WO2003000730A1 - Compounds that modulate estrogen actions - Google Patents

Abstract

The invention features the use of CITED nucleic acids and proteins in improved methods for detecting compounds that modulate estrogen receptor-dependent activity. These methods provide a sensitive assay for the detection of environmental estrogen mimics and, therefore, are useful in assessing the potential health risks associated with compounds present in our surroundings. In addition, these methods may be used to identify compounds beneficial for the treatment or prevention of an estrogen-related condition. The invention also features diagnostic and prognostic methods for estrogen-related disorders that involve CITED protein or nucleic acid assays.

Description

COMPOUNDS THAT MODULATE ESTROGEN ACTIONS

Statement as to Federally Sponsored Research The present research was supported by a grant from the National Institutes of Health (Number R01-CA82230-01). The U.S. government has certain rights to this invention.

Field of the Invention

The field of the invention is estrogen regulation.

Background of the Invention Estrogen is a steroid hormone that plays important roles in a diverse set of biological processes including development, reproduction, bone metabolism, depression, and in the prevention of cardiovascular disease, addition to regulating normal processes in the organism, estrogen has been shown to play a role in certain disorders, such as the growth of breast and prostate cancers. The effects of estrogen on gene regulation are mediated by the estrogen receptor, which is part of a large family of nuclear receptors (NRs). Both estrogen receptors α and β are involved in the progression of a number of human diseases. For example, estrogen receptor β (ERβ) has been shown to play a role in Alzheimer's Disease, Parkinson's Disease, endometrial and breast cancer, and in disorders of the urogenital tract as is described in Wang et al. (Proc. Natl. Acad. Sci. USA 98:2792-

2796, 2001), Takama et al. (Br. J. Cancer 84:545-549, 2001), Gustafsson and Warner (J Steroid Biochem. Mol. Biol. 74:245-248, 2000), and Kuiper et al. (Front. Neuroendocrinol. 19:253-258, 1998).

Estrogen receptors (ERα and ERβ) are expressed in more than 70% of primary human breast cancers, and the growth and survival of such ER-positive breast cancer cells is usually dependent on estrogen (Bouras et al., Cancer Res. 61:903-907, 2001; Roger et al., Cancer Res. 61:2537-2541, 2001; Ciocca et al., Endocrine 13:1-10, 2000). On the other hand, up to 70% of breast cancers can convert androgen to estrogen using tumor aromatase, and this intra-tumor production of estrogen plays a major role in the growth of breast cancers (Brodie et ah, J. Steroid Biochem. Mol. Biol. 61:281-286, 1997; Goss et al., J. Clin. Oncol. 19:881-894, 2001). Chemical agents that inhibit local production of estrogen, or intracellular estrogen signaling, have been used for therapeutic regimens, which are in general referred to as hormone therapy (Ciocca et al., Endocrine 13:1-10, 2000; Johnston, Anticancer Drugs 8:911- 930, 1997; Osborne et al, J. Clin. Oncol. 18:3172-3186, 2000). For example, aromatase inhibitors, such as Anastrozole and Letrozole, block intratumor estrogen production and have been clinically used as the second-line therapy for breast cancers (reviewed in Goss et al., J. Clin. Oncol. 19:881-894, 2001). h addition, Selective Estrogen Receptor Modifiers (SERMs), such as Tamoxifen and Raloxifene, bind to the ERs and act as competitive inhibitors of estrogen signaling (reviewed in Osborne et al., J. Clin. Oncol. 18:3172-3186, 2000). The successful clinical applications of these hormonal agents to the treatment and prevention of breast cancers have demonstrated the importance of estrogen signaling as a target for therapeutic drugs. Unfortunately, a substantial percentage (30-70%) of breast cancers eventually acquire resistance to hormone therapy despite, in most cases, fully functional ERs still being expressed at the time of cancer relapse (Johnston, Anticancer Drugs 8:911-930, 1997; Osborne et α/., J Clin. Oncol. 18:3172-3186, 2000). A possible mechanism for this acquired resistance is that the sensitivity of ER-positive cancer cells to estrogen may be enhanced by overexpression of ER co-activators (reviewed in Johnston,

Anticancer Drugs 8:911-930, 1997). Supporting this statement is data showing that increased expression of an ER coactivator, AIBl (Amplified in Breast Cancers 1), is found in 30-60% of ER-positive breast cancers, and the AIBl gene is amplified in 5- 10% of all human breast cancers (Bouras et al, Cancer Res. 61:903-907, 2001; Anzick et al, Science 277:965-968, 1997; Bautista et al, Clin. Cancer Res. 4:2925- 2929, 1998; Kurebayashi et al, Clin. Cancer Res. 6:512-518, 2000; Thenot et al, Mol. Cell Endocrinol 156:85-93, 1999; Murphy et al, Cancer Res. 60:6266-6271, 2000). h addition, a significant increase in the expression of other ER co-activators (such as SRC-1, TIF2, or CBP) has also been observed during the tumorigenesis of breast cancers (Kurebayashi et al, Clin. Cancer Res. 6:512-518, 2000; Murphy et al, Cancer Res. 60:6266-6271, 2000). Since the growth of many cancers is regulated by the level of estrogen, any compound that affects estrogen-mediated signaling can have an impact on human health. While the human body produces estrogen to regulate a variety of processes, estrogen-like compounds are also commonly found in the environment. For example, endocrine-disrupting contaminants (EDCs) are natural or synthetic chemical compounds found in, for instance, the water, soil, food, plants (e.g., phytoestrogens) or animal bodies, and can exert significant influences on the endocrine systems of humans or animals (Guillette Jr., L. and D. Crain, eds. Environmental Endocrine Disrupters, Taylor & Francis, 2000). Many EDCs show weak estrogenic or anti- estrogenic activities in vivo and/or in vitro, and numerous studies have demonstrated their detrimental effects on wildlife (Crain et al, Endocrine-Disrupting Contaminants and Hormone Dynamics: Lessons from Wildlife, in Environmental Endocrine Disrupters, D. Crain and L. Guillette Jr., Editors 1-21, 2000; Hayes, The Importance of Comparative Endocrinology in Examining the Endocrine Disrupter Problem, in Environmental Endocrine Disrupters, D. Crain and L. Guillette Jr., Editors, Taylor & Francis: New York 22-51, 2000). a specific example, exposure to very low levels of the pesticide dichlorodiphenyltrichloroethane (DDT), or to industrial chemicals, such as polychlorinated biphenyls (PCBs), can induce production of female-specific protein vitellogenin in male fishes, (Anderson et al, Toxicol. Appl. Pharmacol. 137:210-218, 1996; Flouriot et al, J. Mol. Endocrinol 15:143-151, 1995), toads, and reptiles (Palmer et al, Environ. Health Perspect 4: 19-25). hi addition, estrogenic EDC contamination of Lake Apoka of Florida resulted in marked abnormality in gonadal development, serum steroid levels, and phallus size of male alligators (Guillette et al, Environ. Health Perspect. 102:680-688, 1994). Possible influences of DDT on estrogen-related human cancers have also been predicted (Adami et al, Cancer Causes Control 6:551-566, 1995; Soto et al, Environ. Health Perspect 102:380-383, 1994).

Furthermore, EDCs present in the environment are likely to impact human health, as is evidenced by Bisphenol A, a plasticizer widely used in production of polycarbonate plastics, and p-Nonylphenol, a commonly used industrial lubricant, being leached out of plastics into water when heated, and, at very low concentrations, by these chemicals supporting estrogen-dependent in vitro growth of MCF-7 human breast cancer cells (Krishnan et al, Endocrinology 132:2279-2286, 1993; Soto et al, Environ. Health Perspect. 92:167-173, 1991).

The effects of environmental estrogens on human health have not been definitively determined, but the current data suggest that estrogen-like compounds may significantly influence prevention, tumorigenesis, and progression of human proliferative diseases, including breast cancer. Accordingly, there is a significant need for a sensitive assay system that quantitatively detects estrogen-like compounds specifically and efficiently. Preferably, such an assay may be used in high throughput evaluations of environmental estrogens present in dairy food, water, and other materials that may come into direct contact with humans.

Summary of the Invention The present invention features the use of CITED nucleic acids and proteins in improved methods for detecting compounds that modulate estrogen receptor- dependent activity. These methods provide a sensitive assay for the detection of environmental estrogen mimics and, therefore, are useful in assessing the potential health risks associated with compounds present in our surroundings. In addition, these methods may be used to identify compounds beneficial for the diagnosis or prevention of an estrogen-related condition.

Accordingly, the first aspect of the invention features a screening method for determining whether a candidate compound modulates estrogen receptor activity. This method involves: (a) contacting a cell or in vitro sample expressing a greater than naturally-occurring amount of CITED protein, or CITED activity, and an estrogen receptor target gene with a candidate compound; and (b) measuring expression of the estrogen receptor target gene, whereby the candidate compound is determined to modulate estrogen receptor activity if the candidate compound causes a change in expression of the estrogen receptor target gene.

In the second aspect, the invention features another screening method for determining whether a candidate compound modulates estrogen receptor activity.

This method includes: (a) contacting a cell or in vitro sample expressing a CITED2, CITED3, or CITED4 protein and an estrogen receptor target gene with a candidate compound; and (b) measuring expression of the estrogen receptor target gene, whereby the candidate compound is determined to modulate estrogen receptor activity if the candidate compound causes a change in expression of the estrogen receptor target gene, hi preferred embodiments of this aspect of the invention, the cell or sample also expresses a CITED 1 protein, or expresses multiple CITED proteins. h addition, it may also be desirable to use cells or samples expressing additional estrogen receptor co-activators, for example, ATBl, in the methods of these aspects of the invention.

A preferred embodiment of the first or second aspect of the invention encompasses further contacting the cell or sample with an estrogen receptor agonist, for example, estrogen, h an additional preferred embodiment, the candidate compound activates the estrogen receptor target gene, and may be useful, for example, in the treatment or prevention of depression, cardiovascular disease, infertility, or osteoporosis. Samples may be obtained, for example, by means of a biopsy. In another embodiment of these aspects of the invention, the candidate compound inhibits the estrogen receptor target gene and may be useful for the treatment or prevention of cancer (e.g., breast, prostate, or ovarian cancer), a gynecological disorder, endometriosis, or the symptoms of menopause.

Additionally, in further preferred embodiments, the CITED protein, for example, a mammalian protein, is expressed under the control of a heterologous promoter or the CITED protein is encoded by a heterologous nucleic acid. The CITED protein maybe CITED1, CITED2, CITED3, or CITED4.

In another preferred embodiment of these aspects of the invention, the estrogen receptor target gene is TGF-α. h addition, the estrogen receptor target gene may be a reporter gene operably linked to a promoter, for example, a TGF-α promoter, and includes an estrogen response element. hi further embodiments, the estrogen receptor target gene is stably integrated into the genome of the cell, or is transiently transfected into the cell, and may express, for example, a reporter gene, such as green fluorescent protein, firefly luciferase, or alkaline phosphatase.

A third aspect of the invention features another screening method for determining whether a candidate compound modulates estrogen receptor activity. This method involves: (a) contacting a cell expressing an estrogen receptor target gene and a greater than naturally-occurring amount of CITED 1, CITED2, CITED3, or CITED4 protein, or CITED activity, with a candidate compound; and (b) measuring aggregation of the cell, whereby the candidate compound is determined to modulate estrogen receptor activity if the candidate compound causes a change in aggregation in the cell.

In one embodiment of this aspect, the amount of aggregation is compared to the amount of aggregation in control samples from both subjects having an estrogen- related condition and subjects not having an estrogen-related condition. h another embodiment, the cell may be contacted with an estrogen receptor agonist, for example, estrogen, and the candidate compound may increase aggregation. This candidate compound may be useful for the treatment or prevention of depression, cardiovascular disease, infertility, or osteoporosis. hi an additional embodiment, the candidate compound that decreases cell aggregation, and may be useful for the treatment or prevention of cancer (e.g., breast, prostate, or ovarian cancer), a gynecological disorder, endometriosis, or the symptoms of menopause.

Furthermore, in this aspect of the invention, the CITED protein, for example, a mammalian CITED protein, may be expressed under the control of a heterologous promoter, or may be encoded by a heterologous nucleic acid. h another embodiment of this aspect, aggregation may be measured using time-lapse videomicroscopy.

A fourth aspect of the invention features yet another screening method for determining whether a candidate compound modulates expression of a CITED mRNA or protein. This method includes: (a) contacting a cell or in vitro sample expressing a CITED mRNA or protein with a candidate compound; and (b) measuring expression of the CITED mRNA or protein, thereby determining whether the candidate compound modulates expression. one embodiment the candidate compound increases expression of CITED mRNA or protein and is useful for the treatment or prevention of depression, cardiovascular disease, infertility, or osteoporosis. In another embodiment, the candidate compound decreases expression of CITED mRNA or protein and is useful for the treatment or prevention of cancer (e.g., breast, prostate, or ovarian cancer), a gynecological disorder, endometriosis, or the symptoms of menopause. In an additional embodiment, the CITED protein, for example, a mammalian protein, is encoded by a heterologous nucleic acid. Furthermore, the CITED protein maybe CITED 1, CITED2, CITED3, or CITED4.

In a fifth aspect, the invention features yet another screening method for determining whether a candidate compound modulates CITED expression. This method includes: (a) contacting a cell or in vitro sample expressing a reporter gene under the control of a CITED promoter with a candidate compound; and (b) measuring expression of the reporter gene, thereby determining whether the candidate compound modulates expression mediated by the CITED promoter.

In one embodiment of this aspect, the compound increases CITED expression and may be useful for the treatment or prevention of depression, cardiovascular disease, infertility, or osteoporosis. hi another embodiment of this aspect, the candidate compound decreases CITED expression and may be useful for the treatment or prevention of cancer (e.g., breast, prostate, or ovarian cancer), a gynecological disorder, endometriosis, or the symptoms of menopause. h an additional embodiment, the CITED promoter, for example, a mammalian CITED promoter, is a heterologous nucleic acid. The CITED promoter may also be a CITED 1, CITED2, CITED3, or CITED4 promoter. Any reporter gene may be utilized (for example, any of the reporter genes listed above). A sixth aspect of the invention features a method of diagnosing an estrogen- related condition or a propensity thereto in a subject, for example, a human. This method involves measuring the amount of a CITED mRNA or protein in a sample from a subject, where an increase or decrease in the CITED mRNA or protein in the sample relative to a control sample indicates that the subject has the estrogen-related condition or a propensity thereto.

A seventh aspect of the invention features a method of determining the prognosis for treatment of an estrogen-related condition or a propensity thereto in a subject, for example, a human. This method involves measuring the amount of a CITED mRNA or protein in a sample from a subject, where an increase or decrease in the CITED mRNA or protein in the sample relative to a control sample determines the prognosis for treatment of an estrogen-related condition or a propensity thereto in the subject. hi one embodiment of these aspects of the invention, the CITED mRNA or protein in the sample is compared to the amount of CITED mRNA or protein in control samples from both subjects having the estrogen-related condition and subjects not having with the estrogen-related condition. For example, in the sixth aspect of the invention, a decrease in the amount of a CITED mRNA or protein indicates that the estrogen-related condition may be infertility or osteoporosis, and an increase in the amount of a CITED mRNA or protein indicates that the estrogen-related condition may be cancer (e.g., breast, prostate, or ovarian cancer), a gynecological disorder, or endometriosis. In embodiments of the seventh aspect of the invention, a decrease in the amount of a CITED mRNA or protein indicates a negative prognosis for the treatment of infertility or osteoporosis, and an increase in the amount of a CITED mRNA or protein indicates a negative prognosis for the treatment of cancer (e.g., breast, prostate, or ovarian cancer), a gynecological disorder, or endometriosis. The sample may be obtained from the affected tissue, for example, by means of a biopsy. In addition, the level of CITED mRNA or protein may be determined in a postmortem analysis, such as an autopsy, using the methods of the invention, on a patient who died, for example, from a cardiovascular disease, a depression-related disorder, or cancer.

