TRANSCRIPTIONAL CO-REPRESSOR THAT INTERACTS WITH NUCLEAR HORMONE RECEPTORS AND USES THEREFOR

Abstract: In accordance with the present invention, there are provided novel receptor interacting factors, referred to herein as "SMRT", i.e., a Silencing Mediator (co-repressor) for Retionic Acid Receptor (RAR) and Thyroid hormone Receptor (TR). SMRT is anovel protein whose association with RAR and TR both in solution and on DNA response elements is destabilized by ligand. The interaction of SMRT with mutant receptors correlates with the transcriptional silencing activites of receptors. in vivo, SMRT functions as a potent co-repressor. A GAL4 DNA binding domain (DBD) fusion of SMRT behaves as a frank repressor of a GAL4-dependent reporter. Together, these data identify a novel class of cofactor which is believed to represent an important mediator of hormone action.

Description: Transcriptional Co-repressor that Interacts with Nuclear Hormone Receptors and Uses Therefor FIELD OF THE INVENTION The present invention relates to intracellular receptors, methods for the modulation thereof, and methods for the identification of novel ligands therefor. In a particular aspect, the present invention relates to methods for the identification of compounds which function as ligands (or ligand precursors) for intracellular receptors. In another aspect, the present invention relates to novel chimeric constructs and uses therefor. BACKGROUND OF THE INVENTION A central problem in eukaryotic molecular biology continues to be the elucidation of molecules and mechanisms that mediate specific gene regulation. As part of the scientific attack on this problem, a great deal of work has been done in efforts to identify ligands (i.e., exogenous inducers) which are capable of mediating specific gene regulation. Additional work has been done in efforts to identify other molecules involved in εpecific gene regulation. Although much remains to be learned about the specifics of gene regulation, it is known that ligands modulate gene transcription by acting in concert with intracellular components, including intracellular receptors and discrete DNA sequences known as hormone response elements (HREs) . The identification of compounds which directly or indirectly interact with intracellular receptors, and thereby affect transcription of hormone-responsive genes, would be of significant value, e.g., for therapeutic applications. Transcriptional silencing mediated by nuclear receptors plays an important role in development, cell differentiation, and is directly linked to the oncogenic activity of v-erbA. The mechanism underlying this effect is unknown but is one key to understanding the molecular basis of hormone action. Accordingly, the identification of components involved in transcriptional silencing would represent a great advance in current understanding of mechanisms that mediate specific gene regulation. Other information helpful in the understanding and practice of the present invention can be found in commonly assigned United States Patent Nos. 5,071,773 and 4,981,784; and United States Patent Application Nos. 325,240, filed March 17, 1989; 370,407, filed June 22, 1989; and 438,757, filed November 16, 1989, all of which are hereby incorporated herein by reference in their entirety. BRIEF DESCRIPTION OF THE INVENTION In accordance with the present invention, we have discovered a novel receptor interacting factor, referred to herein as "SMRT", i.e., a silencing mediator (co-repressor) for retinoic acid receptor (RAR) and thyroid hormone receptor (TR) . SMRT is a novel protein whose association with RAR and TR both in solution and on DNA response elements is destabilized by ligand. The interaction of SMRT with mutant receptors correlates with the transcriptional silencing activities of receptors. In vivo , SMRT functions as a potent co-repressor. A GAL4 DNA binding domain (DBD) fusion of SMRT behaves as a frank repressor of a GAL4-dependent reporter. Together, these data identify a novel class of cofactor which is believed to represent an important mediator of hormone action. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the quantitation by phosphoi ager of a dose-dependent dissociation of SMRT from RAR or TR by all-trans retinoic acid (atRA) or thyroid hormone (triiodothyronine or T3) . Figure 2 presents amino acid (aa) sequences of SMRT (Genbank accession number XXXXX) . The aa sequence presented in parentheses (i.e., residues 1330-1376) is an alternatively spliced insert which is not present in the original two-hybrid clone (C-SMRT, aa 981 to C-terminal end) . The proline-rich N-terminal domain (aa 1-160) and the glutamine-rich region (aa 1061-1132) , as well as the ERDR and SG regions, are also indicated. The C-terminal region of SMRT (aa 1201 to C-terminal end) shows 48% aa identity to RIP13 (Seol et al., Molecular Endocrinology 9:72-85 (1995)) . The rest of the sequence of RIP13 shows 22% aa identity to SMRT (aa 819-1200) . Figure 3 illustrates mediation of the silencing effect of hRARα and hTR? by SMRT in vivo . Figure 3 (A) illustrates that v-erbA reverses the silencing effect of GAL-RAR (GAL4 DBD-hRAR 156-462) while SMRT restores the silencing effect. Figure 3(B) illustrates that the RAR403 truncation mutant reverses the silencing effect of GAL-TR (GAL4 DBD-hTR? 173-456) while SMRT restores the silencing effect. Figure 3(C) illustrates that v-erbA and full length SMRT or C-SMRT have no effect on GAL-VP16 activity. Figure 3(D) illustrates that a GAL4 DBD fusion of full length SMRT suppresses the thymidine kinase basal promoter activity containing four GAL4 binding sites. The fold of repression was calculated by dividing the normalized luciferase activity transfected with the GAL4 DBD alone by those transfected with indicated amount of GAL DBD fusion constructs. DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, there are provided co-suppressors of steroid/thyroid hormone receptor activity, said co-suppressors having a structure and function characteristic of the silencing mediator for retinoic acid receptor and thyroid hormone receptor. Co-suppressors contemplated by the present invention have substantially the same sequence as residues 1-1329 plus 1376-1495, as set forth in SEQ ID NO:l, optionally further