Leu Ata Thr Thr Ser Arg Phe Arg Glu Leu Lys Lsu Gln Nis Lys Glu Tyr Leu Cys Vat Lys Ala Net file Leu Leu Asn Ssr Ser Net Tyr Pro Leu Ala Ssr Ala Asn Gln Gtu Ale Glu Ser Ser Arg Lys Lau Thr His Leu Leu Asn Ala Val Thr Asp Ala Leu Val Trp Val Ile Ala Lys Ser Gly 3~ 395 400 Ile Ser Ser Gln Gln Gln Ser Vat Arg Leu Ala Asn Leu Leu Net Leu WO 99/11760 ~ PGT/US98J18030 4~ 410 415 Leu Ser His Val Arg His Ile Ser Asn Lys Gly Met Glu His Leu Leu 4~ 425 430 Ser llet Lys Cys Lys Asn Val Val Pro Vat Tyr Asp Leu Leu Leu Gtu Ilet Leu Asn Ala His Thr Leu Arg Gly Tyr Lys Ser Ser Ile Ser Gly Ser Glu Cys Ser Ser Thr Glu Asp Ser Lys Asn Lys Glu Ser Ser Gln Asn Leu Gln Ser 6ln (2) INFORNATIOM FOR SE0 1D 110:5:
(i) SEOUEMCE CHARACTERISTICS:
(A) IEMGTN: 485 amino acids (8) TYPE: amino acid (C) STRANDEDI~SS: uNuwxn (D) TOPOLOGr: ur~nown (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) (8) LOCAT1011: 1..485 (D) OTHER IMFORMATI011: /note. ~Mino acid sequence of huasn ERbeta~
(xi) SEQUENCE DESCRIPTION: SE0 ID N0:5:
Met Thr Phe Tyr Ser Pro Ala Val Met Asn Tyr Ser Ile Pro Ser Asn Vat Thr Asn Lau Gtu Gly Gly Pro Gly Arg Gln Thr Thr Ser Pro Asn Val Leu Trp Pro Thr Pro Gly Nis Leu Ser Pro Leu Val Val His Arg Gln Leu Ser Nis Lau Tyr Ala Glu Pro Gln Lys Ser Pro Trp Cys Glu Ala Arg Ser Leu Glu His Thr Le~u Pro Val Asn Arg Glu Thr Leu Lys Arg Lys Val Ser G~ly Asn Arg Cys Ala ~r Pro Val Thr Gly ~ Gly Ser Lys Arg Asp Ala Nis Phe Cys Ala Vsl Cys Ser Asp Tyr Ala Ser Gly Tyr Nis Tyr Gly Vsl Trp Ser Cys Glu Gly Cys Lys Ala Phe Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Ile Cys Pro Ala Thr Asn Gln Cys Thr ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys Arg Leu Arg Lys Cys Tyr Glu Val Gly Ilet Val Lys Cys Gly Ser Arg Arg Glu Arg Cys Gty Tyr Arg Leu Vsl Arg Arg Gln Arg Ser Ala Asp Glu Gln Leu ;ids, Cys Ala Gly Lys A2l~a Lys Arg Ser Gly Gly Nis Ala Pro Arg Val Arg Glu Leu Leu Leu Asp Ala Leu Ser Pro Glu Gln Leu Yal Leu Thr Leu Leu Glu Ala Glu Pro Pro Nis Vsl leu ile Ser Arg Pro Ser Ala Pro Phe Thr Glu Ala Ser Ilet Met Met Lau Ser Thr Lys Leu Ala Asp Lys Glu leu Val His Met Ile Ser Trp Ala Lys Lys 1le Pro 2~ 265 270 Gly Phe Val Glu Leu Ser Leu Phe Asp Gln Val Arg Lou Leu Glu Ser Cys Trp Met Glu Val Leu Met ltet Gly Leu Met Trp Arg Ser Ile Asp 2~ 295 300 His Pro Gly Lys Leu Ile Phe Ala Pro Asp Lau Val Leu Asp Arg Asp Glu Gly Lys.Cys Vel Glu Gly Ile Leu Glu Ile Phe Asp Met Leu Leu Ala Thr Thr Ser Arg Phe Arg Glu Leu Lys Leu Gln His Lys Glu Tyr Leu Cys Val Lys Ala Met Ile Leu Leu Asn Ser Ser Net Tyr Pro Leu Val Thr Ala Thr Gln Asp Ala Asp Ser Ser Arg Lys Leu Ale Nis Leu Leu Asn Ala Val Thr Asp Ala Leu Vat Trp Vat Ile Ala Lys Ser Gly Ile Ser Ser Gln Gln Gln Ser Met Arg Lau Ale Asn Leu Leu Met Leu Leu Ser Nis Val Arg His Ala Ser Asn Lys Gly Ilet Glu Nis Leu Leu Asn Met Lys Cys Lys Asn Vsl Val Pro Vat Tyr Asp Leu Leu Leu Glu Met Leu Asn Ala His Val Leu Arg Gly Cys Lys Ser Ser Ile Thr Gly Ser Glu Cys Ser Pro Ala Glu Asp Ser Lys Ser Lys Glu Gty Ser Gln Asn Leu Gln Ser Gln (2) 1HFORMATI011 FOR SEQ ID 110:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1460 bees pairs (B) TYPE: nucleic scid (C) STRANDEDHESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genoaic) (fx) FEATURE:
(A) NAME/ICEY: -(B) LOCATIOM: 1..1460 (D) OTHER IMFORMATION: /notes "DNA sequence of huaen ERbeta"