In another preferred embodiment of these aspects, the CITED protein used for diagnosis is CITED1, CITED2, CITED3, or CITED4. hi addition, CITED1 or

CITED4 protein maybe compared using an antibody against CITED 1 or CITED4. h an eighth aspect, the invention features a protein having an amino acid sequence that is at least 80% identical to at least 90 contiguous amino acids of zebrafish CITED3 (SEQ ID NO:2). This amino acid sequence may be identical to SEQ ID NO:2. A ninth aspect features a nucleic acid encoding a protein that is at least 80% identical to at least 90 contiguous amino acids of zebrafish CITED3 (SEQ ID NO:2). h a preferred embodiment of this aspect, the nucleic acid is at least 80% identical to zebrafish CITED3 (SEQ JD NO:2). A tenth aspect of the invention features a protein having an amino acid sequence at least 80% identical to human CITED 4 (SEQ ID NO:4). This amino acid sequence may be identical to SEQ ID NO:4.

An eleventh aspect features a nucleic acid encoding a protein that is at least 80% identical to human CITED4 (SEQ ID NO: 4). A twelfth aspect features a vector containing the nucleic acid of the eighth or tenth aspects of the invention.

A thirteenth aspect of the invention features an antibody that specifically recognizes a human CITED4 protein, and a fourteenth aspect of the invention features an antibody that specifically recognizes a human CITEDl protein.

Definitions

As used herein, by a "CITED nucleic acid" is meant a nucleic acid that encodes a polypeptide that has a CITED biological activity and that is substantially identical to any one of a CITEDl, CITED2, CITED3, or CITED4 protein. For example, a CITEDl nucleic acid may be substantially identical to GenBank Accession Number NM_004143, U65092, or U65091; a CITED2 nucleic acid maybe substantially identical to GenBank Accession Number NM_006079, AF129290, U65093, U86445, or NP_006070; a CITED3 nucleic acid may be substantially identical to GenBank Accession Number AF261079 or AI031460, or to SEQ JD NO:l; and a CITED4 nucleic acid may be substantially identical to GenBank Accession Number AF143369 or AL158843, or to SEQ JD NO:3.

As used herein, by a "CITED polypeptide" or a "CITED protein" is meant an amino acid sequence that has a CITED biological activity and is substantially identical to any one of a CITEDl, CITED2, CITED3, or CITED4 protein. For example, a CITEDl polypeptide may be substantially identical to GenBank Accession Number NP_004134; a CITED2 polypeptide maybe substantially identical to GenBank Accession Number AAF01263, AAF01264, AAG36932, NP_034958, or Q99967; a CITED3 polypeptide may be substantially identical to GenBank Accession Number AAF76148, or to SEQ ID NO:2; and a CITED4 polypeptide may be substantially identical to GenBank Accession Number NP_062509 or NM H9563, or to SEQ ID NO:4. By a "CITED biological activity," as used herein, is meant the ability to specifically bind to an estrogen receptor in an estrogen-dependent manner and also to specifically bind to a CBP/ρ300 protein. Preferably, a protein having a "CITED biological activity" also contains an acidic C-terminal transcription activation domain. In addition, a protein having "CITED biological activity" may also interact with a TGF-α promoter in an estrogen-dependent manner. Interactions with a TGF-α promoter may be measured using techniques known to those skilled in the art, for example, a chromatin immunoprecipitation (ChIP) assay, hi addition, further guidance for assaying protein interactions or function may be found in, for example, Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000).

By an "estrogen receptor target gene" is meant any gene that is regulated by the estrogen receptor, for example, a TGF-α, an α-1 anti-chymotrypsin, a c-Fos, a cdc25, a Ha-Ras, a progesterone receptor, a PS2, a BRCA1, a cathepsin D, an E2F1, an HGM1, an insulin receptor substrate-1, an insulin receptor binding protein-4, a c- Myc, or a WISP-2 gene. Regulation may include modulation of transcription.

By an "estrogen response element" is meant a nucleic acid sequence that modulates expression of another nucleic acid sequence, for example, a gene, in an estrogen-dependent manner.

By "modulate estrogen receptor activity" is meant to either increase or decrease the activity of an estrogen receptor relative to that observed under control conditions. For example, estrogen receptor activity may be measured by determining the level of expression of estrogen receptor target genes, or by expression of a reporter gene that is under the control of an estrogen response element. The modulation in estrogen receptor activity is preferably an increase or decrease of at least 20%, 40%, 50%, 75%, 90%, 100%, 200%, 500%, or even 1000%. By "modulate expression" is meant to either increase or decrease expression, for example, of a protein or nucleic acid sequence, relative to control conditions. The modulation in expression is preferably an increase or decrease of at least 20%, 40%, 50%, 75%, 90%, 100%, 200%, 500%, or even 1000%. By an "estrogen receptor agonist" is meant a compound that increases an estrogen receptor activity relative to control conditions. This increase in estrogen receptor activity may be, for example, an increase of 20%, 40%, 50%, 75%, 90%, 100%, 200%, 500%, or even 1000%.

By a "candidate compound" or "test compound" is meant a chemical, be it naturally-occurring or artificially-derived, that is surveyed for its ability to modulate estrogen receptor activity, for example, in one of the assay methods described herein. Candidate compounds may include, for example, peptides, polypeptides, synthetic organic molecules, naturally-occurring organic molecules, nucleic acid molecules, and components thereof. By a "greater than naturally-occurring amount of CITED protein or CITED activity" is meant an amount of CITED protein, or CITED activity, that is at least 20%, 50%, 75%, 100%, 200%, 500%, or 1000% greater than the amount of CITED protein or CITED activity that is naturally present within a particular cell or sample. A greater than naturally-occurring amount of CITED protein may be generated by expressing a heterologous CITED protein in a cell or sample. Alternatively, a CITED protein may be expressed in a cell or sample using a heterologous promoter, by using multiple copies of the endogenous gene, or by exploiting a mutation in the CITED gene or elsewhere in a cell chromosome that results in increased CITED expression or activity. By "operably linked" is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).

By a "heterologous promoter" is meant a promoter that regulates the expression of a nucleic acid with which it is not normally associated. A "heterologous promoter" may be, for example, a viral promoter (e.g., a cytomegalovirus (CMV) promoter, a herpes simplex virus thymidine kinase promoter, or an adenovirus E1B promoter), a bacterial promoter, or a mammalian promoter, such as the β-globin promoter.

By a "heterologous nucleic acid" is meant a nucleic acid, for example, a DNA or RNA molecule, that is not normally present in a cell or sample obtained from a cell. This nucleic acid may be from another organism, or it may be, for example, an mRNA molecule that is not normally expressed in a cell or sample.

By "reporter gene" is meant a gene whose expression may be assayed; such genes include, without limitation, those encoding glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), alkaline phosphatase, and β-galactosidase.

By a "CITED promoter" is meant a nucleic acid sequence that is normally positioned adjacent to a CITED nucleic acid sequence and regulates its transcription. A CITED promoter may be, for example, a human CITED4 promoter substantially similar to the nucleic acid sequence of SEQ JD NO:5, or it may be a mouse CITED4 promoter substantially similar to the one shown in panel B of Figure 15. The mouse CITEDl promoter is described in Fenner et al. (Genomics 51 :401-407, 1998) and the human CITED2 promoter is described in Leung et al (Genomics 61:307-313, 1999). The promoter region of a known gene may be readily determined, using standard techniques, by one skilled in the art.

By an "estrogen-related condition" is meant a condition that is modulated by estrogen. Examples of "estrogen-related conditions" include, without limitation, depression, cardiovascular disease, infertility, osteoporosis, breast cancer, prostate cancer, ovarian cancer, a gynecological disorder, endometriosis, or the symptoms of menopause.

By a "gynecological disorder" is meant an abnormal condition of a mammalian, for example, human, reproductive tract or any organ or gland associated with the reproductive system.

By a "change in cell aggregation," as used herein, is meant an increase or decrease in cell aggregation. Such a change may involve at least a 20%, 30%, 50%, 75%, 100%, 200%., 500%, or even 1000% change in cell aggregation when compared to control cells. Cell aggregation may be measured by the assays described herein or by any other standard method.

By "specifically recognizes," as used herein in reference to an antibody, is meant an increased affinity of an antibody for the protein against which it was raised, relative to an equal amount of any other protein. For example, an anti-CITED4 antibody preferably has an affinity for CITED4 that is least 2-fold, 5-fold, 10-fold, 30- fold, or 100-fold greater than for an equal amount of any other protein, including other CITED proteins. Similarly, an anti-CITEDl antibody preferably has an affinity for CITEDl that is least 2-fold, 5-fold, 10-fold, 30-fold, or 100-fold greater than for an equal amount of any other protein, including other CITED proteins.

By "substantially identical" is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 60%, 70%, 75%, or 80%, more preferably 85%, 90% or 95%, and most preferably 99% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 15 amino acids, preferably at least 20 contiguous ammo acids, more preferably at least 25, 50, 75, 90, 100, 150, 200, 250, 300, or 350 contiguous amino acids, and most preferably the full-length amino acid sequence. For nucleic acids, the length of comparison sequences will generally be at least 45 contiguous nucleotides, preferably at least 60 contiguous nucleotides, more preferably at least 75, 150, 225, 275, 300, 450, 600, 750, 900, or 1000 contiguous nucleotides, and most preferably the full- length nucleotide sequence.

Sequence identity may be measured using sequence analysis software on the default setting (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705). Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

Multiple sequences may also be aligned using the Clustal W(1.4) program (produced by Julie D. Thompson and Toby Gibson of the European Molecular Biology Laboratory, Germany and Desmond Higgins of European Bioinformatics Institute, Cambridge, UK) by setting the pairwise alignment mode to "slow," the pairwise alignment parameters to include an open gap penalty of 10.0 and an extend gap penalty of 0.1 , as well as setting the similarity matrix to "blosuni." hi addition, the multiple alignment parameters may include an open gap penalty of 10.0, an extend gap penalty of 0.1 , as well as setting the similarity matrix to "blosuni," the delay divergent to 40%, and the gap distance to 8.

By "purified" is meant separated from other components that naturally accompany it. Typically, a factor is substantially pure when it is at least 50%, by weight, free from proteins, antibodies, and naturally-occurring organic molecules with which it is naturally associated. Preferably, the factor is at least 75%, more preferably, at least 90%, and most preferably, at least 99%, by weight, pure. A substantially pure factor maybe obtained by chemical synthesis, separation of the factor from natural sources, or production of the factor in a recombinant host cell that does not naturally produce the factor. Proteins, vesicles, and organelles may be purified by one skilled in the art using standard techniques, such as those described by Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000). The factor is preferably at least 2, 5, or 10 times as pure as the starting material, as measured using polyacrylamide gel electrophoresis, column chromatography, optical density, HPLC analysis, or Western analysis (Ausubel et al. , Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000). Preferred methods of purification include immunoprecipitation, column chromatography such as immunoaffinity chromatography, magnetic bead immunoaffinity purification, and panning with a plate-bound antibody. By "mutation" is meant an alteration in a naturally-occurring or reference nucleic acid sequence, such as an insertion, deletion, frameshift mutation, silent mutation, nonsense mutation, or missense mutation. Preferably, the amino acid sequence encoded by the nucleic acid sequence has at least one amino acid alteration from a naturally-occurring sequence. Examples of recombinant DNA techniques for altering the genomic sequence of a cell, embryo, fetus, or mammal include inserting a DNA sequence from another organism (e.g., a human) into the genome, deleting one or more DNA sequences, and introducing one or more base mutations (e.g., site- directed or random mutations) into a target DNA sequence.

Advantages

The present invention provides in vitro assay systems that can be used to detect estrogenic compounds specifically and efficiently. Estrogenic activity of compounds cannot normally be predicted based on structure alone, making an experimental approach necessary. Prior to the present invention, the lack of predictable and reliable in vitro assay systems for evaluating potential sources and strengths of environmental estrogens was a major obstacle in furthering their isolation. Previously, human mammary epithelial cells, normal or malignant, were used in in vitro assays for estrogen dependent growth. However, the use of human mammary epithelium cells for such assays generally is time-consuming and requires both carefully selected reagents and extensive training of technicians. Moreover, when crude specimens are to be analyzed for potential contamination with unknown estrogenic compounds, such assays suffer from a possibility that specimen-induced growth of breast cancer cells may not be correlated with the existence of an estrogenic compound; instead growth may be induced by another mechanism. hi addition, prior to the present invention, the most sensitive biological assays for compounds that bind estrogen receptors and exhibit estrogen-like activities involved an estrogen-binding domain capable of activating transcription fused to a heterologous GAL-4 DNA binding domain. The ability of this fusion protein to activate transcription of a GAL-4-dependent gene was evaluated, for instance, by measuring the enzymatic activity of the gene product. However, these assays suffered from a low sensitivity for estrogen-induced transcription, thereby proving insufficient for high throughput screens for compounds that modulate estrogen receptor-dependent transcription.

The present invention uses CITED proteins, for example, in a green fluorescent protein (GFP)-based mammalian two-hybrid system, and provides significant advantages over the prior in vitro assay systems. The use of a CITED protein as a transcription co-activator enhances estrogen receptor-mediated transcription in mammalian cells by up to ten-fold without affecting other nuclear receptors. Accordingly, the specific and strong activation of estrogen receptor- mediated transcription enables high throughput screens for identifying compounds that modulate estrogen receptor-dependent activity by assaying for the estrogen receptor-mediated expression of a reporter gene.

Furthermore, the CITED mRNA or protein described in the present invention provides sensitive diagnostic assays for determining if a subject has, or has a propensity for acquiring, an estrogen-related condition, as well as for determining the prognosis for treatment of an estrogen-related condition. By measuring the amount of CITED mRNA or protein in a subject, for example, by standard Northern or Western analysis, one may determine if a subject has, or has a propensity for acquiring infertility, osteoporosis, cancer (e.g., breast, prostate, or ovarian cancer), a gynecological disorder, endometriosis, or the symptoms of menopause.

Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

Brief Description of the Drawings Figure IA is a schematic diagram of an assay used to show that the CITED proteins enhance estrogen-dependent transcription mediated by the LBD/AF2 domain of ERs.

Figures IB-IE are graphs of data showing that CITED proteins selectively enhance estrogen-dependent transcription.

Figure 2 is a series of graphs showing that CITEDl enhances estrogen- dependent transcription mediated by the endogenous ERs. Figure 3 A is an alignment of the alanine scan mutations in the CR2 domain of

CITEDl (SEQ ID NOS:6-14).

Figure 3B is a Western blot showing protein expression of the alanine scan mutants.

Figure 3C is a graph showing the requirement of amino acids 157-158 "VL" for the ERα-enhancing activity of CITED 1.

Figure 3D is a graph showing that the mut2 CITEDl mutant retains the p300- dependent transactivating activity. Figure 4A is a Western blot showing that CITEDl interacts with the LBD/AF2 domain of ERα in an estrogen-dependent manner.

Figure 4B is a series of two Western blots showing that CITEDl interacts with endogenous ERα in an estrogen-dependent manner. Figure 5 A is a Western blot showing estrogen-dependent binding of CITED 1 to ERα.

Figure 5B is a GST pull-down assay and a Western blot showing the loss of ERα-binding activity of a function-dead CITEDl mutant.

Figures 6A-6D are a series of immunohistochemical staining images showing CITEDl and ERα expression in mouse mammary epithelial cells.

Figure 7 A is a Western blot showing the expression of CITEDl in MCF7 cells by retrovirus transduction.

Figure 7B is a Western blot showing stabilization of the estrogen-dependent interaction of ERα and p300 by CITEDl in vivo. Figure 7C is a graph showing the enhancement of estrogen-dependent growth of MCF-7 cells by CITEDl.

Figure 7D is a series of phase contrast photomicrographs showing the enhancement of estrogen-induced cell aggregation of MCF-7 cells by CITEDl.

Figure 8 A is a gel showing semi-quantitative RT-PCR analysis of estrogen- inducible genes.

Figure 8B is a series of two graphs showing a quantitative representation of the amounts of mRNA transcripts analyzed in Figure 8 A.

Figure 8C is a Northern blot showing the estrogen-induced expression of the TGF-α mRNA transcript. Figure 8D is a graph showing a quantitative representation of the amounts of

TGF-α mRNA transcript analyzed in Figure 8C.