comprising the amino acid residues set forth in SEQ ID NO:2 (i. e., residues 1330-1375 of SEQ ID NO:l) . The phrase "substantially the same" is used herein in reference to the nucleotide sequence of DNA, the ribonucleotide sequence of RNA, or the amino acid sequence of protein, that have slight and non-consequential sequence variations from the actual sequences disclosed herein. Species that are substantially the same are considered to be equivalent to the disclosed sequences and as such are within the scope of the appended claims. In this regard, "slight and non-consequential sequence variations" mean that sequences that are substantially the same as the DNA, RNA, or proteins disclosed and claimed herein are functionally equivalent to the sequences disclosed and claimed herein. Functionally equivalent sequences will function in substantially the same manner to produce substantially the same compositions as the nucleic acid and amino acid compositions disclosed and claimed herein. In particular, functionally equivalent DNAs encode proteins that are the same as those disclosed herein or that have conservative amino acid variations, such as substitution of a non-polar residue for another non-polar residue or a charged residue for a similarly charged residue. These changes include those recognized by those of skill in the art as those that do not substantially alter the tertiary structure of the protein. In accordance with another aspect of the present invention, there are provided antibodies raised against the above-described co-suppressor. Such antibodies can be employed for studying tissue localization of invention co- repressor, the structure of functional domains, the purification of receptors, as well as in diagnostic applications, therapeutic applications, and the like. Preferably, for therapeutic applications, the antibodies employed will be monoclonal antibodies. The above-described antibodies can be prepared employing standard techniques, as are well known to those of skill in the art, using the invention co-repressor or portions thereof as antigens for antibody production. Both anti-peptide and anti-fusion protein antibodies can be used [see, for example, Bahouth et al. (1991) Trends Pharmacol Sci. vol. 11:338-343; Current Protocols in Molecular Biology (Ausubel et al., eds.) John Wiley and Sons, New York (1989)]. Factors to consider in selecting portions of invention co-repressor for use as immunogen (as either a synthetic peptide or a recombinantly produced bacterial fusion protein) include antigenicity, accessibility (i.e., where the selected portion is derived from, e.g., the ligand binding domain, DNA binding domain, dimerization domain, and the like) , uniqueness of the particular portion selected (relative to known receptors and co-suppressors therefor) , and the like. In accordance with yet another aspect of the present invention, there are provided methods to block the repressing effect of invention co-suppressors, said method comprising administering an effective amount of an antibody as described herein. Alternatively, a silencing domain of a nuclear receptor can be employed. Those of skill in the art can readily determine suitable methods for administering said antibodies, and suitable quantities for administration, which will vary depending on numerous factors, such as the indication being treated, the condition of the subject, and the like. In accordance with a still further aspect of the invention, there are provided isolated polynucleic acids encoding the above-described co-suppressor. In addition, there are also provided vectors containing the above- described polynucleic acid. In accordance with a still further aspect of the present invention, there are provided complexes comprising the above-described co-suppressor and a homodimeric or heterodimeric member of the steroid/thyroid hormone superfamily of receptors, wherein said member contains a silencing domain which represses basal level promoter activity of target genes. Homodimeric or heterodimeric members of the steroid/thyroid hormone superfamily of receptors contemplated for use herein include thyroid hormone receptor homodimer, thyroid hormone receptor- retinoid X receptor heterodimer, retinoic acid receptor homodimer, retinoic acid receptor-retinoid X receptor heterodimer, retinoid X receptor homodimer, and the like. The above-described complexes optionally further comprise a response element for the member of the steroid/thyroid hormone superfamily of receptors. Such response elements are well known in the art. Thus, for example, RAR response elements are composed of at least one direct repeat of two or more half sites separated by a spacer of five nucleotides. The spacer nucleotides can independently be selected from any one of A, C, G or T. Each half site of response elements contemplated for use in the practice of the invention comprises the sequence -RGBNNM-, wherein R is selected from A or G; B iε selected from G, C, or T; each N is independently selected from A, T, C, or G; and M iε selected from A or C; with the proviso that at least 4 nucleotides of said -RGBNNM- sequence are identical with the nucleotideε at correεponding positions of the sequence -AGGTCA-. Response elements employed in the practice of the present invention can optionally be preceded by N χ , wherein x falls in the range of 0 up to 5. Similarly, TR response elements can be composed of the same half site repeats, with a spacer of four nucleotides. Alternatively, palindromic constructs as have been described in the art are alεo functional aε TR reεponεe elementε. The above-described co-represεor/dimeric receptor complexeε can be diεsociated by contacting complex with a ligand for the member of the steroid/thyroid hormone εuperfamily of receptors. As employed herein, the term "ligand (or ligand precurεor) for a member of the steroid/thyroid hormone superfamily of receptors" (i.e., intracellular receptor) refers to a substance or compound which, in its unmodified form (or after conversion to its "active" form) , inside a cell, binds to receptor protein, thereby creating a ligand/receptor complex, which in turn can activate an appropriate hormone response element. A ligand therefore iε a compound which acts to modulate gene transcription for