(xi) SEDIIENCE DEixRIPTI0fl:, SEa tD
N0:6:

TCCGGCMT GTCACTAACT

TGG~AGGTGG GCCTGGTCGG GGIICCACAA GCCCAAIITGT120 GTTGTGGCG ACACCTGGGC

TGTAAACAGA GAGAGCTGA

ATATGCTAT GGAGTCTGGT

AGGAGTAAT GATTATATTT

CAAGAGCTGC GGGCCTGCC

CTCCCGGAGA GAGAGIITGTG

GCTGGCTGT GCCGGGAGG

CGTGTGCTG ATGGCCGCC

GACCAAGTTG GCCGACAAGG

CTTTGTGGAG CTGGCCTGT

AGATCTTGTT CTGGAGGGG

CTGTGTCAAG GCGTGATCC

TTGGGTGATT GCCAAGAGCG

CCTGATGCTC CTGTCCGCG

GTGAAGTGC AAAAATGTGG

CGTGCTTCGC GGGTGCAAGT

TAAMGCAAA GAGGGCTCCC

AGAACCTAG GTCTGGTGA

Claims (71)

WHAT IS CLAIMED IS:

1. A method of screening a test compound for differential ER.alpha.-mediated and ER.beta.-mediated activation at an AP1 site, said method comprising the steps of:
a) providing a first cell comprising an estrogen receptor .beta.(ER.beta.), an protein, and a construct comprising a promoter comprising an AP1 site which regulates expression of a first reporter gene;
b) contacting said first cell with said test compound; and c) comparing the expression of said first reporter gene with the ER.alpha.-mediated expression of a gene at an AP1 site.

2. The method of claim 1, wherein said first cell contains a heterologous estrogen receptor beta (ER.beta.).

3. The method of claim 1, wherein said ER.beta. comprises an amino acid seqeunce of SEQ ID NO:3 or SEQ ID NO: 4.

4. The method of claim 1, wherein said cell contains a heterologous AP1 protein.

5. The method of claim 1, wherein said reporter gene is selected from the goup consisting of chloramphenicol acetyl transferase (CAT),luciferase, .beta.-galactosidase (.beta.-gal), alkaline phosphatase, horse radish peroxidase (HRP), growth hormone (GH), and green fluorescent protein (GFP).