Figure 9 is the Zebrafish CITED3 nucleic acid sequence (SEQ JD NO:l).

Figure 10 is the Zebrafish CITED3 amino acid sequence (SEQ ID NO:2).

Figure 11 is the Human CITED4 nucleic acid sequence (SEQ ID NO:3). Figure 12 is the Human CITED4 amino acid sequence (SEQ JD NO:4).

Figure 13 is the Human CITED4 promoter sequence (SEQ JD NO: 5). Figure 14A is the nucleotide sequence of the mouse CITED4 cDNA (SEQ ID NO: 16). The dashed square indicates the consensus sequence for translational initiation, and the polyadenylation signal is underlined. The sequence corresponding to an EST clone (GenBank Accession Number AA839758) that was used as probe for screening a cDNA library is boxed. Nucleotides in CITED4 open reading frame are shown in capitals, and deduced amino acids are shown below each codon (SEQ ID NO: 18).

Figure 14B is a phylogenetic analysis of the CITED family proteins, where the numbers indicate distances in phylogenetic units. CITED4 is equally distant from three other members of the CITED family.

Figure 14C is an alignment of amino acid sequences of the CITED family proteins (SEQ ID NOS:2 and 18-25). The conserved regions 1, 2, and 3 (CR1, CR2, and CR3, respectively) are underlined. Amino acids showing similarity are boxed and lightly shaded; identical amino acids are densely shaded. Figure 15A is a schematic diagram of the mouse cited4 genomic DNA. The mouse cited4 gene consists of a single exon (open box), including an open reading frame (closed box), and 5'- and 3'- untranslated sequences of the mRNA transcript. Several restriction sites are indicated, where "RI" stands for EcoRI, "RV" for EcoRV, "Pv" for Pvuπ "B" for BarnHI, "K" for Kpnl, "N" for Notl, and "X" for Xhol. Figure 15B is the nucleotide sequence of the promoter region of mouse cited4 gene (SEQ ID NO:26). The transcriptional initiation site was determined using the 5'- RACE method and is defined as nucleotide +1. The TATA box is indicated by a rectangle.

Figure 16A is a Northern blot depicting the distribution of the CITED4 mRNA transcript in adult mouse tissues. Poly(A)-rich RNA mouse tissue blot was hybridized sequentially with a ³²P-labeled mouse CITED4 cDNA probe and then with a β-actin probe as a loading control. A single species of the CITED4 mRNA transcript (~1.5 kb) was detected in all tissues with particularly strong expression in the heart and spleen. Figure 16B is a Northern blot showing expression of the CITED 1 , CITED2, and CITED4 mRNA transcripts in mammary glands of pregnant mice. Total RNA blot (5 μg/lane) of 11.5 dpc mouse mammary glands was sequentially hybridized with cDNA probes for mouse CITEDl, CITED2, and CITED4 and the positions of the 28S and 18S ribosomal RNAs are indicated.

Figure 16C is a Western blot showing expression of CITEDl and CITED4 proteins in the mouse mammary gland. Equal amounts (50 μg) of total tissue proteins were subjected to immunoblotting using anti-CITEDl and anti-CITED4 antibodies. Placenta isolated at age 11.5 dpc expressed both CITEDl and CITED4 proteins and were used as a positive control. CITEDl protein is strongly expressed in the mammary glands of nulliparous mice, but is only weakly expressed in pregnant mice (11.5 dpc). hi contrast, CITED4 protein is strongly expressed in mammary glands of pregnant mice, but is undetectable in nulliparous mice.

Figure 16D is a Northern blot showing the induction of CITED4 mRNA transcript during prolactin-induced differentiation of SCp2 mouse mammary epithelial cells. SCp2 cells were cultured in the differentiation medium containing 3 μg/ml prolectin (PRL) for the indicated number of days. Equal amounts (5 μg/lane; ethidium bromide (EtBr) staining of the RNA gel is shown) of total RNA isolated from the culture were subjected to RNA blot hybridization using P-labeled mouse CITED4 cDNA probe.

Figure 17A is a Western blot showing that CITED4 binds to p300 in vivo. COS-1 cells were transfected with HA-tagged mouse CITED4 (lanes marked 1 and 2) or its mutant lacking the CR2 region (lanes marked 3 and 4). Cell lysates were prepared 48 hours after transfection and subjected to immunoprecipitation using an anti-p300 rabbit polyclonal antibody to precipitate endogenous p300, or using non- immunized rabbit IgG as a control. Co-precipitated HA-CITED4 was detected by immunoblotting using an anti-HA antibody.

Figure 17B is the result of an enzymatic CAT assay showing that CITED4 activates transcription in a CBP/p300-dependent manner. NTH3T3 cells were transfected with the following GAL4 DNA-binding domain (GAL4DB) fusion proteins: "GAL4DB" (no fusion); "GAL4DB-CITED1" (fused to human CITEDl), and "GAL4DB-CITED4" (fused to mouse CITED4). A GAL4-dependent CAT reporter plasmid as well as adeno virus El A protein and its mutants were cotransfected. Cell lysates were prepared 24 hours after transfection and subjected to an enzymatic CAT assay. In this figure, "RelCAT" stands for relative CAT activity, "wt" for wild-type El A, "Δ2-36" for an El A mutant that suppresses RB function but not CBP/ρ300 function, and "m928" for an El A mutant that suppresses CBP/ρ300 function but not RB function. Figure 17C is the result of an enzymatic CAT assay showing that CITED4 does not co-activate Smad4. NTH3T3 cells were transfected with GAL4DB-fusion Smad4 C-terminal transactivating domain (GAL4DB-Smad4C), human CITEDl or mouse CITED4, together with a GAL4-dependent CAT reporter plasmid. Cell lysates were prepared 24 hours after transfection and subjected to an enzymatic CAT assay. Fig. 18 is a series of photomicrographs showing expression of ERα, CITEDl and CITED4 in mouse mammary glands by immunohistochemistry. The antigen was visualized using a DAB chromogen with counterstaining by methyl green.

Fig. 19 is a Western blot showing expression of CITEDl and CITED4 in the mouse mammary glands. Total tissue homogenate of mammary glands from nulliparous mice and from 11.5 dpc pregnant mice (50 μg/lane) was subjected to anti- CITEDl Western blotting. After the anti-CITEDl antibody was stripped off, the same blot was subjected to anti-CITED4 Western blotting.

Fig. 20 is a schematic diagram of the structure of linearized pG5GFP-Hyg. The GFP reporter gene is inserted downstream of five repeats of the GAL4-binding elements and the EIB proximal promoter sequence containing the TATA box. The hygromycin resistance gene (Hyg*) is inserted upstream of the reporter expression unit and driven by the thymidine kinase (TK) promoter.

Detailed Description We have developed in vitro methods for detecting compounds that modulate estrogen receptor-dependent activity, which can be readily adapted to high throughput screening. The method of the invention uses CITED (CBP/p300-Interacting Transactivators with glutamic acid (E)/aspartic acid (D)-rich carboxyl-terminal domain) proteins to increase the sensitivity of the estrogen receptor to estrogen or estrogen-like compounds.

The CITED family consists of four nuclear proteins, CITEDl (formerly MSG1) (Shioda et al, Proc. Natl. Acad. Sci. USA 93:12298-12303, 1996; Dunwoodie et al., Mech. Dev. 72:27-40, 1998), CITED2 (formerly MRG1 or ρ35srj) (Shioda et al, Gene 204:235-241, 1997; Sun et al, Proc. Natl. Acad. Sci. USA 95:13555-13560, 1998; Bhattacharya et al, Genes & Dev. 13:64-75, 1999; Schlange et al, Mech. Dev. 98:157-160, 2000), CITED3 (Andrews et al, Mech. Dev. 95:305-308, 2000), and CITED4 (formerly MRG2) (Yahata et al, J. Biol. Chem. 275:8825-8834, 2000), that share a strictly-conserved C-terminal transcriptional activating domain (designated as the conserved region 2 (CR2) domain). The CR2 domain functions in binding to the CBP/p300 transcriptional integrators (Bhattacharya et al, Genes & Dev. 13:64-75, 1999; Yahata et al, J. Biol. Chem. 275:8825-8834, 2000). When tethered to heterologous DNA binding domains, all CITED proteins strongly activate transcription in a manner dependent on the CR2 domain and CBP/p300 (Shioda et al. Proc. Natl. Acad. Sci. USA, 95:9785-9790 1998; Yahata et al., J. Biol. Chem. 275:8825-8834 2000). However, no CITED protein has any significant DNA-binding motifs or detectable affinity to DNA, indicating that the CITED proteins may interact with DNA-binding proteins and function as transcriptional co-activators. Data showing that CITEDl enhances transcription mediated by the SMAD transcription factors in a manner dependent on CBP/p300 support this observation (Shioda et al, Proc. Natl. Acad. Sci. USA 95:9785-9790, 1998). hi addition, CITED2 binds to the LJM domain of the Lhx2 transcription factor and enhances Lhx2-dependent transcription of the LH/FSH glycoprotein α-subunit gene (Glenn and Maurer, J. Biol. Chem. 274:36159-36167, 1999). Furthermore, data from Bhattacharya et al. (Genes & Development 13:64-75, 1999) demonstrated that the binding of CITED2 to CBP/p300 competes with the interaction between the HIF-1 transcription factor and CBP/p300, resulting in suppression of HIF-1 -dependent transcription. Since expression of the CITED proteins shows remarkable tissue specificity and cytokine- dependency, CITED proteins may play roles in tissue-specific and/or cytokine- dependent regulation of CBP/p300-dependent gene expression (Dunwoodie et al, Mech. Dev. 72:27-40, 1998; Fenner et al, Genomics 51:401-407, 1998; Li et al, Exp. Cell Res. 242:478-486, 1998; Sun et al, Proc. Natl Acad. Sci. USA 95:13555-13560, 1998; Bhattacharya et al, Genes & Dev. 13 :64-75, 1999; Andrews et al, Mech. Dev. 95:305-308, 2000). We examined possible effects of the CITED proteins on ligand-induced transcriptional activation mediated by nuclear receptors (NRs). The NR superfamily of transcription factors regulates transcriptional activation and suppression in a manner dependent on binding to specific ligands such as steroid hormones. A conserved amphipathic α-helical structure within their ligand-binding domain (LBD), referred to as activation function 2 (AF2), is required for their ligand-dependent transcriptional activation, while another region, activation function 1 (AF1), is responsible for their ligand-independent transactivating activity. NRs require co- activator proteins for their transcriptional activating activities (Freedman, Cell 97:5-8, 1999; Glass and Rosenfeld, Genes & Dev. 14:121-141, 2000). Such co-activators, which usually function as multiprotein complexes, include histone acetyltransferases (the pl60 family co-activators (SRC-l/NcoA-1, TIF2/GRIPl/NcoA-2, ρCJ /ACTR/AIBl/RAC3/NCoA-3), the ρ300 and CBP transcriptional integrators, and the CBP/p300-associated factor, pCAF), ATP-dependent chromatin remodeling complexes (SWI/SNF/BRG complexes), and mediator-like protein complexes (DRIP/TRAP/ARC/PBP complexes).

The functional importance of CBP/p300 in NR-mediated transcriptional activation has been demonstrated in vivo (Chakravarti et al, Nature 383:99-103, 1996) and in vitro (Kraus and Kadonaga, Genes & Development 12:331-342, 1998). The histone acetyltransferases and the ATP-dependent chromatin remodeling complexes may relieve chromatin-mediated transcriptional repression, while the mediator-like complexes may recruit general transcription factors and RNA polymerase II holoenzyme to promoters. Dynamic and cyclic recruitment of estrogen- bound ERα and these co-activators to ER target promoters have been recently demonstrated, supporting a model that these co-activator complexes act in a combinatorial manner rather than independently (Shang et al, Cell 103:843-852 2000).

In addition, there are a number of additional proteins that have been proposed as NR co-activators based on the simple criteria of ligand-dependent binding to NRs and/or the ability to synergize NR-mediated transactivation, as evaluated by transfection-based assays. These other NR co-activators include HMG factors, MEF2, cyclin D, ARA5Y, SNURF, NcoA-62, PC2, and PC4 (Glass and Rosenfeld, Genes & Development 14:121-141, 2000). Several co-activators have relative preferences for a subset of NRs, e.g., ARA70 selectively enhances transcription mediated by androgen receptor (Yeh et al, Endocrine 11:195-202 1999), and NRIF3 interacts selectively with thyroid hormone receptor and retinoid X receptor (Li et al, Mol. Cell Biol. 19:7191-7202 1999). However, most of the ligand-dependent co-activators do not show strong selectivity in target NRs.

As is described in the following non-limiting examples, we show that CITED proteins function as selective estrogen receptor co-activators and are therefore likely to function in cell- and gene-specific regulation of estrogen dependent transcription.

These examples are provided for the purpose of illustrating the invention and should not be construed as limiting.

Example 1 CITED Proteins Enhance Estrogen Receptor-Mediated Gene Expression

To determine whether the CITED proteins affect transcriptional functions of NRs, we evaluated effects of the CITED proteins on transcription activated by the AF1 or LBD/AF2 domains of NRs. AFl or LBD/AF2 domains from selected NRs were fused to the GAL4 DNA-binding domain (GAL4DBD), and their transactivating activities were evaluated by transfection-based reporter assays in the presence or absence of co-transfection with CITED proteins. Figure 1 A shows a schematic representation of the assay used. hi these experiments, NIH3T3 cells were transfected with GAL4DBD fusion of AFl or AF2 domain of nuclear receptors (NR-AF1/AF2), the CITED proteins, together with luciferase reporter plasmid pGLUC8 harboring eight repeats of GAL4 binding element (GAL4BE) followed by the EIB TATA box. Cells were cultured in hormone-free medium for 24 hours after transfection, followed by culture in the presence or absence of ligands for additional 24 hours before luciferase assay. As is shown in Figure 2B, the CITED proteins selectively enhance estrogen-dependent transcription. Cells were incubated in the presence (solid bars) or absence (open bars) of cognate ligands: 100 nM 17β-estradiol for ERα and ERβ, 10 nM dihydroxyandrostendione for androgen receptor (AR); 100 nM ail-trans retinoic acid for retinoic acid receptor α (RARα); 100 nM 9-cis retinoic acid for retinoid X receptor α (RXRα); 100 nM -aldosterone for mineralocorticoid receptor (MR); and 10 nM 1,25-dihydroxy vitamin D3 for vitamin D receptor (VDR).

The CITED proteins showed no effect on the ligand-independent transactivation mediated by the AFl of the ERα, the androgen receptor, or the retinoic acid receptor α. In addition, the effect on ligand-dependent transactivation mediated by the LBD/AF2 domains of androgen receptor, retinoic acid receptor α, retinoid X receptor, mineralocorticoid receptor, or vitamin D3 receptor was weak. However, all of the CITED proteins significantly enhanced estrogen-dependent transactivation mediated by the LBD/AF2 domain of both ERα and ERβ without affecting the basal transcription observed in the absence of estrogen. The relatively weaker ER-co- activating activities of CITED2 and CITED3 when compared to CITEDl or CITED4 are likely due to their extremely short protein half-lives in mammalian cells, resulting in poor protein expression in these experiments (Bhattacharya et al, Genes & Development 13:64-75, 1999).

Further support for this observation comes from data showing that the ER-co- activating effect of CITEDl is dependent on the amount of CITEDl expression. To show that CITEDl enhances estrogen-dependent transcription by the LBD/AF2 domain of ERα in a dose-dependent manner, cells were co-transfected with pGLUC8, GAL4DBD-ERα- AF2, and varying amounts of CITED 1 , then stimulated with 100 nM 17β-estradiol. The results of these experiments indicate that the CITED proteins may function as selective co-activators for the LBD/AF2 domain of ERs.