a gene maintained under the control of a hormone response element, and includes compounds such as hormones, growth substanceε, non-hormone compoundε that modulate growth, and the like. Ligandε include steroid or steroid-like hormone, retinoidε, thyroid hormoneε, pharmaceutically active compounds, and the like. Individual ligands may have the ability to bind to multiple receptors. Accordingly, as employed herein, "putative ligand" (also referred to aε "test compound") refers to compoundε such as εteroid or steroid-like hormones, pharmaceutically active compounds, and the like, which are suspected to have the ability to bind to the receptor of intereεt, and to modulate tranεcription of genes maintained under the control of reεponεe elementε recognized by εuch receptor. Exampleε of known ligandε include all-trans- retinoic acid (ligand for retinoic acid receptor) , 9-cis- retinoic acid (ligand for retinoid X receptor) , thyroid hormone (ligand for thyroid hormone receptor), 1,25- dihydroxy vitamin D 3 (ligand for vitamin D 3 receptor) , and the like. In accordance with another aεpect of the present invention, there iε provided a method to repress the activity of a member of the steroid/thyroid hormone superfamily of receptors containing a silencing domain which represεeε baεal level promoter activity of target genes, said method compriεing contacting εaid member of the steroid/thyroid hormone εuperfamily of receptorε with a sufficient quantity of a co-εuppreεεor aε described hereinabove so as to repress the activity of said member. Members of the superfamily contemplated for repression in accordance with thiε aεpect of the preεent invention include thyroid hormone receptor, retinoic acid receptor, vitamin D receptor, peroxiεome proliferator activated receptor, and the like. In accordance with yet another aεpect of the present invention, there is provided a method to identify compounds which relieve the suppression of steroid/thyroid hormone receptor activity caused by a co-suppressor as described hereinabove, said method comprising comparing the size of the above-described co-suppressor/dimeric receptor complex (i.e., complexeε compriεing the above-deεcribed co- suppressor and a homodimeric or heterodimeric member of the steroid/thyroid hormone superfamily of receptors) upon exposure to test compound, relative to the size of said complex in the absence of test compound. An observed size corresponding to intact complex iε indicative of an inactive compound, while an obεerved size that reflects dissociation of the complex is indicative of a compound that diεrupts the complex, thereby relieving the εuppreεεion caused thereby. Optionally, the complex employed in this assay further compriseε a response element for said member of the steroid/thyroid hormone superfamily of receptors. The εize of the above-described complex can readily be determined employing various techniques available in the art. For example, electrophoretic mobility εhift aεsayε (EMSA) can be employed (wherein receptor alone or receptor-co-suppreεsor complex is bound to target DNA and the relative mobility thereof determined) . Those of skill in the art can readily identify other methodology which can be employed to determine the εize of the complex aε a result of exposure to putative ligand. In accordance with a still further aspect of the present invention, there iε provided a method to identify compoundε which relieve the εuppreεsion of steroid/thyroid hormone receptor activity cauεed by a co-εuppressor as described hereinabove, without subεtantially activating said receptor, said method comprising: comparing the reporter εignal produced by two different expreεεion εyεtemε in the abεence and preεence of teεt compound, wherein εaid firεt expreεεion εyεtem compriεes a complex comprising: a homodimeric or heterodimeric member of the steroid/thyroid hormone superfamily of receptorε εelected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer, or retinoic acid receptor-retinoid X receptor heterodimer, a response element for said member of the εteroid/thyroid hormone εuperfamily of receptorε, wherein εaid reεponεe element is operatively linked to a reporter, and optionally, invention co-εuppreεεor, and wherein εaid second expression system comprises a complex comprising: a homodimeric or heterodimeric form of the same member of the εteroid/thyroid hormone εuperfamily of receptors as employed in said firεt expreεεion εystem, wherein said member is mutated such that it retains hormone dependent activation activity but has lost itε ability to repress baεal level promoter activity of target genes, the same responεe element-reporter combination aε employed in εaid first expresεion εyεtem, and optionally, invention co-εuppreεεor, and thereafter εelecting thoεe compounds which provide: a higher reporter signal upon exposure of said compound to said first expression system, relative to reporter signal in the absence of said compound, and substantially the same reporter signal upon exposure of said compound to said second expression system, relative to reporter signal in the absence of said compound, wherein said selected compoundε are capable of relieving the εuppreεεion of steroid/thyroid hormone receptor activity caused by a co-suppressor having a structure and function characteriεtic of the silencing mediator for retinoic acid and thyroid receptors, but εubεtantially lacking the ability to activate εteroid/thyroid hormone receptor activity. The addition of invention co-εuppreεsor iε optional in the above-deεcribed aεεay because it is present endogenously in most host cells employed for such assays. It is preferred, to enεure the presence of a fairly conεtant amount of co-suppressor, and to ensure that co- suppreεεor iε not a limiting reagent, that co-εuppressor be supplied exogenously to the above-deεcribed aεεayε. Mutant receptors contemplated for use in the practice of the present invention are conveniently produced by expression plasmids, introduced into the host cell by transfection. Mutant receptors contemplated for use herein include RAR403 homodimers, RAR403-containing heterodimers, TR160 homodimers, TR160-containing heterodimers, and the like. Reporter constructs contemplated for use in the practice of the present invention comprise: (a) a promoter that is operable in the host cell, (b) a hormone response element, and (c) a DNA segment encoding a reporter protein, wherein the reporter protein-encoding DNA segment is operatively linked to the promoter for tranεcription of the DNA εegment, and wherein the hormone reεponεe element iε operatively linked to the promoter for activation thereof. Hormone reεponεe elementε contemplated for uεe in the practice of the preεent invention are well known in the art, as has been noted previouεly. Exemplary reporter genes include chloramphenicol transferase (CAT) , luciferase (LUC) , beta-galactosidase (β-gal ) , and the like. Exemplary promoters include the simian virus (SV) promoter or modified form thereof (e.g., ΔSV) , the thymidine kinase (TK) promoter, the mammary tumor virus (MTV) promoter or modified form thereof (e.g., ΔMTV) , and the like [see, for example, Mangelεdorf et al., in Nature 115:224-229 (1990) , Mangelεdorf et al., in Cell j56_:555-561 (1991) , and Berger et al., in J. Steroid Biochem. Molec. Biol. 4J.:733-738 (1992) ]. As used herein in the phrase "operative response element functionally linked to an operative reporter gene", the word "operative" means that the respective DNA sequences (represented by the termε "GAL4 reεponεe element" and "reporter gene") are operational, i.e., work for their intended purpoεeε; the word "functionally" means that after the two segments are linked, upon appropriate activation by a ligand-receptor complex, the reporter gene will be expressed as the result of the fact that the "GAL4 response element" was "turned on" or otherwise activated. In practicing the above-described functional bioassay, the expresεion plaεmid and the reporter plasmid are co-transfected into εuitable host cells. The transfected host cells are then cultured in the presence and absence of a teεt compound to determine if the teεt compound is able to produce activation of the promoter operatively linked to the response element of the reporter plasmid. Thereafter, the transfected and cultured hoεt cells are monitored for induction (i.e., the presence) of the product of the reporter gene sequence. Any cell line can be uεed as a suitable "hoεt" for the functional bioaεεay contemplated for uεe in the practice of the preεent invention. Thuε, cells contemplated for use in the practice of the present invention include transformed cells, non-transformed cells, neoplastic cellε, primary cultures of different cell types, and the like. Exemplary cells which can be employed in the practice of the present invention include Schneider cells, CV-1 cells, HuTuδO cells, F9 cellε, NTERA2 cells, NB4 cells, HL-60 cells, 293 cells, Hela cells, yeast cellε, and the like. Preferred host cellε for uεe in the functional bioassay syεtem are COS cellε and CV-l cellε. COS-l (referred to aε COS) cellε are monkey kidney cellε that expreεε SV40 T antigen (Tag) ; while CV-1 cells do not express SV40 Tag. The presence of Tag in the COS-l derivative lines allows the introduced expresεion plaεmid to replicate and provideε a relative increaεe in the amount of receptor produced during the assay period. CV-1 cells are presently preferred because they are particularly convenient for gene transfer εtudies and provide a sensitive and well-described host cell system. The above-described cellε (or fractionε thereof) are maintained under physiological conditions when contacted with physiologically active compound. "Physiological conditions" are readily understood by those of skill in the art to comprise an isotonic, aqueouε nutrient medium at a temperature of about 37°C. In accordance with yet another aεpect of the present invention, there is provided a method to identify compounds which activate steroid/thyroid hormone receptor activity, but substantially lack the ability to relieve the suppression caused by a co-εuppressor as described hereinabove, said method comprising: comparing the reporter signal produced by two different expression εyεtems in the absence and presence of test compound, wherein εaid firεt expreεεion εyεtem compriεes a complex compriεing: a homodimeric or heterodimeric member of the εteroid/thyroid hormone εuperfamily of receptorε εelected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer, or retinoic acid receptor-retinoid X receptor heterodimer, a reεponεe element for εaid member of the εteroid/thyroid hormone εuperfamily of receptors, wherein εaid response element is operatively linked to a reporter, and optionally, invention co-suppressor, and wherein said second expreεεion εystem comprises a complex comprising: a homodimeric or heterodimeric form of the same member of the steroid/thyroid hormone superfamily of receptors as employed in said first expresεion εyεtem, wherein said member is mutated such that it retains hormone dependent activation activity but has lost its ability to repreεε baεal level promoter activity of target geneε, the εame reεponεe element-reporter combination as employed in said first expression εyεtem, and optionally, invention co-suppressor, and thereafter selecting thoεe compounds which provide: a higher reporter signal upon exposure of said compound to said second expresεion εyεtem, relative to reporter εignal in the absence of compound, and εubεtantially the same reporter signal upon exposure of said compound to said firεt expreεεion syεtem, relative to reporter εignal in the abεence of εaid compound, wherein said selected compounds are capable of activating steroid/thyroid hormone receptor activity, but εubstantially lacking the ability to relieve the suppression caused by a co-suppreεsor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptors. In accordance with a still further aspect of the present invention, there is provided a method to identify compounds which relieve the suppression of steroid/thyroid hormone receptor activity caused by a co-suppressor as described hereinabove, and activate said receptor, said method comprising: comparing the reporter signal produced by two different expression systems in the absence and presence of test compound, wherein said first expression syεtem comprises a complex comprising: a homodimeric or heterodimeric member of the steroid/thyroid hormone εuperfamily of receptors selected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer, or