6. The method of claim 5, wherein said reporter gene encodes a luciferase or a green fluorescent protein (GFP).

7. The method of claim 1, wherein said test compound is a test compound known to have anti-estrogenic activity.

8. The method of claim 1, wherein said ER.alpha.-mediated expression of a gene at an AP1 site is determined by:
d) providing a second cell comprising an estrogen receptor a (ER.alpha.), AP1 proteins, and a construct comprising a promoter comprising an AP1 site which regulates expression of a second reporter gene;
e) contacting said second cell with said test compound; and f) detecting expression of said second reporter gene

9. The method of claim 8, wherein said standard estrogen response element is from the Xenopus vitellogenin A2 gene.

10. The method of claim 8, wherein said second reporter gene and said first reporter gene are the same reporter genes.

11. The method of claim 8, wherein said first cell and said second cell are the same cell.

12. A method of screening a test compound for the ability to activate or inhibit estrogen receptor .beta. (ER.beta.) mediated gene activation at an AP1 site, said method comprising the steps of:
a) providing a first cell comprising an estrogen receptor .beta. (ER.beta.), proteins; and a construct comprising a promoter comprising an AP1 site which regulates expression of a first reporter gene;
b) contacting said first cell with said test compound; and c) detecting expression of said first reporter gene.

13 . The method of claim 12, wherein said first cell contains a heterologous estrogen receptor .beta. (ER.beta.).

14. The method of claim 12, wherein said ER.beta. comprises the amino acid sequence of Seq ID No: 3 or Seq ID NO: 5.

15. The method of claim 14, wherein said first cell contains a heterologous AP1 protein.

16. The method of claim 12, wherein said reporter gene is selected from the group consisting of chloramphenicol acetyl transferase (CAT), luciferase, .beta.-galactosidase (.beta.-gal), alkaline phosphatase, horse radish peroxidase (HRP), growth hormone (GH), and green fluorescent protein (GFP).

17. The method of claim 16, wherein said reporter gene encodes a luciferase or a green fluorescent protein (GFP).

18. The method of claim 12, wherein said test compound is a test compound known to have anti-estrogenic activity.

19. The method of claim 12, further comprising the steps of:

d) providing a second cell comprising an estrogen receptor .alpha.(ER.alpha.), proteins, and a construct comprising a promoter comprising an AP1 site which regulates expression of a second reporter gene;
e) contacting said second cell with said test compound; and f) detecting expression of said second reporter gene.

20. The method of claim 12, further comprising the steps of:
d) providing a third cell comprising an estrogen receptor .alpha. (ER.alpha.), and a construct comprising a promoter comprising a standard estrogen response element (ERE) which regulates expression of a third reporter gene;
e) contacting said third cell with said test compound; and f) detesting expression of said third reporter gene.

21. The method of claim 20, wherein said standard estrogen response element is from the Xenopus vitellogenin A2 gene.

22. The method of claim 12, further comprising the steps of:
d) providing a fourth cell comprising an estrogen receptor .beta. (ER.beta.), and a construct comprising a promoter comprising a standard estrogen response element (ERE) which regulates expression of a fourth reporter gene;
e) contacting said fourth cell with said test compound; and f) detecting expression of said fourth reporter gene.

23. The method of claim 22, wherein said standard estrogen response element is from the Xenopus vitellogenin A2 gene.

24. The method of claim 20, wherein said first cell and said third cell are the same cell.

25. The method of claim 22, wherein said first cell and said fourth cell are the same cell.

26. The method of claim 12, further comprising contacting said first cell with a second compound, in addition to said test compound, wherein said second compound is known to activate transcription through estrogen receptor .beta.
(ER.beta.) mediated gene activation at an AP1 site;
wherein said detecting comprises detecting test compound mediated decrease in said estrogen receptor .beta. (ER.beta.) mediated gene activation at an AP1 site.

27. The method of claim 26, wherein said detecting comprises comparing the expression of said first reporter gene in the presence of the test compound and the second compound with the expression of said first reporter gene in the presence of the second compound without the test compound.

28. The method of claim 26, wherein said second compound known to activate transcription through estrogen receptor .beta. (ER.beta.) mediated gene activation at an AP1 site is identified by a method comprising the steps of:
a) providing a second cell comprising an estrogen receptor .beta. (ER.beta.), and AP1 protein, and a construct comprising a promoter comprising an AP1 site that regulates expression of a second reporter gene;
b) contacting said second cell with second compound; and c) detecting the expression of said second reporter gene, wherein an increase in expression of said second reporter gene produced by said compound indicates that said second compound activates transcription through ER.beta. at said AP1 site.