Recent studies have revealed that binding of estrogen agonists to the LBD of ERs induces major conformational changes that stabilize interactions between ERs and LBD-binding co-activators. Estrogen antagonists, such as tamoxifen and raloxifen (partial agonists) or ICI164384 (pure antagonist), also bind to the LBD of ERs, but induce aberrant conformational changes that preclude co-activator binding (Freedman, Cell 97:5-8, 1999; Glass and Rosenfeld, Genes & Development 14:121- 141, 2000). As is shown in Figure ID, these estrogen antagonists failed to induce LBD/AF2-mediated transactivation in the presence or absence of CITED 1 , whereas transcription induced by the natural estrogen agonist (17β-estradiol) was remarkably enhanced by CITEDl. For these experiments, cells were co-transfected with pGLUCδ, GAL4DBD-ERα-AF2, CITEDl (solid bars) or a control vector (open bars), followed by stimulation with ligands (100 nM 17β-estradiol, 100 nM ICI164384, 100 nM tamoxifen, or 100 nM raloxifen). The results obtained (Fig. ID) indicate that the functional interaction of CITEDl with ERα-LBD/AF2 requires the correct, active form, conformation of ligand-bound ER.

To investigate whether CBP/p300 is required for CITEDl to enhance transcriptional activation mediated by ERα-LBD/AF2, we determined effects of p300 on this transcriptional activation by transfection-based assays, in which cells were co- transfected with pGLUC8, GAL4DBD-ERα-AF2, with or without CITEDl or p300, and then stimulated with 100 nM 17β-estradiol (E2) (Fig. IE). The results obtained from these assays show that, in the absence of CITEDl, the effect of p300 transfection on transcriptional activation mediated by ERα-LBD/AF2 is minimal. However, when a limited amount of CITEDl alone was transfected, the transcriptional activation was enhanced by about 4-fold. By transfecting p300 in addition to CITED 1 , transcriptional activation is further augmented by about 1.8-fold. Accordingly, these data demonstrate a synergistic effect of p300 on the ERα-LBD/AF2 co-activation by CITEDl, and indicate an involvement of p300, and likely CBP as well, in the mechanism of ER-co-activation by CITEDl.

CITEDl enhances estrogen-dependent transactivation mediated by endogenous ERs in a manner dependent on core promoters located downstream of estrogen response elements

We next wanted to determine whether CITEDl enhances estrogen-dependent transcription mediated by full-length, endogenous ERs. To address this question, we transfected MCF-7 cells (ER-positive) or COS-1 cells (ER-negative) with ER- dependent luciferase reporter plasmids harboring ER binding elements (ERE) located upstream of core promoters (globin, thymidilate kinase (tk), or adenovirus EIB) and varying amounts of CITEDl. The transfected cells were either incubated in the absence of estrogen, or stimulated with 100 nM 17β-estradiol (E2) for 24 hours before being used in a luciferase assay. We evaluated the possible promoter-dependency of the ER-coactivating activity of CITEDl by using three ER-dependent luciferase reporter plasmids with different core promoters: pEREG-Luc that harbors one copy of estrogen response element (ERE) followed by β-globin promoter (ERE-globin promoter) (Eckner et al, Genes & Development 8:869-884, 1994); pERE-tk harboring one copy of ERE followed by herpes simplex virus thymidine kinase promoter (ERE- tk promoter); and p3XERE-ElBTATA harboring three copies of ERE followed by adenovirus EIB TATA box (ERE-E1B promoter) (Kalkhoven et al, EMBO J. 17:232-243, 1998). h MCF-7 cells, co-rransfection of CITEDl enhanced estrogen- dependent transcription from the ERE-tk and ERE-E1B promoters in a manner dependent on the dose of CITEDl. In contrast, COS-1 cells showed no effect of CITEDl co-transfection on estrogen-dependent transcription from any of these promoters. Furthermore, estrogen-induced transcription from the ERE-globin promoter was not at all affected by CITEDl cotransfection in either MCF-7 or COS-1 cells. These data demonstrate that CITEDl is capable of enhancing estrogen- dependent transactivation mediated by endogenous ERs, as well as indicating that the ER-co-activating activity of CITED 1 may be promoter-dependent.

The N-terminal region of the conserved CR2 domain of CITEDl is required for functional interaction with ERs but not with CBP/p300

We also determined which amino acid sequences of CITEDl are required for the functional interaction with ERs. None of the four CITED proteins possesses the LXXLL (SEQ JD NO: 15) signature motif that was previously shown to be necessary and sufficient for binding of many ligand-dependent co-activators to NRs (Glass and Rosenfeld, Genes &Development 14:121-141, 2000). Given that all members of the CITED family show ER-co-activating activity (Fig. IB), we looked at amino acid sequences that are conserved among, and unique to, the CITED proteins with which ERs could interact. All CITED proteins have a conserved CR2 domain, which we chose to further characterize (Bhattacharya et al, Genes & Development 13:64-75, 1999; Yahata et al, J. Biol. Chem. 275:8825-8834, 2000). We tested whether a deletion of the CR2 domain from CITEDl results in loss of its ER-co-activating activity and found that such a mutation (ΔCR2) completely suppressed ER-co- activating activity of CITEDl (Fig. 3C) without affecting expression of CITEDl protein (Fig. 3B). We mapped the ER-interacting regions of CITEDl more precisely by introducing systematic alanine-scan mutations in the CR2 domain and evaluating the effects of these mutations on the ER-co-activating activity (Fig. 3A). To determine if such mutations in the N-terminal region of the CR2 domain (amino acids 155-167) affect the level of CITEDl protein expression, we transfected CITEDl mutants into COS-1 cells and evaluated their expression by anti-HA Western blotting (Fig. 3B, where ΔCR2 is a CITEDl deletion mutant lacking the CR2 domain). None of the mutants in the N-terminal region of the CR2 domain affected protein expression in this assay. However, mutations in the C-terminal region of the CR2 domain are frequently associated with marked reduction in the amount of CITED 1 protein expression, making it difficult to obtain systematic evaluations in this region. hi addition, among the mutations in the N-terminal region of the CR2 domain, only one (mut2, which has alanines substituted for valine and leucine at positions 157 and 158) completely suppresses the ER-co-activating activity, whereas others affected this activity only marginally (Fig. 3C). For these experiments, cells were co- transfected withpGLUC8, GAL4DBD-ERα-AF2, and the CITEDl mutants, followed by stimulation with 100 nM 17β-estradiol (E2) for 24 hours before perfonning the luciferase assay. While these data indicate the N-terminal region of the CR2 domain plays a critical role in the functional interaction between CITEDl and ERs, other regions of CITEDl may also contribute to this interaction. In addition, it should be noted that the amino acid sequence around the critical mut2 mutation did not resemble the canonical LXXLL motif (SEQ JD NO:15; Fig. 3A).

Since the CITED proteins bind directly to CBP/p300 through the CR2 domain (Bhattacharya et al, Genes & Dev. 13:64-75, 1999; Yahata et al, J. Biol. Chem. 275 : 8825-8834, 2000), we considered a possibility that the observed functional interaction between CITEDl and ERs might be indirect, involving CBP/p300 as an intermediate protein. Accordingly, we investigated whether the mutation that suppressed the CITED1-ER interaction concomitantly suppresses the CITEDl-p300 interaction. For these experiments, cells were co-transfected with pGLUC8 and GAL4DBD-fusion CITED 1 mutants. The CITED 1 mutants were tethered directly to the GAL4DBD, and their transcriptional activating activities, which were demonstrated to be totally dependent on interaction with CBP/p300 (Yahata et al, Cell Immunol 171:269-276, 1996), were evaluated (Fig. 3D). The results from these experiments showed that the ΔCR2 mutation completely suppresses the CBP/p300- dependent transactivation by GAL4DBD-CITED1. However, the mut2 mutation, as well as other alanine-scan mutations tested, did not affect this transactivation at all. These results indicate that the functional CITED1-ER and CITED l-CBP/ρ300 interactions are separable, showing that ERα and CBP/p300 likely interact with discrete regions within the CR2 domain.

CITEDl binds to the LBD/AF2 domain of ERα in vivo and in vitro in an estrogen- dependent manner

We investigated whether CITEDl physically interacts with ERα by transfection-based immunoprecipitation analyses. In these experiments, COS-1 cells were transfected with HA epitope-tagged CITEDl and FLAG-tagged transactivating domains of ERα (AFl and LBD/AF2) and cultured for 24 hours in the absence of estrogen. The cells were then stimulated with 100 nM 17β-estradiol E2 or vehicle (ethanol) for one hour. The ERα transactivating domains were immunoprecipitated from cell lysates using an anti-FLAG antibody, and co-precipitated CITEDl was detected by anti-HA Western blotting (Fig. 4A, top panel). When expressed in COS-1 cells, CITEDl co-precipitated with the LBD/AF2 domain in a manner strictly dependent on the presence of estrogen in the culture as seen on Western blots (Fig. 4A, bottom panel). No CITEDl co-precipitation was observed with the AFl domain in the presence or absence of estrogen. Accordingly, CITEDl likely physically and specifically interacts with the estrogen-bound LBD/AF2 domain of ERα in vivo. In addition, we wanted to determine whether CITEDl binds to endogenous (hence, full-length) ERα in vivo. We transfected ERα-positive MCF-7 cells with HA- tagged CITEDl and cultured them for 24 hours in the absence of estrogen. The cells were then stimulated with 100 nM 17β-estradiol (E2) or vehicle (ethanol) for one hour, and CITEDl was immunoprecipitated from the cell lysate by using an anti-HA antibody (Fig. 4B, top panel). Co-precipitated endogenous ERα was detected by Western blotting using an anti-ERα antibody (Fig. 4B, bottom panel). The data show that endogenous ERα co-precipitates with CITEDl when cells are stimulated with estrogen. Based on these results, we conclude that CITEDl interacts, and likely forms a complex with ERα in vivo.

We next determined whether CITEDl binds directly to estrogen-bound ERα in vitro by using a GST (glutathione S-transferase) pull-down assay. For this assay, we prepared purified protein reagents including polyhistidine epitope-tagged CITEDl (His-CITEDl) and GST-tagged AFl or LBD/AF2 domains of ERα. We verified that these protein reagents were not contaminated with CBP or p300 by Western blotting. As is shown in Fig. 5 A, CITEDl bound to the GST-fusion LBD/AF2 domain in vitro in a manner strictly dependent on the presence of estrogen in the binding reaction. These experiments were performed by immobilizing equal amounts of GST or GST- fusion proteins of ERα domains (AFl or LBD/AF2) on glutathione-conjugated Sepharose beads and incubating the beads with polyhistidine epitope-tagged CITEDl (His-CITEDl) in the presence or absence of 100 nM 17β-estradiol. After extensive washing of the beads, co-precipitated CITEDl was detected by anti-His Western blotting. There was no interaction of CITEDl with the GST-fusion AFl domain or with GST alone in the presence or absence of estrogen. Therefore, it is likely that CITEDl physically and directly binds to the LBD/AF2 domain of ERα in an estrogen- dependent manner. The functional interaction of CITED 1 with the LBD/AF2 domain of ERα requires the N-terminal region of the CR2 domain (see Fig. 3). To characterize the molecular basis of this requirement, we determined whether or not the mut2 mutation of CITEDl, which abolished this functional interaction, binds to the LBD/AF2 domain of ERα in vitro. For these experiments, equal amounts of GST-fusion LBD/AF2 of ERα were immobilized on beads and incubated with His-CITED 1 or His-CITEDl (mut2) in the presence or absence of 100 nM 17β-estradiol. After washing of the beads, co-precipitated CITEDl was detected by anti-His Western blotting. The results of these experiments show that the mut2 mutant CITEDl does not at all bind to the LBD/AF2 domain with or without estrogen (Fig. 5B). Accordingly, the lack of the transcriptional enhancing activity of the mut2 mutant likely is due to its inability to bind to ER, confirming the critical importance of the N- terminal region of the CR2 domain of CITEDl in the CITEDl -ERα interaction.

CITEDl protein is expressed in nulliparous mouse mammary gland epithelial cells but rapidly disappears with pregnancy

To obtain insights into the possible biological significance of the ER-co- activating activity of CITEDl, we examined, by immunohistochemistry, if CITEDl protein is expressed in the major ER-target organs of mice. Although we did not detect significant amounts of CITEDl in uterus or ovary, strong expression of CITEDl was observed in 12 week-old nulliparous mouse mammary epithelial cells (Fig. 6A). Practically all mammary epithelial cells were positive for CITEDl, and most of them showed nuclear localization of the protein with some cells also showing cytosolic staining. These results are in accord with a staining pattern previously reported for human melanoma tissue (Li et al, Exp. Cell Res. 242:478-486, 1998). hi addition, the mammary epithelial cells that expressed CITEDl, seen in Figure 6A, also strongly expressed ERα (Fig. 6B).

Further analysis of CITEDl -positive cells showed that these cells disappeared rapidly when mice became pregnant (Summarized in Table 1). Only a small percentage of mammary epithelial cells expressed CITEDl in 11.5 dpc pregnant mice (Fig. 6C and Table 1), and no CITEDl expression was detected in lactating mammary gland (Fig. 6D and Table 1). The strong expression of CITEDl protein in nulliparous mouse mammary epithelial cells and its rapid disappearance upon pregnancy was also confirmed by anti-CITEDl Western blotting (Figure 19). These results indicate possible biological roles of CITEDl in nulliparous mammary epithelial cells.

Table 1: Expression of CITED proteins in mouse mammary epithelial cells

Stage nulliparPregnant Delivery LactatWeaning Weaning ous 11.5 dpc ing 3 weeks 5 weeks after after delivery delivery

CITED1 ++ +/- +/- +/- + CITED4 + ++ +++ + +/- CITEDl stabilizes the estrogen-dependent interaction of ERα and p300 in MCF-7 breast cancer cells and enhances cellular sensitivity to estro en

Plasma estrogen levels are far lower in nulliparous mice than in pregnant mice. Given our observation that the expression of CITED 1 protein is stronger in nulliparous mice, but is rapidly lost with pregnancy, we investigated whether CITEDl may a play role in the regulation of cellular sensitivity to estrogen. Accordingly, we compared the estrogen sensitivity of ERα-positive MCF-7 human breast cancer cells expressing exogenous CITEDl to those not expressing CITEDl. As is shown in Fig. 7 A, a moderate amount of CITEDl was expressed stably in MCF-7 cells infected with a CITEDl -expressing retro virus, while no CITEDl protein was detected in cells infected with a control virus.

Using these retrovirus-infected MCF-7 cells, we wanted to determine whether the estrogen-induced interaction of ERα with p300, a critical step of estrogen-induced transcription (Shang et al, Cell 103:843-852, 2000), is modulated by the presence of CITEDl. In these experiments, we infected two independent batches of MCF-7 cells with CITEDl -expressing retrovirus or vector and cultured these cells in hormone-free medium for 16 hours before stimulating them with 100 nM 17β-estradiol (E2(+)), or vehicle (E2(- ), for 3 hours and performing an anti-ERα immunoprecipitation. The expression level of endogenous ERα and p300 protein was not affected by the presence of CITEDl (Fig. 7B, bottom panel). In addition, when cells were starved for estrogen, no ERα-p300 interaction was detected by immunoprecipitation with or without CITEDl. However, when cells were stimulated with 100 nM 17β-estradiol, three to five-times more p300 was co-precipitated with ERα from lysates of CITED 1- expressing MCF-7 cells than from lysates of control cells (Fig. 7B, top panel). These results indicate that the estrogen-induced interaction of ERα with ρ300 is stabilized when CITEDl is expressed in cells.

To examine if CITEDl enhances cellular responses to estrogen, we determined whether expression of CITEDl in MCF-7 cells affects their estrogen-dependent growth. CITEDl -expressing MCF-7 cells (Fig. 7C, solid bar) grew significantly better than the control cells (Fig. 7C, open bar), especially when estrogen concentration in the culture medium was low. In these experiments, MCF-7 cells were cultured in hormone-free medium for 2 days before an equal number of cells was used to inoculate dishes, which were then cultured for 8 days in the absence or presence of varying concentrations of 17β-estradiol (E2) followed by cell counting using a hemocytometer. Each bar in Figure 7C represents the mean ± SEM (Standard Error of the Mean) of the fold increase in cell number over the starting number as calculated from three independent cultures. As is also seen in Figure 7C, when cultured in the complete absence of estrogen, both CITEDl -expressing and control cells grew only marginally, indicating that the growth-facilitating activity of CITEDl is dependent on the presence of estrogen. Thus, our data show that CITEDl enhances estrogen-dependent growth of MCF-7 cells.