retinoic acid receptor-retinoid X receptor heterodimer, a response element for said member of the steroid/thyroid hormone εuperfamily of receptors, wherein said reεponεe element is operatively linked to a reporter, and optionally, invention co-suppreεεor, and wherein εaid εecond expreεεion system comprises a complex comprising: a homodimeric or heterodimeric form of the εame member of the εteroid/thyroid hormone superfamily of receptors as employed in εaid firεt expression εyεtem, wherein said member is mutated εuch that it retains hormone dependent activation activity but has lost itε ability to repreεs basal level promoter activity of target genes, the same responεe element-reporter combination aε employed in said first expresεion system, and optionally, invention co-εuppreεεor, and thereafter εelecting thoεe compounds which provide: increased reporter signal upon exposure of said compound to said εecond expreεsion system, relative to reporter signal in the absence of said compound, and substantially increased reporter signal upon exposure of said compound to said first expresεion εyεtem, relative to reporter εignal in the abεence of εaid compound, wherein εaid εelected compoundε are capable of relieving the εuppreεεion of εteroid/thyroid hormone receptor activity caused by a co-suppreεεor having a εtructure and function characteriεtic of the εilencing mediator for retinoic acid and thyroid receptors, and activating said receptor. In accordance with still another embodiment of the present invention, there are provided modified forms of the above-described co-suppreεsor, including: full length silencing mediator for retinoic acid and thyroid receptors pluε GAL4 DNA binding domain, full length silencing mediator for retinoic acid and thyroid receptors plus GAL4 activation domain, full length εilencing mediator for retinoic acid and thyroid receptorε plus giutathione S-transferaεe (GST) tag, and the like. The above-described modified forms of invention co-suppreεεor can be uεed in a variety of wayε, e.g., in the assays described herein. An especially preferred modified co-suppresεor of the invention compriεeε full length silencing mediator for retinoic acid and thyroid receptors plus GAL4 activation domain. In accordance with a still further embodiment of the present invention, there is provided a method to identify compounds which disrupt the ability of a co- suppressor as described hereinabove to complex with steroid/thyroid hormone receptors, without substantially activating said receptor, εaid method comprising: comparing the reporter signal produced by two different expreεsion εystems in the absence and preεence of teεt compound, wherein εaid firεt expreεsion syεtem compriεeε a complex compriεing: a modified co-εuppreεsor as described above, a homodimeric or heterodimeric member of the steroid/thyroid hormone superfamily of receptorε εelected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer or retinoic acid receptor-retinoid X receptor heterodimer, and a response element for said member of the steroid/thyroid hormone superfamily of receptors, wherein said responεe element is operatively linked to a reporter, and wherein said εecond expression system comprises a complex comprising: said modified co-suppressor, a homodimeric or heterodimeric form of the same member of the steroid/thyroid hormone superfamily of receptors as employed in said first expresεion εystem, wherein said member is mutated such that it retains hormone dependent activation activity but haε lost its ability to represε baεal level promoter activity of target genes, and the same response element-reporter combination as employed in said first expresεion εystem, and thereafter selecting those compounds which provide: a lower reporter signal upon exposure of said compound to said first expression system, relative to reporter signal in the abεence of said compound, and substantially the same reporter signal upon exposure of said compound to said second expression system, relative to reporter signal in the absence of said compound, wherein said selected compoundε are capable of disrupting the ability of a co-suppressor having a structure and function characteristic of the εilencing mediator for retinoic acid and thyroid receptorε to complex with εteroid/thyroid hormone receptorε, without εubεtantially activating εaid receptor. Mutant receptorε contemplated for use in this embodiment of the preεent invention include RAR403 homodimerε, RAR403-containing heterodimerε, TR160 homodimers, TR160-containing heterodimers, and the like. Suitable host cells for uεe in this embodiment of the present invention include mammalian cells as well as yeast cells. Yeast cells are presently preferred because they introduce no background since SMRT (i.e., εilencing mediator (co-repressor) for retinoic acid receptor (RAR) and thyroid hormone receptor (TR) ) iε not endogenous to yeast. In accordance with yet another embodiment of the present invention, there is provided a method to identify compounds which activate steroid/thyroid hormone receptor activity, but εubεtantially lack the ability to diεrupt a complex compriεing a εteroid/thyroid hormone receptor and a co-suppresεor aε deεcribed hereinabove, εaid method comprising: comparing the reporter signal produced by two different expression systemε in the absence and presence of test compound, wherein said first expresεion system comprises a complex comprising: a modified co-suppresεor aε deεcribed above, a homodimeric or heterodimeric member of the steroid/thyroid hormone superfamily of receptors selected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer or retinoic acid receptor-retinoid X receptor heterodimer, and a response element for εaid member of the εteroid/thyroid hormone εuperfamily of receptorε, wherein εaid response element is operatively linked to a reporter, and wherein εaid εecond expreεεion εystem comprises: said modified co-suppressor, a homodimeric or heterodimeric form of the same member of the steroid/thyroid hormone superfamily of receptorε as employed in said first expresεion εyεtem, wherein said member is mutated such that it retains hormone dependent activation activity but has lost its ability to repress basal level promoter activity