29. The method of claim 12, further comprising contacting said first cell with a second compound, in addition to said test compound, wherein said second compound is known to inhibit transcription through estrogen receptor .beta.
(ER.beta.) mediated activity at an AP1 site; and wherein said detecting comprises detecting test compound mediated increase in estrogen receptor .beta. (ER.beta.) mediated gene activation at an AP1 site.

30. The method of claim 29, wherein said detecting comprises comparing the expression of said first reporter gene in the presence of said second compound and said test compound with the expression of said first reporter gene in the presence of said second compound without said test compound.

31. The method of claim 29, wherein said second compound known to inhibit transcription through estrogen receptor .beta. (ER.beta.) mediated gene activation at an AP1 site is identified by a method comprising the steps of:
a) providing a second cell comprising an estrogen receptor .beta. (ER.beta.), and AP1 protein, and a construct comprising a promoter comprising an AP1 site that regulates expression of a second reporter gene;
b) contacting said second cell with second compound; and c) detecting the expression of said second reporter gene, wherein a decrease in expression of said second reporter gene produced by said compound indicates that said second compound inhibits transcription through ER.beta. at said AP1 site.

32. A cell comprising an estrogen receptor .beta. (ER.beta.), AP1 proteins, and a construct comprising a promoter comprising an AP1 site which regulates expression of a first reporter gene.

33. The cell of claim 32, wherein said cell further contains a receptor for a nuclear transcription factor ligand other than estrogen.

34. The cell of claim 32, wherein said cell contains a heterologous estrogen receptor a (ER.beta.).

35. The cell of claim 32, wherein said cell contains a heterologous AP1 protein.

36. The cell of claim 32, wherein said heterologous AP1 protein is c-jun.

37. The cell of claim 32, wherein said first reporter gene is selected from the group consisting of chloramphenicol acetyl transferase (CAT), luciferase, .beta.-galactosidase (.beta.-gal), alkaline phosphatase, horse radish peroxidase (HRP), growth hormone (GH), and green fluorescent protein (GFP).

38. The cell of claim 37, wherein said reporter gene encodes a luciferase or a green fluorescent protein (GFP).

39. The cell of claim 38, wherein said cell further comprises a construct comprising a promoter comprising a standard estrogen response element (ERE) which regulates expression of a second reporter gene.

40. The cell of claim 39, wherein said standard estrogen response element is from the Xenopus vitellogenin A2 gene.

41. The cell of claim 32, wherein said cell is a mammalian cell.

42. The cell of claim 41, wherein said cell is derived from breast tissue or from uterine tissue.

43. A method of screening a nuclear transcription factor ligand for the ability to modulate estrogen receptor .beta. mediated activation or inactivation of transcription at an AP1 site, said method comprising the steps of:

a) providing a first cell containing an estrogen receptor .beta. (ER.beta.), an AP1 protein, a receptor for said nuclear transcription factor ligand, and a construct comprising a promoter comprising an AP1 site which regulates expression of a first reporter gene;
b) contacting said first cell with said transcription factor ligand and with a compound having ER.beta. mediated activity at said AP1 site; and c) detecting expression of said first reporter gene.

44. The method of claim 43, further comprising the steps of:
d) providing a second cell containing an estrogen receptor .beta. (ER.beta.), a receptor for said nuclear transcription factor ligand, and a construct comprising a promoter comprising an estrogen response element (ERE) that regulates expression of a second reporter gene;
e) contacting said second cell with said transcription factor ligand and with said compound having AP-1 mediated estrogenic activity; and f) detecting expression of said second reporter gene.

45. The method of claim 44, wherein said first cell and said second cell are the same cell.

46. The method of claim 43, further comprising the steps of:
d) providing a second cell containing a cognate receptor of said transcription factor ligand, and a promoter comprising a response element for said cognate receptor that regulates expression of a second reporter gene;
e) contacting said second cell with said transcription factor ligand and with said compound having compound having ER.beta. mediated activity at said AP1 site; and f) detecting expression of said second reporter gene.

47. The method of claim 46, wherein said first cell and said second cell are the same cell.

48. The method of claim 43, wherein said nuclear transcription factor ligand is selected from the group consisting of a glucocorticoid; a progestin, vitamin D, retinoic acid, a an androgen, a mineralcorticoid, and a prostaglandin.