We next determined whether CITEDl affects estrogen-induced aggregation of MCF-7 cells (Olea et al, Int. J. Cancer 50:112-117, 1992). An equal number of hormone-starved CITEDl -expressing and control MCF-7 cells were used to inoculate dishes and were cultured in the presence of varying concentrations of 17β-estradiol (E2) for 5 days. In the complete absence of estrogen, neither CITEDl -expressing nor control cells showed significant aggregation (Fig. 7D, top panels). However, when a very low concentration (1 pM) of 17β-estradiol was added to the culture medium, CITEDl -expressing cells showed marked aggregation, while control cells showed minimal morphological changes (Fig. 7D, middle panels). At a higher estrogen concentration (10 nM), both CITEDl -expressing MCF-7 cells and control cells showed comparable amounts of aggregation (Fig. 7D, bottom panels). (All panels of Figure 7D show phase contrast photomicrographs of 200-fold magnification.) These data provide added evidence that CITEDl enhances cellular sensitivity to estrogen.

CITEDl enhances estrogen-induced expression of the transfonning growth factor-α mRNA transcript in MCF-7 cells

To further characterize effects of CITEDl expression in MCF-7 cells, we next determined whether expression of known estrogen target genes in this cell line is affected by CITEDl. In these experiments, we compared the level of the mRNA transcripts for selected estrogen-inducible genes present in CITEDl -expressing and control MCF-7 cells after stimulation with estrogen using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique as described in Shioda et al. (Am. J. Pathol. 150:2099-2112, 1997). The estrogen-inducible genes analyzed included a-1 antichymotrypsin; c-Fos; cdc25; Ha-Ras; progesterone receptor (PR); PS2; transforming growth factor- (TGF-a); BRCA1; cathepsin D; E2F1; HMG1; insulin receptor substrate-1; insulin receptor binding protein-4; c-Myc; and WISP-2. Among these fifteen genes only two, TGF-α and c-Myc were affected by CITEDl expression, hi the presence of CITEDl, estrogen-induced expression of the TGF-α mRNA transcript was significantly enhanced without changes in its basal expression level observed in the absence of estrogen, while both basal and estrogen-induced expression of the c-Myc mRNA transcript was significantly suppressed (Fig. 8 A and 8B). In addition, we did not observe significant effects of CITEDl on basal or estrogen-induced expression of other estrogen-target genes (PR data is shown in Fig. 8A and 8B). hi these experiments, MCF-7 cells infected with vector (V) or CITED 1- expressing (C) retroviruses were cultured in hormone-free medium for 24 hours, followed by stimulation with 10 nM of 17β-estradiol for indicated periods. Total RNA was then isolated and subjected to semi-quantitative RT-PCR analysis of estrogen-inducible genes in the presence of α- PdCTP. In Figure 8A, "PR" stands for progesterone receptor and "GAPDH" stands for glyceroaldehyde 3-phosphate dehydrogenase (a control gene not inducible by estrogen). Figure 8B shows a quantitative representation of the amounts of the mRNA transcripts analyzed in panel A. For this quantitative analysis, the bands of the radiolabeled PCR products were excised from gels (shown in panel A), and their radioactivity was counted by liquid scintillation. The calculated relative amount of the PCR product, normalized to the GAPDH amount, is shown.

The enhancement of estrogen-induced expression of the TGF-α mRNA transcript by CITEDl was further confirmed by Northern blot hybridization (Fig. 8C and 8D). Total RNA analyzed in panel A (5 μg per lane) was subjected to Northern blot hybridization using a radiolabeled cDNA probe specific to human TGF-α. To confirm that we loaded equal amounts of RNA, we stained the RNA staining with ethidium bromide. In addition, we performed a quantitative analysis of amounts of the TGF-α mRNA transcript analyzed in Figure 7C using densitometry.

We also determined that CITEDl is recruited to the endogenous TGF-α promoter in MCF-7 cells in an estrogen-dependent manner, strongly supporting participation of CITEDl in transcriptional activation of this promoter. A Chromatin rmmuno Precipitation (ChIP) assay may be used to show that a factor is involved in conditionally induced transcriptional activation of an endogenous promoter. For example, a ChIP assay may be used to detect protein factors that are recruited to the endogenous TGF-α promoter in an estrogen-dependent or -independent manner in human breast cancer cells. In addition, this technique may also be used to determine the dynamic profile of estrogen-induced recruitment of transcription factors and components of transcriptional complexes to a promoter.

Based on our results, we find that CITEDl likely co-activates endogenous ER- target genes in a gene-specific manner. The above experiments were carried out using the following materials and methods.

Materials and Methods

Cell culture, transfection, and reporter assay MCF-7 human breast cancer cells and COS-1 cells were purchased from the

American Type Culture Collection and maintained as described by Yahata et al. (J.

Biol. Chem. 8825-8834, 2000). For hormone-free culture, phenol red-free Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% charcoal/dextran-treated fetal calf serum (FCS) (HyClone) was used. Transfection and luciferase reporter assay were performed as described by Yahata et al. (J. Biol.

Chem. 8825-8834, 2000). The described results represent the mean of at least three independent experiments.

Retrovirus transduction Dualotropic LNCX (control) and LNCX-CITED1 (CITEDl -expressing) retro viruses were generated by inserting human CITEDl cDNA into the multiple cloning site of the pLNCX vector, and transfecting the vector into RETROPACK PT67 packaging cells (CLONTECH). We established CITEDl -expressing MCF-7 cells by infecting MCF-7 cells with the LNCX-CITED1 and LNCX viruses, followed by G418 selection. One round of infection generated more than 100 independent G418-resistant clones, which were pooled before conducting the experiments. The reproducibility of experiments using these retrovirus-infected cells was confirmed using at least two independent batches of infected cells.

Plasmids

Mammalian expression plasmids for hemagglutinin (HA) epitope-tagged human CITEDl and CITED2 were previously described by Yahata et al (J. Biol.

Chem. 275:8825-8834, 2000), and plasmids for HA-tagged chick CITED3 (Andrews et al, Mech. Dev. 95:305-308, 2000) and mouse CITED4 (Yahata et al, J. Biol. Chem. 275:8825-8834, 2000) were constructed similarly. The plasmid for HA-tagged p300 is described in Eckner et al (Genes & Development 8:869-884, 1994) and plasmids for GAL4DBD fusion nuclear receptors and full-length human ERα are described in Kobayashi et al. (J. Biol. Chem. 275:15645-15651, 2000). hi addition, plasmids for the FLAG epitope-tagged AFl or AF2 domain of ERα were constructed using a PCR-based standard protocol and confirmed by sequencing. Luciferase reporter plasmid pERE-tk-Luc is described in White et al. (Endocrinology 135:175-182, 1994) and plasmid p3XERE-ElbTATA-Luc in Kalkhoven et al. (EMBO J. 17:232-243, 1998). A GAL4-dependent luciferase reporter plasmid pGLUC8 was constructed by inserting eight tandem repeats of GAL4 binding elements into pGV-B2 luciferase reporter vector (Tokyo Ink, Japan) before the EIB TATA box. Furthermore, bacterial expression plasmids for glutathione S- transferase (GST) fusion ERα domains were described in Endoh et al (Mol. Cell Biol. 19:5363-5372, 1999).

Antibodies, immunoprecipitation. Western blotting, and immunohistochemistry Western blots may be performed using techniques standard in the art. For example, the blocking buffer may be 5% nonfat dry milk and 0.05% Tween-20 in Phosphate Buffered Saline (pH 7.0); the washing buffer maybe 0.05% Tween-20 in Phosphate Buffered Saline and the primary and secondary antibodies may be diluted in the Blocking buffer. The blot may be blocked, in blocking buffer, at room temperature for 2 hours. The primary antibody, diluted 1 :3000 in blocking buffer, may be added, and the blot may be incubated at room temperature for 1 hour. After the incubation, the blot maybe washed for 15 minutes, followed by two 5-minute washes, at room temperature in washing buffer. The secondary antibody, for example, an anti-rabbit or anti-mouse IgG antibody conjugated with horseradish peroxidase (Amersham) diluted 1 :3000 in blocking buffer, may then be added and the blot may be incubated at room temperature for 1 hour. Following the incubation with the secondary antibody, the blot maybe washed once for 15 minutes and three times for 5 minutes at room temperature in washing buffer. The Western blot may be developed using ECL chemiluminescence (Amersham) for approximately 10 seconds to 3 minutes.

An anti-CITEDl rabbit polyclonal antibody that reacts with both human and mouse CITEDl is described by Li etal (Exp. Cell Res. 242:478-486,1998) and by Shioda et al. (Proc. Natl. Acad. Sci. USA 93:12298-12303, 1996). An anti-CITED2 monoclonal antibody (clone JA22) is described in Bhattacharya et al. (Genes and Development 13:64-75, 1999). The anti-HA monoclonal antibody (12CA5) was purchased from Roche Molecular Biochemicals. The anti-FLAG monoclonal antibody (M2) was obtained from Sigma and the anti-His (H-15), anti-ERα (sc-786), and anti-p300 (N-15) antibodies were purchased from Santa Cruz Biotechnology.

In addition, we raised anti-human CITEDl rabbit polyclonal antibodies against synthetic peptides LGQNEFDFTADFPSG (SEQ ID NO:29) and AYSNLAVKDRKAV (SEQ ID NO:30). The anti-human CITEDl polyclonal antibodies were generated either against a unique sequence found only in human CITEDl and not in the other CITED family members, or against the human CITEDl CR2 region. Polyclonal antibodies were generated by Research Genetics (Huntsville, Alabama) using peptides designed by us. The standard protocol used by Research Genetics is provided in Table 2, below. Table 2: Generation of Polyclonal Antibodies

Two New Zealand white female rabbits are utilized for each MAP peptide immunogen and are housed in individual cages.

Pe tide S nthesis

However, any standard method known in the art for generating polyclonal antibodies may also be used, for example, as described in Ausubel et al. (Current Protocols in Molecular Biology, Wiley hiterscience, New York, 2000).

Furthermore, we raised monoclonal antibodies against human CITED 1, using the full-length human CITEDl protein. The antibodies antibodies with the greatest affinity for human CITED- 1 were selected using ELISAs. hi general, monoclonal antibodies against CITED proteins may be prepared using the standard hybridoma technology (see, e.g., Kohler et al, Nature 256:495, 1975; Kohler et al, Eur. J. Immunol. 6:511, 1976; Kohler et al, Eur. J. Immunol. 6:292, 1976; Ha merling t al, Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981; and Ausubel et al., Current Protocols in Molecular Biology, Wiley hiterscience, New York, 2000). For transfection-based co-immunoprecipitation assays, 1 x IO⁶ cells (per 6 cm dish) were transfected with expression plasmids and cultured for 24 hour in hormone- free medium, followed by stimulation with hormones. Cells were then lysed in JP buffer (50 mM Hepes/NaOH (pH 7.5), 120 mM NaCl, 2.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and 0.5% NP-40) supplemented with phosphatase inhibitors (50 mM sodium fluoride, 25 mM sodium glycerophosphate, 1 mM orthovanadate) and proteinase inhibitors (10 μg/ml aprotinin, 10 μg/ml leupeptin). Immunoprecipitation and Western blotting were performed as described by Yahata et al. (J. Biol. Chem. 275:8825-8834, 2000) or by Harlow and Lane (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, NY, 1988). Preparation of histo logical slides and immunohistochemical staining was performed using an anti-CITEDl polyclonal antibody, as described by Li et al. (Exp. Cell Res. 242:478-486, 1998), and an anti-ERα (sc-786) antibody.

hi vitro protein binding assay

Polyhistidine epitope-tagged wild-type and mutant human CITEDl (His- CITEDl) and was expressed in Sf9 insect cells using recombinant baculoviruses and purified to homogeneity using a metal chelating column (Yahata et al, J. Biol. Chem. 275:8825-8834, 2000). Glutathione S-transferase (GST) fusion proteins of the AFl or LBD/AF2 domain of ERα (GST- AFl and GST-AF2) were expressed in E. coli and purified using glutathione-conjugated Sepharose beads (Amersham). For the GST pull-down assay, wild-type or mutant forms of His-CITEDl were incubated with GST, GST-AFl, or GST-AF2 immobilized on glutathione-conjugated Sepharose beads in binding buffer (20 mM Tris/HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, and O.P/o NP-40) for 2 hours at 4°C in the presence or absence of 100 nM 17β-estradiol. Beads were then washed four times with the binding buffer and boiled in standard SDS-PAGE loading buffer for 5 minutes. Wild-type or mutant His-CITEDl protein that bound the GST fusion proteins was detected by anti-His Western blotting. Reverse transcription - polymerase chain reaction (RT-PCR) and Northern blotting Total RNA was isolated from MCF-7 cultures using the RNEASY RNA isolation kit (QIAGEN) according to the manufacturer's instructions. Equal amounts of total RNA (100 ng per reaction) were subjected to semi-quantitative RT-PCR analysis in the presence of α-³ PdCTP, as described in Shioda et al. (Am. J. Pathol

150:2099-2112, 1997).

For Northern blot hybridization of the TGF-α mRNA transcript, 5 μg per lane of total RNA was separated by 1% formaldehyde agarose gel electrophoresis, transferred to a nylon membrane, and hybridized with the radiolabeled RT-PCR product of human TGF-α as described in Shioda et al. (Proc. Natl. Acad. Sci. USA

91:11919-11923, 1994). The amount of TGF-α mRNA transcript may be quantified by densitometry of the radioactive bands on the X-ray fihns.

Example 2 Cloning and Characterization of CITED4

A BLAST search of the EST (expressed sequence tag) database with the amino acid sequence of the CBP/p300-binding domain of human CITEDl revealed an existence of a highly homologous novel peptide encoded by several independent EST clones. Many of such EST clones, including EST 1247350 (GenBank AA839758), had been isolated from a cDNA library for mouse mammary gland. A preliminary RNA blot hybridization using EST 1247350 clone as probe demonstrated a strong expression of the corresponding mRNA transcript (~1.5 kb) in 10.5 dpc pregnant female mouse mammary gland (see Fig. 16B). A cDNA library was constructed from the same mammary gland RNA and screened with the EST127350 cDNA probe, resulting in isolation of 18 independent positive cDNA clones. Seven of these clones were almost identical in size (-1.25 kbp) and harbored a complete open reading frame (546 nucleotides encoding a 182 amino acids protein), 3' untranslated sequence with a polyadenylation signal followed by a short fragment of poly(A) tail, and -210 nucleotides of 5' untranslated sequence with minor variations in length (Fig. 14A). The predicted translation start codon located within a context of the consensus translational initiation sequence (Kozak, J. Cell Biol. 115:887-903, 1996); effective generation of the predicted protein with molecular mass of 21 kDa was confirmed by in vitro translation using a rabbit reticulocyte lysate system. An alignment of the deduced amino acid sequence with known CITED proteins revealed a strong similarity (31 amino acids identity with the consensus profile consisting of 44 amino acids) at the C-terminal CBP/p300-binding domain (Fig. 14C). This C-terminal domain is the signature motif for the CITED family proteins and referred as

Conserved Region 2 (CR2) (Shioda et al, Gene 204:235-241, 1997; Yahata et al, J. Biol. Chem. 275:8825-8834, 2000). Therefore, we conclude that we isolated full- length mouse cDNA clones representing a novel member of the CITED family proteins, which we designate as CITED4. Interestingly, the Conserved Region 1 (CR1), another signature motif for the

CITED protein (Shioda et al, Gene 204:235-241, 1997; Yahata et al, J. Biol. Chem. 275:8825-8834, 2000), is not conserved well in CITED4, making CITED4 unique within the CITED proteins. Instead, CITED4 shares a well-conserved N-terminal sequence with CITED2 and CITED3, thus defining Conserved Region 3 (CR3) as a novel signature motif for the CITED proteins. Phylogenetic analysis of amino acid sequences of the CITED proteins revealed that CITED4 is equally distant from other three CITED proteins (Fig. 14B). As is evident from Figure 14C, CITEDl proteins do not have well conserved CR3 domains, but do contain highly conserved CR2 domains. CITED2 and CITED3 proteins are distinct family members since several organisms, including Xenopus and Zebrafish, have been shown to contain both a CITED2 and a CITED3 protein, hi addition, the chicken CITED3 protein is more similar to the other CITED3 proteins than to any other CITED family members.