of target genes, and the same response element-reporter combination as employed in said first expresεion εystem, and thereafter selecting thoεe compoundε which provide: a higher reporter εignal upon exposure of said compound to said second expression system, relative to reporter signal in the absence of compound, and subεtantially the same reporter signal upon exposure of εaid compound to εaid first expression system, relative to reporter signal in the absence of compound, wherein εaid εelected comoundε are capable of activating εteroid/thyroid hormone receptor activity, but εubεtantially lack the ability to diεrupt the complex of a co-εuppreεεor having a εtructure and function characteriεtic of the εilencing mediator for retinoic acid and thyroid receptorε and a steroid/thyroid hormone receptor. Suitable hoεt cellε for uεe in thiε embodiment of the present invention include mammalian cells as well as yeast cells. Yeast cells are presently preferred because they introduce no background since SMRT is not endogenous to yeast. In accordance with a εtill further embodiment of the present invention, there is provided a method to identify compounds which activate a steroid/thyroid hormone receptor, and disrupt the ability of a co-εuppreεεor as described hereinabove to complex with said receptor, said method comprising: comparing the reporter εignal produced by two different expreεsion systemε in the absence and preεence of teεt compound, wherein εaid firεt expression system comprises a complex comprising: a modified co-εuppressor as described above, a homodimeric or heterodimeric member of the steroid/thyroid hormone εuperfamily of receptorε εelected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer or retinoic acid receptor-retinoid X receptor heterodimer, and a reεponεe element for said member of the steroid/thyroid hormone εuperfamily of receptorε, wherein εaid response element is operatively linked to a reporter, and wherein said second expression system comprises a complex comprising: said modified co-suppresεor, the εame homodimeric or heterodimeric member of the εteroid/thyroid hormone superfamily of receptors as employed in said first expression system, wherein said member is mutated εuch that it retainε hormone dependent activation activity but has lost its ability to repreεε baεal level promoter activity of target genes, and the same responεe element-reporter combination aε employed in said first expresεion εyεtem, and thereafter selecting thoεe compoundε which provide: a reduction in reporter signal upon exposure of compound to said first expression system, relative to reporter signal in the abεence of εaid compound, and increased reporter εignal upon expoεure of compound to said second expression system, relative to reporter signal in the absence of said compound, wherein said selected compounds are capable of activating a steroid/thyroid hormone receptor and diεrupting a complex compriεing εteroid/thyroid hormone receptor and a co-εuppreεεor having a εtructure and function characteristic of the silencing mediator for retinoic acid and thyroid receptorε. Suitable hoεt cellε for uεe in this embodiment of the preεent invention include mammalian cells as well as yeast cells. Yeaεt cellε are presently preferred because they introduce no background since SMRT iε not endogenous to yeast. In accordance with yet another aεpect of the preεent invention, there iε provided a method to identify compoundε which activate a εteroid/thyroid hormone receptor and/or diεrupt the ability of a co-εuppreεsor aε deεcribed hereinabove to complex with εaid receptor, said method compriεing: comparing the reporter signals produced by a combination expresεion system in the absence and presence of test compound, wherein said combination expression system comprises: a first homodimeric or heterodimeric member of the steroid/thyroid hormone superfamily of receptors selected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer, or retinoic acid receptor- retinoid X receptor heterodimer, a εecond homodimeric or heterodimeric form of the same member of the steroid/thyroid hormone εuperfamily of receptorε aε employed in εaid first homodimer or heterodimer, wherein said member is mutated εuch that it retains hormone dependent activation activity but haε loεt itε ability to repreεε baεal level promoter activity of target geneε (i.e., provides basal level expression) , wherein either said first homodimer (or heterodimer) or said second homodimer (or heterodimer) is operatively linked to a GAL4 DNA binding domain, a responεe element for εaid member of the εteroid/thyroid hormone superfamily of receptorε, wherein said responεe element is operatively linked to a first reporter, a GAL4 responεe element, wherein εaid reεponse element is operatively linked to a second reporter, and optionally a co-suppressor of steroid/thyroid hormone receptor activity, said co-suppressor having a structure and function characteriεtic of the εilencing mediator for retinoic acid and thyroid receptorε, and thereafter identifying aε capable of relieving the suppreεεion of steroid/thyroid hormone receptor activity caused by a co-suppressor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptorε, but subεtantially lacking the ability to activate steroid/thyroid hormone receptor activity those compoundε which provide: a higher reporter εignal from the reporter reεponεive to the firεt member upon exposure of said compound to εaid firεt member, relative to reporter εignal in the absence of said compound, and εubεtantially the εame reporter εignal from the reporter reεponsive to the second member upon exposure of εaid compound to εaid εecond member, relative to reporter εignal in the absence of said compound, or identifying aε capable of activating steroid/thyroid hormone receptor activity, but subεtantially lacking the ability to relieve the suppreεsion cauεed by a co-εuppreεεor having a εtructure and function characteristic of the silencing mediator for retinoic acid and thyroid receptors those compounds which provide: a higher reporter signal from the reporter responsive to the εecond member upon exposure of said compound to said εecond member, relative to reporter εignal in the abεence of compound, and subεtantially the εame reporter εignal