49. The method of claim 46, wherein said cognate receptor is selected from the group consisting of an estrogen receptor, a glucocorticoid receptor, a progestin PR-A receptor, and progestin PR-B receptor, androgen receptor, a mineralcorticoid receptor, and a prostaglandin rector.

50. The method of claim 43, wherein said ER.beta. comprises an amino acid sequence of Seq ID No: 3 or SEQ ID No: 5.

51. The method of claim 43, wherein said estrogen receptor ER.beta. is heterologous to said cell.

52. The method of claim 43, wherein said receptor for said nuclear transcription factor ligand is heterologous to said cell.

53. The method of claim 43, wherein said cell expresses an AP1 protein from a heterologous DNA.

54. The method of claim 53, wherein said AP1 protein is c-jun.

55. The method of claim 43, wherein said nuclear transcription factor is a progestin; and said receptor for said nuclear transcription factor ligand is a progestin receptor.

56. The method of claim 43, wherein said nuclear transcription factor is a glucocorticoid and said receptor for said nuclear transcription factor ligand is a GR
receptor.

57. A method of screening an agent for the ability to alter modulation of estrogen receptor .beta. (ER.beta.) activation or inactivation of transcription at an AP1 site by a nuclear transcription factor ligand, said method comprising the steps of:
a) providing a first cell containing an estrogen receptor .beta. (ER.beta.), an AP1 protein, a receptor for said nuclear transcription factor ligand, and a promoter comprising an AP1 site which regulates expression of a first reporter gene;
b) contacting said first cell with said transcription factor ligand, with a compound having ER.beta. mediated activity at an AP1 site, and with said agent; and c) detecting expression of said first reporter gene.

58. The method of claim 57, further comprising the steps of:
d) providing a second cell containing an estrogen receptor .beta. (ER.beta.), a receptor for said nuclear transcription factor ligand, and a promoter comprising an estrogen response element (ERE) that regulates expression of a second reporter gene;
e) contacting said second cell with said transcription factor ligand and with said compound having AP-1 mediated estrogenic activity; and f) detecting expression of said second reporter gene.

59. The method of claim 58, wherein said first cell and said second cell are the same cell.

60. The method of claim 57, wherein said nuclear transcription factor is selected from the group consisting of a glucocorticoid, a progestin; vitamin D, retinoic acid, an androgen, a mineralcorticoid, a prostaglandin.

61. The method of claim 57, wherein said a receptor for said nuclear transcription factor ligand is selected from the group consisting of an estrogen receptor, a glucocorticoid receptor, a progestin PR-A receptor, progestin PR-B receptor, an androgen receptor, a mineralcorticoid receptor, and a prostaglandin receptor.

62. The method of claim 57, wherein said cell contains a heterologous estrogen receptor .beta. (ER.beta.).

63. The method of claim 57, wherein said cell expresses a heterologous receptor for said nuclear transcription factor ligand.

64. The method of claim 57, wherein said cell contains a heterologous AP1 protein.

65. The method of claim 64, wherein said AP1 protein is c-jun.

66. The method of claim 57, wherein said nuclear transcription factor is a progestin; and said receptor for said nuclear transcription factor ligand is a progestin receptor.

67. The method of claim 57, wherein said nuclear transcription factor is a glucocorticoid and said receptor for said nuclear transcription factor ligand is a GR
receptor.

68. A kit for screening a compound for the ability to activate or inhibit estrogen receptor .beta. (ER.beta.) mediated gene activation at an AP1 site, said kit comprising a container containing a cell comprising an estrogen receptor .beta.
(ER.beta.), an AP1 protein, and a construct comprising a promoter comprising an AP1 site which regulates expression of a first reporter gene.

69. The kit of claim 68, further comprising instruction materials containing protocols for the practice of the assay methods of claims 1, 9, 10, 12, 16, or 18.

70. The kit of claim 68, wherein said cell further comprises a receptor for a nuclear transcription factor ligand other than estrogen.

71. The kit of claim 68, further comprising instruction materials containing protocols for the practice of the assay methods of claims 29, 30, 32, 42, or 43.