When expressed in COS-1 cells and analyzed by SDS-polyacrylamide gel electrophoresis followed by anti-CITED4 immunoblotting, the full-length mouse CITED4 cDNA generated a 21 kDa protein that co-migrated with the in vitro translated CITED4. Endogenous CITED4 protein expressed in mouse mammary gland (see Fig. 16C) also showed identical electrophoretic mobility with COS-1 expressed CITED4. Thus, it appeared that CITED4 does not receive significant post- translational modifications. Mouse CITED4 binds to CBP/p300 in a manner dependent on the CR2 region

The common characteristic feature of the known CITED family proteins is their activity to bind to CBP/p300 through their CR2 region, hi addition, when tethered to a heterologous DNA-binding domain, CITED proteins also activate transcription in a manner dependent on CBP/ρ300 (Bhattacharya et al, Genes and Development 13:64-75, 1999; Shioda et al, Gene 204:235-241, 1997; Yahata et al, J. Biol. Chem. 275:8825-8834, 2000).

As expected, mouse CITED4 bound to p300 in vivo. When expressed in COS- 1 cells, HA-tagged CITED4 was co-precipitated with endogenous p300 by an anti- p300 antibody (Fig. 17A; p300 precipitation is not shown). The specificity of the p300-dependent co-precipitation was confirmed by the absence of CITED4 co- precipitation using IgG from non-immunized rabbits. A CITED4 mutant lacking the CR2 region was not co-precipitated, even by the anti-p300 antibody, demonstrating the requirement of this domain for interaction with p300. When tethered to GAL4 DNA-binding domain (GAL4DB), mouse CITED4 activated transcription in a manner dependent on CBP/p300 (Fig. 17B). In these experiments, and as reported previously for human CITEDl (Yahata et al, J. Biol. Chem. 275:8825-8834, 2000), transcriptional activation mediated by mouse CITED4 is effectively suppressed by wild-type adenovirus El A protein that inhibits functions of both RB tumor suppressor protein and CBP/p300. The Δ2-36 mutant of El A suppresses RB only, while the m928 mutant suppresses CBP/p300 only (Arany et al, Nature 13:64-75, 1999; Yavuzer et al, Oncogene 10:123-134, 1995). Whereas the Δ2-36 mutant failed to suppress CITED4-dependent transcription, the m928 mutant still effectively suppressed it. Taken together, these results demonstrate that mouse CITED4 indeed has functional features that are characteristic to the CITED proteins. We previously reported that transcriptional activation mediated by the Smad4 C-terminal transactivating domain, which is dependent on CBP/p300, was strongly enhanced by co-transfection of CITEDl whereas other CITED proteins showed no effects (Shioda et al, Proc. Natl. Acad. Sci. USA 95:9785-9790, 1998). As shown in Fig. 17C, CITED4 also failed to enhance Smad4C-mediated transcriptional activation. Co-transfection of both CITEDl and CITED4 resulted in marginal decrease in the CITEDl -enhanced transactivation, most likely due to the competition of CITEDl and CITED4 for binding to a limited amount of endogenous CBP/p300 (squelching effect; see (Janknecht and Hunter, Nature 383:22-23, 1996)). These results confirmed that, among the CITED proteins, the Smad4C-enhancing activity is unique to CITEDl, providing further evidence that the CITED proteins do not enhance CBP/p300- dependent transcription nonspecifically but require specific interaction with their target DNA-binding transcription factors (Shioda et al, Proc. Natl. Acad. Sci. USA 95:9785-9790, 1998).

Isolation and characterization of mouse cited4 genomic DNA clones Three independent PAC clones harboring mouse cited4 gene were identified by screening an arrayed library using a CITED4 cDNA probe. These three PAC inserts were subcloned into a vector and mapped for restriction digestion sites (Fig. 15A). Preliminary Southern blot hybridization analysis using high molecular weight mouse genomic DNA revealed that the entire cited4 gene is located within a 10.5 kpb EcoRI fragment, a 9 kbp BamHI fragment, and a 4.5 kbp EcoRJ/BamHI double digestion fragment; all these results matched to those obtained with the PAC inserts. Sequencing of the 4.5 kbp EcoRI/BamHI fragment of a PAC insert showed that the entire mouse cited4 gene, which consisted of only a single exon without any short introns, is contained within this insert (Fig. 15A). The relative location of mouse cited4 gene in the restriction map and its single-exon nature were further confirmed by isolating several independent genomic DNA clones from a library followed by Southern blot hybridization analysis and sequencing. These results validated that mouse CITED4 is an independent member of the CITED family encoded by a discrete gene, eliminating a possibility that it was derived from alternative splicing of a previously reported cited family gene.

To establish the promoter sequence of mouse cited4 gene, its transcription initiation site was determined using 5 '-RACE method, and is indicated as nucleotide +1 in Figure 15B. In addition, a canonical TATAA box was found 26 nucleotides upstream of the transcription initiation site (Fig. 15B, boxed). Pregnancy-induced expression of CITED4 in the mouse mammary gland

An extended screening of adult mouse tissues for expression of the CITED4 mRNA transcript by RNA blot hybridization revealed that it was strongly expressed in the mammary glands of pregnant mice. Furthermore, the mRNA transcripts for CITEDl, CITED2, and CITED4 were expressed simultaneously in the mammary glands of 11.5 dpc pregnant mice and readily detectable by hybridization of a total RNA blot (Fig. 16B).

Since the mRNA transcripts of three members of the CITED family were strongly and simultaneously expressed in the mammary glands of pregnant mice, we next attempted to determine amounts of expression of these proteins in this tissue by immunoblotting. CITED2 protein was not detectable in any stages of mammary gland by this method using an anti-CITED2 antibody (Bhattacharya et al, Genes and Development 13:64-75, 1999), probably because of its very short in vivo half-life (Bhattacharya et al, Genes and Development 13:64-75, 1999). However, both CITEDl and CITED4 proteins were detected but at different stages of this tissue, for example, CITED4 protein was readily detected in the mammary glands of pregnant mice, but undetectable in the mammary glands of nulliparous mice. In contrast, CITEDl protein was expressed strongly in the mammary glands of nulliparous mice, but undetectable in the mammary glands of pregnant mice (Fig. 16C). The expression profile of CITED4 protein in mouse mammary glands suggests that this protein may be induced along with differentiation of mammary epithelial cells. To characterize CITED4 further, we determined the amount of CITED4 mRNA transcript in mammary epithelial cells of pregnant SCp2 mice, during their prolactin- induced differentiation (Desprez et al, Mol. Cell Biol. 15:3398-3404, 1995), by RNA blot hybridization. As is shown in Fig. 16D, expression of the CITED4 mRNA transcript in SCp2 cells was dramatically augmented during the gradual induction of differentiation of these cells cultured in a prolactin-containing medium. These results support that expression of CITED4 in mouse mammary glands is dependent on the differentiation of mammary epithelial cells. The above experiments were carried out using the following materials and methods. Materials and Methods Cell culture

COS-1 and NTH3T3 cells were maintained in Dulbecco's Minimum Essential Medium supplemented with 10% fetal calf serum and antibiotics. Cells were transfected using LipofectAMINE Plus reagent (Life Technologies) following manufacturer's instructions.

Plasmids

Mammalian expression plasmids for hemagglutinin (HA) epitope-tagged mouse CITED4 and its deletion mutants, and GAL4 DNA-binding domain

(GAL4DB) fusion of CITED4, were constructed using standard techniques. The plasmid for HA-tagged p300 is described in Eckner et al. (Genes & Development 8:869-884, 1994) and plasmids for GAL4DBD fusion nuclear receptors and full- length human ERα are described in Kobayashi'et al. (J. Biol. Chem. 275:15645- 15651, 2000).

Expression plasmids for adeno virus El A protein and its mutants, GAL4DB- fusion CITEDl, and GAL4DB-fusion Smad4 C-terminal domain were described previously (Yahata et al, J. Biol Chem. 275:8825-8834, 2000).

Northern blotting. Western blotting, and immunohistochemistry

Western blots may be performed using techniques standard in the art. For example, the blocking buffer may be 5% nonfat dry milk and 0.05% Tween-20 in Phosphate Buffered Saline (pH 7.0); the washing buffer may be 0.05% Tween-20 in Phosphate Buffered Saline and the primary and secondary antibodies may be diluted in the Blocking buffer. The blot may be blocked, in blocking buffer, at room temperature for 2 hours. The primary antibody, diluted 1 :3000 in blocking buffer, may be added, and the blot may be incubated at room temperature for 1 hour. After the incubation, the blot may be washed for 15 minutes, followed by two 5-minute washes, at room temperature in washing buffer. The secondary antibody, for example, an anti-rabbit or anti-mouse IgG antibody conjugated with horseradish peroxidase (Amersham) diluted 1 :3000 in blocking buffer, may then be added and the blot may be incubated at room temperature for 1 hour. Following the incubation with the secondary antibody, the blot may be washed once for 15 minutes and three times for 5 minutes at room temperature in washing buffer. The Western blot may be developed using ECL chemiluminescence (Amersham) for approximately 10 seconds to 3 minutes. An anti-CITEDl rabbit polyclonal antibody that reacts with both human and mouse CITEDl is described by Li et al. (Exp. Cell Res. 242:478-486,1998) and by Shioda et al. (Proc. Natl. Acad. Sci. USA 93:12298-12303, 1996). An anti-CITED2 monoclonal antibody (clone JA22) is described in Bhattacharya et al. (Genes and Development 13:64-75, 1999). We raised anti-mouse CITED4 rabbit polyclonal antibodies against synthetic peptides YAGPGMDSGLRPRGA (SEQ ID NO:27) and FDCFSDLGSAPAAGS (SEQ ID NO:28). The anti-CITED4 cross-reacts with the human CITED4, but does not cross-react with other known CITED proteins overexpressed in COS-1 cells, when evaluated by immunoblotting. h addition, we generated an anti-human CITED4 polyclonal antibody by immunizing rabbits with a GST-CITED4 fusion protein. Polyclonal antibodies were generated by Research Genetics (Huntsville, Alabama) using peptides designed by us, as described above. However, any standard method known in the art for generating polyclonal antibodies may also be used, for example, as described in Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2000). The anti-HA monoclonal antibody (12CA5) was purchased from Roche

Molecular Biochemicals. The anti-FLAG monoclonal antibody (M2) was obtained from Sigma and the anti-His (H-15), anti-ERα (sc-786), and anti-p300 (N-15) antibodies were purchased from Santa Cruz Biotechnology.

A poly(A)-rich RNA blot for adult mouse tissues was purchased from CLONTECH. Isolation of total RNA from the mammary glands of pregnant mice or from SCp2 cells, gel separation of RNA and transfer to nylon membrane, and hybridization with mouse CITEDl, CITED2, and CITED4 cDNA probes were performed following standard protocols and as described in Sambroo et al. (Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Laboratory Press, New York, 1989) and Shioda et al. (Proc. Natl Acad. Sci. USA 93 : 12298- 12303, 1996). Mammary epithelial cells from pregnant SCp2 mice were obtained from M. Bissell (Life Sciences Division, Lawrence Berkeley National Laboratory, CA). Their maintenance and in vitro induction of epithelial differentiation by prolactin-containing medium were performed as described in Desprez et al. (Mol. Cell Biol. 15:3398-3404, 1995).

Example 3 Use of CITED Proteins in Estrogen-Related Screens The demonstration that CITED proteins, e.g., CITEDl and CITED4, strongly enhance the estrogen-dependent transcriptional activation mediated by estrogen receptors, indicates that CITED proteins may be used in in vitro systems to assay for estrogen-like activities to greatly increase the sensitivity of the system. Accordingly, the use of CITED proteins enables in vitro assay systems that quantitatively and specifically detect estrogenic compounds. These systems provide highly sensitive high throughput evaluations of environmental estrogens or estrogen mimics, even if these compounds are only weak transcriptional activators. Such assay systems typically consist of: (i) a CITED protein, for example, CITEDl, CITED2, CITED3, or CITED4; (ii) an estrogen receptor-regulated gene; and (iii) a host cell.

The host cells may transiently or stably express either a full-length estrogen receptor, or the estrogen-binding/transcriptional activating domains derived from human estrogen receptors α and/or β fused to a heterologous DNA binding domain, such as a GAL-4 DNA binding domain. The human estrogen receptor used in this method may be the human ERα (e.g., GenBank Accession Numbers AF258449, AF258450, AF258451, or NM000125) or the human ERβ (e.g., GenBank Accession Number NM001437). However, estrogen receptors from other species may also be used, for example, the zebrafish ERα (e.g., GenBank Accession Number AF349412), the zebrafish ERβ (e.g., GenBank Accession Numbers AF349414 or AJ275911), the chicken ERβ (e.g., GenBank Accession Number AB036415), the mouse ERα (e.g., GenBank Accession Numbers NM007953 or NM007956), or the mouse ERβ (e.g., GenBank Accession Number NM010157). In addition, the host cells may be human cells, such as normal or malignant human mammary epithelial cells. For example, the host cells maybe CV-l/EBNA-1 cells, and maybe engineered to harbor in their chromosomes, or to transiently express, a GAL4-dependent reporter gene expressing GFP when activated by GAL4-fusion transactivators (e.g., the GB133 cell line (Shioda et al, Proc. Natl. Acad. Sci. USA 97:5220-5224, 1997)).

In addition, the reporter gene used in this system may include an estrogen responsive element. Such a reporter may be derived from certain natural estrogen target genes, for example, the TGF-α, the α-1 anti-chymotrypsin, the c-Fos, the cdc25, the Ha-Ras, the progesterone receptor, the PS2, the BRCA1, the cathepsin D, the E2F1, the HGM1, the insulin receptor substrate-1, the insulin receptor binding protein-4, the c-Myc, or the WISP-2 gene. However, the reporter also may be synthetically generated by those skilled in the art. h addition, the reporter may also contain binding sites for a heterologous DNA binding domain (e.g., the GAL-4 DNA binding domain). Preferably the products of the reporter gene are easily detectable. For example, a reporter gene may express firefly luciferase, alkaline phosphatase, CAT, or green fluorescent protein. The CITED protein used in this system may be, for example, a CITED 1 polypeptide substantially identical to GenBank Accession Number NP_004134; a CITED2 polypeptide substantially identical to GenBank Accession Number AAF01263, AAF01264, AAG36932, NP_034958, or Q99967; a CITED3 polypeptide substantially identical to GenBank Accession Number AAF76148, or to SEQ JD NO:2; or a CITED4 polypeptide substantially identical to GenBank Accession Number NP_062509 or NM 319563, or to SEQ JD NO:4.

As described above, CITED proteins enhance estrogen receptor-mediated transcription in mammalian cells by up to 10-fold and, therefore, significantly increase the sensitivity of the assay. The use of CITED proteins is also advantageous since these proteins enhance transcriptional functions of both estrogen receptor α and β very selectively without affecting any other nuclear receptors so far tested. For example, in a mammalian assay system using a GFP reporter, when estrogenic compounds are added to the culture, the engineered cells express GFP with enhanced sensitivity due to the presence of the CITED protein. By enhancing the level of GFP expression the use of CITED proteins enables automated reading of fluorescence intensity. In addition, by enabling automated screening of cells for compounds that affect estrogen receptor-dependent transcription, the use of CITED proteins also makes it possible to screen for such compounds in a high throughput manner. Consequently, such an assay system establishes a new avenue for identifying the environmental presence of estrogen-like compounds and their potential influences on human health.