from the reporter reεponεive to the firεt member upon expoεure of said compound to said first member, relative to reporter signal in the absence of said compound, or identifying as capable of relieving the suppresεion of steroid/thyroid hormone receptor activity caused by a co-suppresεor having a structure and function characteriεtic of the silencing mediator for retinoic acid and thyroid receptorε, and activating εaid receptor those compounds which provide: a higher reporter εignal from the reporter reεponεive to the εecond member upon exposure of said compound to said second member, relative to reporter signal in the absence of said compound, and a greater increase in reporter signal from the reporter reεponsive to the first member upon expoεure of εaid compound to εaid firεt member, relative to reporter signal in the absence of said compound. Thus, the change in expression level of the two different reporters introduced in a single transfection can be monitored εimultaneouεly. Baεed on the reεultε of this single transfection, one can readily identify the mode of interaction of test compound with the receptor/SMRT complex. Exemplary GAL4 response elements are thoεe containing the palindromic 17-mer: 5'-CGGAGGACTGTCCTCCG-3 ' (SEQ ID NO: 3) , εuch aε, for example, 17MX, aε deεcribed by Webεter et al., in Cell 5_2: 169-178 (1988) , aε well aε derivativeε thereof. Additional examples of suitable responεe elements include those deεcribed by Hollenberg and Evanε in Cell 55:899-906 (1988) ; or Webster et al. in Cell .54:199-207 (1988) . In accordance with still another embodiment of the preεent invention, there iε provided a method to identify compounds which activate a steroid/thyroid hormone receptor and/or diεrupt the ability of a co-suppresεor aε described hereinabove to complex with said receptor, said method comprising: comparing the reporter signals produced by a combination expression system in the absence and presence of test compound, wherein εaid combination expreεεion εyεtem compriεeε: a modified co-εuppreεsor aε deεcribed above, a first homodimeric or heterodimeric member of the steroid/thyroid hormone superfamily of receptors selected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer, or retinoic acid receptor- retinoid X receptor heterodimer, a second homodimeric or heterodimeric form of the εame member of the εteroid/thyroid hormone superfamily of receptors as employed in said first homodimer or heterodimer, wherein εaid member iε mutated εuch that it retainε hormone dependent activation activity but haε lost its ability to represε baεal level promoter activity of target geneε, wherein either εaid firεt homodimer (or heterodimer) or εaid εecond homodimer (or heterodimer) is operatively linked to a GAL4 DNA binding domain, a responεe element for εaid member of the εteroid/thyroid hormone εuperfamily of receptorε, wherein said responεe element is operatively linked to a first reporter, a GAL4 response element, wherein said response element is operatively linked to a εecond reporter, and thereafter identifying aε capable of diεrupting the ability of a co-suppressor having a structure and function characteriεtic of the εilencing mediator for retinoic acid and thyroid receptorε to complex with a steroid/thyroid hormone receptor, without substantially activating steroid/thyroid hormone receptor, those compoundε which provide: a lower reporter εignal from the reporter responsive to the first member upon exposure of said compound to εaid firεt member, relative to reporter εignal in the absence of said compound, and subεtantially the same reporter signal from the reporter responεive to the second member upon exposure of said compound to said εecond member, relative to reporter εignal in the abεence of said compound, or identifying as capable of activating steroid/thyroid hormone receptor activity, but εubεtantially lacking the ability to diεrupt a complex compriεing a εteroid/thyroid hormone receptor and a co- suppressor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptorε, those compounds which provide: a higher reporter signal from the reporter responsive to the second member upon exposure of said compound to said εecond member, relative to reporter signal in the absence of compound, and substantially the same reporter signal from the reporter responεive to the firεt member upon expoεure of said compound to said first member, relative to reporter εignal in the absence of said compound, or identifying as capable of disrupting a complex comprising a εteroid/thyroid hormone receptor and a co- suppressor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptorε, and activating said receptor those compounds which provide: a reduction in reporter signal from the reporter responsive to the first member upon exposure of said compound to εaid firεt member, relative to reporter εignal in the absence of said compound, and increased reporter εignal from the reporter reεponsive to the second member upon exposure of said compound to εaid second member, relative to reporter εignal in the abεence of said compound. In accordance with a still further aspect of the present invention, there is provided a method to identify compounds which relieve the suppresεion of steroid/thyroid hormone receptor activity caused by a co-suppressor as described hereinabove, said method comprising determining the effect of adding test compound to an expression system compriεing: a modified member of the εteroid/thyroid hormone superfamily of receptors, wherein said modified member contains an activation domain which renders said receptor constitutively active, a fusion protein comprising the receptor interaction domain of SMRT operatively linked to the GAL4 DNA binding domain, and a GAL4 responεe element operatively linked to a reporter. Prior to addition of an effective ligand for the member of the εteroid/thyroid hormone εuperfamily of receptorε employed herein, the association of the modified member and the fusion protein will be effective to bind the GAL4 responεe element and activate tranεcription of the reporter. The presence of an effective ligand is indicated by a reduction of reporter εignal upon exposure to ligand, which disrupts the interaction of the modified member and fusion protein. Activation domainε contemplated for uεe in the practice of the present invention are well known in the art and can readily be identified by the artisan. Examples include the GAL4 activation domain, BP64 , and the like. To summarize, a novel nuclear receptor co-repreεεor which mediates the transcriptional silencing of RAR and TR has been identified. This discovery is of great interest becauεe tranεcriptional εilencing haε been shown to play an important role in development, cell differentiation and the oncogenic activity of v-erbA (Baniah ad et al., EMBO J . 