Compounds that increase estrogen receptor activity may be useful in the treatment or prevention of depression, cardiovascular disease, infertility, and osteoporosis. In addition, compounds that decrease estrogen receptor activity may be useful in the treatment or prevention of breast cancer, prostate cancer, ovarian cancer, a gynecological disorder, endometriosis, and the symptoms of menopause. Such compounds may be identified and isolated using the screens and assays described herein.

The following methods may be used to identify compounds that are estrogenic or anti-estrogenic.

Detection of estrogenic and anti-estrogenic compounds and their precursors by GFP- reported two-hybrid in MCF-7 cells

MCF-7 cells may be purchased from American Type Culture Collection and may be maintained in regular high-glucose Dulbecco's Miminum Essential Medium

(DMEM) supplemented with non-stripped 10% fetal calf serum (FCS). Under these culture conditions, FCS-derived estrogen sufficiently supports the estrogen-dependent growth of MCF-7 cells. In addition, the weak estrogenic activity of phenol red, the pH indicator of DMEM, may also contribute to MCF-7 cell growth (Hopp et al, J.

CellPhysiol 157:594-602, l993;

et al, Anticancer Res. 12:789-794,

1992; Grady et al, Endocrinology 129:3321-3330, 1991; Revillion et al, Anticancer

Res. 10:1067-1070, 1990; Ortmann et al, J. Steroid Biochem. 35:17-22, 1990; Ernst et al, J. Steroid Biochem. 33 :907-914, 1989; Dumesic et al, Life Sci. 44:397-406,

1989; Bindal et al, J. Steroid Biochem. 31:287-293, 1988; Glover et al, Cancer Res.

48:3693-3697, 1988; Welshons et al, Mol. Cell Endocrinol. 57:169-178, 1988;

Welshons et al, Eur. J. Cancer Clin. Oncol. 23:1935-1939, 1987; Hubert et al,

Biochem. Biophys. Res. Commun. 141:885-891, 1986). MCF-7 cells express ERα and ERβ (Burow et al, Int. J. Oncol 16:1179-1187,

2000). They also express CITEDl weakly, but CITED4 expression is not detectable.

Prior to transfection, MCF-7 cells may be cultured in a steroid hormone-free condition (phenol red-free DMEM supplemented with 5% charcoal/dextran-stripped FCS) for approximately 24-48 hours. Cells may be transfected with a pG5GFP reporter plasmid (Fig. 20) (closed circular covalent form) together with expression plasmids for GAL4DB-AF2 (ERα or ERβ) and the CITED proteins (e.g., CITEDl and/or CITED4) by a standard method using the LipofectAMJNE reagent (Gibco/BRL). After transfection, cells maybe cultured in the presence or absence of known concentrations of estrogen 17β-estradiol (approximately 10-10,000 pM; a recent study reported the EC50 of 17β-estradiol in supporting growth of MCF-7 cells to be 100 pM (Chiarenza et al, Cancer Res. 61:3002-3008, 2001)), or androgen dehydroepiandrosterone (approximately 1-100 nM), which is converted to estrogen intracellularly by tumor aromatase (Schmitt et al, Mol. Cell Endocrinol. 173:1-13, 2001). To determine the time course of estrogen or androgen-dependent expression of GFP, cells may be observed at every 6 hours up to 72 hours after transfection by a conventional fluorescence microscope and a multi-well fluorometer. To determine whether this system may be suitable for detection of anti-estrogen compounds as well, transfected cells may be cultured in the presence of low concentrations (e.g., approximately 10-100 pM) of 17β-estradiol plus varying concentrations (approximately 0.1-100 nM) of Tamoxifen, Raloxifene, or ICI164384, followed by evaluation of decrease in GFP expression by these anti-estrogens. Having demonstrated detection of authentic estrogen, this system can be used to detect two representative known estrogenic EDCs, for example, Bisphenol A and p- Nonylphenol (Krishnan et al, Endocrinology 132:2279-2286, 1993; Soto et al, Environ. Health Perspect. 92:167-173, 1991). Estrogenic activities of known amounts of these compounds (approximately 1-100 nM) maybe evaluated as described above for authentic estrogen. Attempts may also be made to detect those chemicals in plastic extracts prepared following the published protocols (Krishnan et al, Endocrinology 132:2279-2286, 1993; Soto et al, Environ. Health Perspect. 92:167-173, 1991).

MCF-7 cells maybe transfected with linearized pG5GFP-Hyg (Fig. 20) by a standard lipofection method (e.g., LIPOFECTAMJNE), and stable clones may be selected in the presence of hygromycin B (e.g., 0.5 mg/ml) for 2 weeks. Individual colonies may be isolated and tested for their ability to detect 17β-estradiol (estrogen) and dehydroepiandrosterone (androgen) by the GFP reporter in the presence of transiently transfected GAL4DB-AF2s and the CITED proteins as described above. High responder clones with a minimum leakage of GFP expression in the absence of estrogen (designated as MCF-7/GFP cells) may be amplified in the presence of a low concentration (e.g., 0.1 mg/ml) of hygromycin B and subjected to the next step of engineering.

MCF-7/GFP cells may be infected by amphotropic retroviruses expressing GAL4DB-fusion AF2 domains of ERα or ERβ using a standard technique. Construction of GAL4DB-estrogen receptor AF2 domain fusion proteins is described in Kobayashi et al. (J. Biol. Chem. 275:15645-15651, 2000). The retrovirus backbone maybe LPCX (CLONTECH, CA), a derivative of the standard LNCX virus expressing a puromycin-resistance marker gene. Infected cells may be selected by puromycin (approximately 0.05-0.1 mg/ml) for 2 weeks to obtain stable clones. Efficiency of the GFP-reported detection of estrogen and androgen, as well as leak expression of GFP in the absence of hormones may be evaluated for each clone in the presence of co-transfected CITED proteins as described above. High responder clones with a minimum leakage GFP expression (designated as MCF-7/GFP-ERα or -ERβ cells) may be amplified in the presence of low concentrations of hygromycin B and puromycin (approximately 0.01-0.02 mg/ml) and subjected to the next step of engineering.

MCF-7/GFP-ERα and -ERβ cells may be further infected by amphotropic retroviruses expressing CITEDl or CITED4 using a standard technique. The retrovirus backbone may be the standard LNCX virus expressing a neomycin- resistance marker gene. Infected cells maybe selected in the presence of G418 (e.g., 1 mg/ml) for 2 weeks to obtain stable clones. Efficiency of the GFP-reported detection of estrogen and androgen as well as leakage of GFP expression may be evaluated for each clone without co-transfection. High responder clones with a minimum leakage of GFP expression may then be selected. Those final products may be designated as Fluorescence Estrogen Detector cells, or FED cells, followed by indication of the origin of the AF2 domain for GAL4DB fusion (α and β for ERα and ERβ, respectively) and the CITED proteins expressed (e.g., 1 or 4): such as FED-α4. The FED cells maybe amplified in the presence of low concentrations of hygromycin B, puromycin, and G418 (e.g., 0.2 mg/ml) and maybe subjected to further characterization.

The FED cells may be inoculated in multi-well plates (e.g., 96 to 384 wells per plate), steroid hormone-starved, and exposed to varying concentrations estrogens, androgens, anti-estrogens, and known estrogen-related EDCs as described above, followed by monitoring their GFP expression by a multi-well plate fluorometer.

The mammalian two-hybrid system described above may also be used to identify and study protein-protein interactions, for instance the direct binding of transcription factors to a CITED protein.

Evaluation of estrogen-dependent cell growth

The engineered cancer cells (including the control cells) may be inoculated in 35 mm culture dishes (e.g., 50,000 cells/5 ml medium/dish) and incubated in the regular DMEM supplemented with 10% FCS for 12 hours. Cells attached to the dishes may be washed with serum-free DMEM three times and then cultured in the phenol red-free DMEM supplemented with 1-5%) stripped FCS in the presence of graded concentrations of 17β-estradiol (for example: 0, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, and 100 nM): a recent study reported the EC50 of 17β-estradiol in stimulating growth of MCF-7 cells to be 100 pM (Chiarenza et al, Cancer Res. 61: 3002-8, 2001). Cells may be grown for 7 days and counted daily. Adherent cells may be detached by trypsin/EDTA, suspended in trypan blue-containing medium, and counted using a hemocytometer.

To evaluate effects of the SERMs on estrogen-dependent growth of the engineered breast cancer cells, the same growth assay may be performed in the presence of moderate concentrations of 17β-estradiol (10 pM, 100 pM, and 1 nM) plus graded concentrations of two representative SERMs that are used for hormone therapy of breast cancers, for example, tamoxifen and raloxifene (0.1 nM, 1 nM, 10 nM, and 100 nM; maximum in vitro effects are usually observed at around 60 nM concentration). Effects of anti-estrogen ICIl 82780, which is commonly used as a positive control agent for in vitro assays of the SERM effects, may also be examined. Time-dependent growth-profiles of MCF-7 cells in the presence of 17β-estradiol and/or tamoxifen were published by Chiarenza et al. (Cancer Res. 61 : 3002-8, 2001). hi addition, to evaluate effects of the aromatase inhibitors on estrogen- dependent growth of the engineered cancer cells, the growth assay may be performed in the complete absence of estrogen but the presence of moderate concentrations of androgen (dehydroepiandrosterone, 1 nM, 10 nM, and 100 nM) plus graded concentrations of aromatase inhibitors Letrozole and Anastrozole (0.01 nM, 0.1 nM, 1 nM, and 10 nM). These two agents are the third generation non-steroidal aromatase inhibitors that have now become standard second-line treatment agents after tamoxifen treatment for advanced breast cancers. Suppression of MCF-7 cell growth by aromatase inhibitors in culture has been reported by many studies (reviewed in Goss et al, JClin Oncol 19: 881-94, 2001).

Cancer cell apoptosis may be evaluated by a standard protocol of propidium iodide/annexin V-FITC double staining followed by flow cytometry. The engineered cancer cells and the control cells maybe inoculated in 10 cm culture dishes (1x10 cells/10 ml medium/dish) and incubated in the regular DMEM supplemented with 1- 5% FCS for 12 hours. To induce apoptosis, cells attached to the dishes may be washed with serum-free DMEM three times and then cultured in the phenol red-free DMEM without serum for 72 hours. After serum and hormone starvation, cells may be cultured for an additional 48 hours in the same medium in the presence of graded concentrations of 17β-estradiol or dehydroepiandrosterone. Cells may then be washed and suspended in propidium iodide/Annexin V-FITC dual staining solution (TAGS Annexin V kit: Trevigen, Gaithersburg, MD) followed by flow cytometry. Cells positive for Annexin V staining may be counted as apoptotic cells; propidium iodide staining may be used to distinguish apoptotic and necrotic cells.

Quantitative evaluation of estrogen effects on cell migration and aggregation of cultured breast cancer cells In addition to uses in screening for compounds that affect transcription of estrogen-dependent genes, CITED proteins may also be used in screens for compounds that affect estrogen-dependent aggregation and migration. Estrogen induces aggregation of MCF-7 cells while suppressing their migration (Olea et al, Int J Cancer 50: 112-7, 1992). To quantitatively evaluate migration and aggregation of the engineered breast cancer cells in culture, one may use time-lapse recorded videomicroscopy linked to computer-aided cell tracking and measurement of cell aggregation as described in Munn et al (J. Immunol. Methods 166: 11-25, 1993). The engineered cancer cells and the control cells may be cultured in the presence or absence of estrogen, androgen, and hormonal agents. Time-lapse digital images of the migrating and aggregating cell population in culture dishes may be obtained by an RGB camera through a phase contrast microscope and stored on a computer and/or on a VCR tape. The incubator attached to the microscope stage can maintain cell cultures in air containing 5% carbon dioxide at 37 ± 0.1 °C. Migration of each cell in the images may automatically be tracked by a specifically designed software, which provides raw data of mean migration velocity of each of the automatically selected cells as well as estimated overall mean migration rate of the whole culture population. The size distribution and the shape of cell aggregates may also be evaluated automatically, and the accurate rate of aggregation maybe calculated using techniques known to one skilled in the art.

Test extracts and compounds hi general, compounds that affect estrogen receptor-dependent transcription are identified from large libraries of both natural products, synthetic (or semi- synthetic) extracts or chemical libraries, according to methods known in the art.

Those skilled in the art will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal- based extracts, fermentation broths, and synthetic compounds, as well as modifications of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from, for example, Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, Wl).

Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including, but not limited to, Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art (e.g., by combinatorial chemistry methods or standard extraction and fractionation methods). Furthermore, if desired, any library or compound may be readily modified using standard chemical, physical, or biochemical methods.

In addition, those skilled in the art readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their effects on compounds associated with estrogen regulation should be employed whenever possible.

When a crude extract is found to affect estrogen receptor-dependent transcription, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having activities that affect estrogen receptor-dependent transcription. The same in vivo and in vitro assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogenous extracts are known in the art. Assays to be used for identifying compounds that affect estrogen receptor- dependent transcription may include measuring estrogen receptor-dependent reporter gene expression in a mammalian two-hybrid system. hi addition, compounds identified as affecting estrogen receptor-dependent transcription may be used in treating an estrogen-related condition. Example 4 Use of CITED Proteins and Nucleic Acids in Diagnosis CITED proteins may also be used in diagnosing an estrogen-related condition or a propensity for an estrogen-related condition in an organism, where a decrease or increase in the level of CITED protein or nucleic acid production, relative to a control, may provide an indication of a deleterious condition, such as cancer.

For example, a decrease in CITED mRNA or protein in a subject, relative to a control subject, would be indicative of a diagnosis that the subject has, or has a propensity for acquiring infertility or osteoporosis. In addition, a decrease in CITED mRNA or protein, relative to control, would correlate with a poor prognosis for the treatment of these conditions.

On the other hand, an increase in CITED mRNA or protein in a subject, relative to a control subject, would be indicative of a diagnosis that the subject has, or has a propensity for acquiring cancer (e.g., breast, prostate, or ovarian cancer), a gynecological disorder, endometriosis, or the symptoms of menopause. Furthermore, an increase in CITED mRNA or protein, relative to control, would correlate with a poor prognosis for the treatment of these conditions.

Levels of CITED protein or nucleic acid expression may be assayed by any standard technique and compared to control samples showing normal CITED protein or nucleic acid expression. For example, expression in a biological sample (e.g., a biopsy) may be monitored by standard Northern blot analysis, using, for example, probes designed from a CITED, e.g., a citedl, cited2, citedS, or cited4, nucleic acid sequences, or from nucleic acid sequences that hybridize to a CITED, e.g., a citedl, cited2, cited3, or cited4, nucleic acid sequence. Measurement of such expression may be aided by PCR (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, Wiley h terscience, New York, 2000; PCR Technology: Principles and Applications for DNA Amplification, ed., H.A. Ehrlich, Stockton Press, NY; and Yap and McGee, Nucl Acids Res. 19:4294, 1991).

Alternatively, a patient sample may be analyzed for one or more alterations in a CITED sequence, when compared to a wild-type CITED sequence, for example, by using a mismatch detection approach. A wild-type CITEDl nucleic acid sequence may be substantially identical to GenBank Accession Number NM_004143, U65092, or U65091; a wild-type CITED2 nucleic acid maybe substantially identical to GenBank Accession Number NM_006079, AF129290, U65093, U86445, or NP_006070; a wild-type CITED3 nucleic acid may be substantially identical to GenBank Accession Number AF261079, AI031460, or to SEQ ID NO:l; and a wild- type CITED4 nucleic acid may be substantially identical to GenBank Accession

Number AF143369, AL158843, or to SEQ JD NO:3. For instance, an alteration may be in the N-terminal region of a CR2 domain and may include a mutation corresponding to one described in Figure 3A, e.g., mut2.

Generally, these techniques involve PCR amplification of nucleic acid from the patient sample, followed by identification of the mutation (i.e., mismatch) by either altered hybridization, aberrant electrophoretic gel migration, binding or cleavage mediated by mismatch binding proteins, or direct nucleic acid sequencing. Any of these techniques maybe used to facilitate mutant CITED detection, and each is well known in the art (see, for example, Orita et al, Proc. Natl. Acad. Sci. USA 86:2766-2770, 1989; and Sheffield et al, Proc. Natl. Acad. Sci. USA 86:232-236, 1989).

Mismatch detection assays may be used to provide the opportunity to diagnose a CITED nucleic acid-mediated predisposition to estrogen-related conditions. For example, a patient heterozygous for a CITED mutation may show no clinical symptoms and yet possess a higher than normal probability of developing one or more types of diseases, for example, depression, cardiovascular diseases, infertility, or osteoporosis. Given this diagnosis, a patient may take precautions to control their exposure to adverse environmental factors (for example, environmental estrogen-like compounds) and to carefully monitor their medical condition (for example, through frequent physical examinations). This type of CITED diagnostic approach may also be used to detect CITED nucleic acid mutations in prenatal screens.

In yet another approach, immunoassays may be used to detect or monitor a CITED, e.g., a CITEDl, CITED2, CITED3, or CITED4, polypeptide in a biological sample. CITED specific polyclonal or monoclonal antibodies may be used in any standard immunoassay format (e.g., ELISA, Western blot, or RIA assay) to measure CITED polypeptide levels; again comparison is to wild-type CITED levels, and an increase or decrease in CITED production is indicative of a condition involving modulation of estrogen receptor-dependent transcription. Examples of immunoassays are described, e.g., in Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000). hnmunohistochemical techniques may also be utilized for CITED detection. For example, a tissue sample may be obtained from a patient, and a section stained for the presence of a CITED protein using an antibody against that protein and any standard detection system (e.g., one which includes a secondary antibody conjugated to horseradish peroxidase). General guidance regarding such techniques can be found in, e.g., Bancroft and Stevens (Theory and Practice of Histological Techniques, Churchill Livingstone, 1982) and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000). Furthermore, the combination of Laser-Capture Microdissection and Real- Time Quantitative RT-PCR may also be used to detect and quantify amounts of the mRNA transcripts for target proteins in pathological specimens, distinguishing different cell types present in the tumor mass (Sgroi et al, Cancer Res. 59:5656-5661, 1999). Analysis using these technologies is very specific and quantitative, as well as yielding very high sensitivity. In addition, these technologies provide the advantage of semi-automated protocols that permit handling of relatively large numbers of specimens in a short period of time with extraordinary quantitative reproducibility.

The frozen tissue specimens of human breast cancers, which may be obtained from a tumor bank, for example are suitable for LCM/RTQ-PCR analysis as was demonstrated by Sgroi et al. (Cancer Res. 59:5656-61, 1999). Frozen cryostat sections (e.g., 8 μm thickness) maybe mounted on uncoated glass slides, fixed in 70% ethanol, and stained with H&E (Hematoxylin and Eosin) followed by dehydration in ethanol series and xylene. Air-dried specimens may be microdissected with a PIXCELL LCM system (Arcus Engineering, Mountain View, CA) to obtain -5 x IO⁴ morphologically homogenous breast cancer cells, normal breast epithelial cells, as well as stromal cells and other components of tumors. Total RNA maybe extracted from the laser-captured cells following a standard RNA microisolation protocol (Emmert-Buck et al, Science 274: 998-1001, 1996) and reverse-transcribed in the presence of oligo(dT) primer. The amounts of each cDNA species may be quantified using RTQ-PCR, for example, TAQMAN 5'-nuclease fluorogenic quantitative PCR (Had et al, Genome Res. 6:986-994, 1996; Gelmini et al, Clin. Chem 43:752-758, 1997), by monitoring the exponential amplification of the PCR products through detecting the liberation of fluorescence dye from primers. The expression of the housekeeping gene cyclophilin 33A may be used as an internal standard to normalize variances in input cDNA (Sgroi et al, Cancer Res. 59:5656-5661, 1999). Amounts of each of the mRNA transcripts may be determined and scored (none=0, very weak=l , weak = 2, moderate=3, strong=4, and very strong = 5) with the strength in normal mammary epithelial cells being defined as "moderate."

To evaluate the correlation of protein amounts with the corresponding mRNA transcripts quantified by the LCM/RTQ-PCR analysis, proteins of interest may be detected and semi-quantified in the tumor specimens by immunohistochemistry and Western blotting. In case that the amounts of certain mRNA transcripts do not correlate with amounts of encoded proteins, both the LCM/RTQ-PCR analysis and the semi-quantitative immunological assays may be performed.

In addition, cryostat sections may be prepared from the frozen tissues and subjected directly to immunohistochemical staining using a kit for Avidin-Biotin Complex-enhanced, horseradish peroxidase (HRP)-based staining (Vector Laboratories, Burlingame, CA). Endogenous peroxidase activity may be suppressed by soaking the slides in diluted hydrogen peroxide solution, and the presence of the antigens may be visualized by using DAB, an HRP substrate generating brown pigment, followed by nuclear counterstaining with methyl green. Alternatively, small pieces of frozen tissues may be fixed in formaldehyde and subjected to standard paraffin-embedding, sectioning, and rehydration, followed by immunohistochemical staining. This method may be preferable when we want to prepare permanent slides or have technical difficulties in cryostat sectioning of tumor samples with massive necrosis or bleeding. When appropriate, antigens may be retrieved by heating the paraffin-embedded slides in sodium citrate solution. Furthermore, specificity of immunostaining may be evaluated by blocking it with peptides or recombinant proteins. Examples of showing expression of CITEDl and CITED4 in the mouse mammary glands are shown in Figure 18. For all antibodies, the intensity of staining and proportion of positive cells may be scored according to a method described by Armes et al. (Cancer Res. 59: 2011-7, 1999). Permanent slides of normal mammary glands stained with each antibody may be used to define the "moderate" intensity. Furthermore, Western blotting may be used to determine a correlation between the amount of RNA transcript and expression of the corresponding protein. In this approach, small pieces (e.g., approximately 0.05 g) of frozen tissues maybe homogenized directly in a microcentrifuge tube with lOx volume (v/w) of Laemmli buffer using a motor-driven disposable plastic pestle and the boiled homogenates may be subjected to Western blotting following a standard protocol. Normal breast tissues, lysates of cultured breast cancer cells, and recombinant proteins may be used as standards to ensure specific detection of the target proteins as well as to define amounts of expression of each protein for the scoring purpose (none=0, weak=l , moderate=2, and strong=3). The CITED diagnostic assays described above may be carried out using any biological sample (for example, any biopsy sample or bodily fluid, such as blood, or tissue) in which CITED nucleic acids or proteins are normally expressed.

Other Embodiments From the foregoing description, it is apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. All publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

What is claimed is:

Claims

Claims

1. A screening method for determining whether a candidate compound modulates estrogen receptor activity, said method comprising the steps of: (a) contacting a cell or in vitro sample expressing a greater than naturally- occurring amount of CITED protein, or CITED activity, and an estrogen receptor target gene with a candidate compound; and

(b) measuring expression of said estrogen receptor target gene, whereby said candidate compound is determined to modulate estrogen receptor activity if said candidate compound causes a change in expression of said estrogen receptor target gene.

2. A screening method for determining whether a candidate compound modulates estrogen receptor activity, said method comprising the steps of: (a) contacting a cell or in vitro sample expressing a CITED2, CITED3, or

CITED4 protein and an estrogen receptor target gene with a candidate compound; and

(b) measuring expression of said estrogen receptor target gene, whereby said candidate compound is determined to modulate estrogen receptor activity if said candidate compound causes a change in expression of said estrogen receptor target gene.

3. The method of claim 2, wherein said cell or sample also expresses a CITEDl protein.

4. The method of claim 1 or 2, further comprising contacting said cell or sample with an estrogen receptor agonist.

5. The method of claim 4, wherein said agonist is estrogen.

6. The method of claim 1 or 2, wherein said candidate compound activates said estrogen receptor target gene.

7. The method of claim 6, wherein said candidate compound is useful for the treatment or prevention of depression, cardiovascular disease, infertility, or osteoporosis.

8. The method of claim 1 or 2, wherein said candidate compound inhibits said estrogen receptor target gene.

9. The method of claim 8, wherein said candidate compound is useful for the treatment or prevention of cancer, a gynecological disorder, or the symptoms of menopause.

10. The method of claim 9, wherein said cancer is breast, prostate, or ovarian cancer.

11. The method of claim 9, wherein said gynecological disorder is endometriosis.

12. The method of claim 1 or 2, wherein said CITED protein is expressed under the control of a heterologous promoter.

13. The method of claim 1 or 2, wherein said CITED protein is encoded by a heterologous nucleic acid.

14. The method of claim 1 or 2, wherein said CITED protein is a mammalian protein.

15. The method of claim 1, wherein said CITED protein is CITEDl, CITED2, CITED3, or CITED4.

16. The method of claim 15, wherein said CITED protein is CITEDl or

CITED4.

17. The method of claim 1 or 2, wherein said estrogen receptor target gene is TGF-α.

18. The method of claim 1 or 2, wherein said estrogen receptor target gene is a reporter gene operably linked to a promoter comprising an estrogen response element.

19. The method of claim 1 or 2, wherein said estrogen receptor target gene is a reporter gene operably linked to a TGF-α promoter.

20. The method of claim 1 or 2, wherein said estrogen receptor target gene is stably integrated into the genome of said cell.

21. The method of claim 1 or 2, wherein said estrogen receptor target gene is transiently transfected into said cell.

22. The method of claim 20 or 21, wherein said estrogen receptor target gene expresses green fluorescent protein, firefly luciferase, or alkaline phosphatase.

23. The method of claim 22, wherein said estrogen receptor target gene expresses green fluorescent protein.

24. A screening method for determining whether a candidate compound modulates estrogen receptor activity, said method comprising the steps of:

(a) contacting a cell expressing a greater than naturally-occurring amount of CITEDl, CITED2, CITED3, or CITED4 protein, or CITED activity, and an estrogen receptor target gene with a candidate compound; and

(b) measuring aggregation of said cell, whereby said candidate compound is determined to modulate estrogen receptor activity if said candidate compound causes a change in aggregation in said cell.

25. The method of claim 24, wherein the amount of said aggregation is compared to the amount of aggregation in control samples from both subjects having an estrogen-related condition and subjects not having an estrogen-related condition.

26. The method of claim 24, further comprising contacting said cell with an estrogen receptor agonist.

27. The method of claim 24, wherein said agonist is estrogen.

28. The method of claim 24, wherein said candidate compound increases said aggregation.

29. The method of claim 28, wherein said candidate compound is useful for the treatment or prevention of depression, cardiovascular disease, infertility, or osteoporosis.

30. The method of claim 24, wherein said candidate compound decreases said aggregation.

31. The method of claim 30, wherein said candidate compound is useful for the treatment or prevention of cancer, a gynecological disorder, or the symptoms of menopause.

32. The method of claim 31, wherein said cancer is breast, prostate, or ovarian cancer.

33. The method of claim 31, wherein said gynecological disorder is endometriosis.

34. The method of claim 24, wherein said CITED protein is expressed under the control of a heterologous promoter.

35. The method of claim 24, wherein said CITED protein is encoded by a heterologous nucleic acid.

36. The method of claim 24, wherein said CITED protein is a mammalian protein.

37. The method of claim 24, wherein said measuring is performed by using time-lapse videomicroscopy.

38. A screening method for determining whether a candidate compound modulates expression of a CITED mRNA or protein, said method comprising the steps of:

(a) contacting a cell or in vitro sample expressing a CITED mRNA or protein with a candidate compound; and (b) measuring expression of said CITED mRNA or protein, thereby determining whether said candidate compound modulates said expression.

39. The method of claim 38, wherein step (b) comprises measuring the expression of said CITED mRNA.

40. The method of claim 38, wherein step (b) comprises measuring the expression of said CITED protein.

41. The method of claim 38, wherein said candidate compound increases said expression.

42. The method of claim 41, wherein said candidate compound is useful for the treatment or prevention of depression, cardiovascular disease, infertility, or osteoporosis.

43. The method of claim 38, wherein said candidate compound decreases said expression.

44. The method of claim 43, wherein said candidate compound is useful for the treatment or prevention of cancer, a gynecological disorder, or the symptoms of menopause.

45. The method of claim 44, wherein said cancer is breast, prostate, or ovarian cancer.

46. The method of claim 44, wherein said gynecological disorder is endometriosis.

47. The method of claim 38, wherein said CITED protein is encoded by a heterologous nucleic acid.

48. The method of claim 38, wherein said CITED protein is a mammalian protein.

49. The method of claim 38, wherein said CITED protein is CITEDl, CITED2, CITED3, or CITED4.

50. The method of claim 38, wherein said CITED protein is CITEDl or CITED4.

51. A screening method for determining whether a candidate compound modulates CITED expression, said method comprising the steps of:

(a) contacting a cell or in vitro sample expressing a reporter gene under the control of a CITED promoter with a candidate compound; and

(b) measuring expression of said reporter gene, thereby determining whether said candidate compound modulates expression mediated by said CITED promoter.

52. The method of claim 51, wherein said candidate compound increases said expression.

53. The method of claim 52, wherein said candidate compound is useful for the treatment or prevention of depression, cardiovascular disease, infertility, or osteoporosis.

54. The method of claim 51, wherein said candidate compound decreases said expression.

55. The method of claim 54, wherein said candidate compound is useful for the treatment or prevention of cancer, a gynecological disorder, or the symptoms of menopause.

56. The method of claim 55, wherein said cancer is breast, prostate, or ovarian cancer.

57. The method of claim 55, wherein said gynecological disorder is endometriosis.

58. The method of claim 51, wherein said CITED promoter is a heterologous nucleic acid.

59. The method of claim 51, wherein said CITED promoter is a mammalian CITED promoter.

60. The method of claim 51 , wherein said CITED promoter is a CITED 1 ,

CITED2, CITED3, or CITED4 promoter.

61. The method of claim 60, wherein said CITED promoter is a CITEDl or CITED4 promoter.

62. A method of diagnosing an estrogen-related condition or a propensity thereto in a subject, said method comprising, measuring the amount of a CITED mRNA or protein in a sample from said subject, an increase or decrease in said CITED mRNA or protein in said sample relative to a control sample indicating that said subject has said estrogen-related condition or a propensity thereto.

63. A method of determining the prognosis for treatment of an estrogen- related condition or a propensity thereto in a subject, said method comprising, measuring the amount of a CITED mRNA or protein in a sample from said subject, an increase or decrease in said CITED mRNA or protein in said sample relative to a control sample determining the prognosis for treatment of an estrogen-related condition or a propensity thereto in said subject.

64. The method of claim 62 or 63, wherein the amount of said CITED mRNA or protein in said sample is compared to the amount of CITED mRNA or protein in control samples from both subjects having said estrogen-related condition and subjects not having said estrogen-related condition.

65. The method of claim 62, wherein a decrease in said amount of a CITED mRNA or protein indicates that said estrogen-related condition is infertility or osteoporosis.

66. The method of claim 62, wherein an increase in said amount of a CITED mRNA or protein indicates that said estrogen-related condition is cancer or a gynecological disorder.

67. The method of claim 63, wherein a decrease in said amount of a CITED mRNA or protein indicates a negative prognosis for the treatment of infertility or osteoporosis.

68. The method of claim 63, wherein an increase in said amount of a CITED mRNA or protein indicates a negative prognosis for the treatment of cancer or a gynecological disorder.

69. The method of claim 66 or 68, wherein said cancer is breast, prostate, or ovarian cancer.

70. The method of claim 62 or 63, wherein said CITED protein is CITEDl, CITED2, CITED3, or CITED4.

71. The method of claim 70, wherein said CITED protein is CITEDl or CITED4.

72. The method of claim 71, wherein said CITED protein is compared using an antibody against CITED 1 or CITED4.

73. The method of claim 62 or 63, wherein said subject is a human.

74. A protein having an amino acid sequence at least 80% identical to at least 90 contiguous amino acids of zebrafish CITED3 (SEQ JD NO:2).

75. The protein of claim 74, having an amino acid sequence identical to SEQ ID NO:2.

76. A nucleic acid encoding said protein of claim 74.

77. A vector comprising said nucleic acid of claim 76.

78. An antibody that specifically recognizes a human CITED4 protein.

79. An antibody that specifically recognizes a human CITEDl protein.