11:1015-1023 (1992)) ; Gandrillon et al., Cell 49:687-697 (1989)) ; Zenke et al., Cell 61:1035-1049 (1990) ; Barlow et al., EMBO J . 13:4241-4250 (1994) ; Levine and Manley, Cell 59:405-408 (1989); Baniahmad et al., Proc . Natl . Acad . Sci . USA 89:10633-10637 (1992b) ; and Saitou et al., Nature 374:159-162 (1995)) . In fact, v-erbA mutants that harbor the Prol60->Arg change in the TR neither suppress basal tranεcription nor are capable of oncogenic tranεformation (Damm and Evanε, (1993) supra) . The function of SMRT as a εilencing mediator (co-repressor) of RAR and TR is analogous to mSin3 in the Mad-Max-Sin3 ternary complex (Schreiber-Agus et al., Cell 80:777-786 (1995) ; and Ayer et al., Cell 80:767-776 (1995)) . Because GAL-SMRT functions as a potent repressor when bound to DNA, it is reaεonable to speculate that the function of the unliganded receptors is to bring with them SMRT to the template via protein-protein interaction. Thus, the repressor function is intrinsic to SMRT as opposed to the TR or RAR itself (Baniahmad et al., Proc . Natl . Acad . Sci . USA 90:8832-8836 (1993); and Fondell et al., Genes Dev 7:1400-1410 (1993)) . It iε demonstrated herein that the ligand triggerε a dissociation of SMRT from the receptor, which would lead to an initial step in the activation procesε. Thiε would be followed (or be coincident) with an induced conformational change in the carboxy-terminal tranεactivation domain (TC, alεo called AF-2) , allowing association with co-activatorε on the transcription machinery (Douarin et al., EMBO J . 14:2020-2033 (1995); Halachmi et al., Science 264:1455-1458 (1994) ; Lee et al., Nature 374:91-94 (1995) ; and Cavailles et al., Proc . Natl . Acad . Sci . USA 91:10009-10013 (1994)) . Thuε, as has previouεly been εuggeεted (Damm and Evans (1993) supra) , the ligand dependent activation of TR would represent two εeparable proceεses including relief of repression and net activation. The isolation of SMRT now provides a basis for dissecting the molecular basis of trans-represεion. The invention will now be deεcribed in greater detail by reference to the following non-limiting exampleε. Example 1 Iεolation of SMRT Using a GAL4 DBD-RXR fusion protein (see, for example, USSN 08/177,740, incorporated by reference herein in its entirety) as a bait in a yeast two-hybrid screening system (Durfee et al. Genes Dev 7:555-569 (1993)), several cDNA clones encoding receptor interacting proteins were isolated. One of these proteins, SMRT, interacts strongly with unliganded RAR and TR but only weakly with RXR or other receptors in yeast. This protein was selected for further characterization. Example 2 Far-western blotting procedure Total bacteria extracts expresεing GST fuεions of hRAR (aa 156-462) or hRXRα LBD (aa 228-462) and control extracts expresεing GST alone or GST-PML fuεion protein were εubjected to SDS/PAGE and electroblotted onto nitrocellulose in transfer buffer (25 M Tris, pH 8.3/ 192 mM glycine/ 0.01% SDS) . After denaturation/renaturation from 6 M to 0.187 M guanidine hydrochloride in HB buffer (25 mM Hepes, pH 7.7/25 mM NaCl/5 mM MgCl 2 /lmM DTT) filters were saturated at 4°C in blocking buffer (5% milk, then 1% milk in HB buffer plus 0.05% NP40) . In vitro translated S-labeled proteinε were diluted into H buffer (20 M Hepeε, pH 7.7/75 mM KCl/0.1 mM EDTA/2.5 mM MgCl 2 /0.05% NP40/ 1% milk/1 mM DTT) and the filters were hybridized overnight at 4°C with (1 μM) or without ligand. After three washes with H buffer, filters were dried and exposed for autoradiography or quantitated by phosphoimager. GST-SMRT is a GST fuεion of the C-SMRT encoded by the yeaεt two hybrid clone. GST-SMRT haε been purified, but containε εeveral degradation productε. For yeast two-hybrid screening, a construct expressing the GAL4 DBD-hRXRα LBD (aa 198-462) fusion protein was used to εcreen a human lymphocyte cDNA library as described (Durfee et al., (1993) supra.. Full length SMRT cDNA was isolated from a human HeLa cDNA library (Clontech) using the two-hybrid insert as a probe. Using the above-deεcribed far-western blotting procedure, S-labeled SMRT preferentially complexes with bacterial extracts expressing the RAR, marginally associates with RXR and shows no association with control extracts. In contrast, S-PPAR selectively associates with its heterodimeric partner, RXR, but not with RAR. In a similar asεay, S-labeled RAR or TR interactε εtrongly with SMRT and their heterodimeric partner, RXR, but not with degraded GST productε, while 35S-RXR interacts only weakly with SMRT. Binding of ligand to RAR or TR reduces their interactions with SMRT but not with RXR, while binding of ligand to RXR haε only εlight effect. Figure 1 shows the quantitation of a dose-dependent dissociation of SMRT from RAR or TR by all-trans retinoic acid (atRA) or thyroid hormone (triiodothyronine or T3) , demonstrating that the amount of ligand required for 50% dissociation in both cases are close to the kds for both ligands (Munoz et al. EMBO J . 7:155-159 (1988); Sap et al., Nature 340:242-244 (1989) ; and Yang et al., Proc . Natl . Acad . Sci . USA 88:3559-3563 (1991) ) . Full length SMRT encodes a polypeptide of 1495 amino acidε rich in proline and serine residues (see Figure 2 and SEQ ID NO:l) . Genbank database compariεon reveals similarity of the C-terminal domain of SMRT to a partial cDNA encoding another receptor interacting protein, RIP13 (Seol et al., (1995) supra) , whose role in receptor signaling is unknown. Within this region, there can be identified εeveral potential heptad repeatε which might mediate protein-protein interaction with the "a-helical sandwich" structure (Bourguet et al., Nature 375:377-382 (1995)) of the ligand binding domain (LBD) of receptors. Example 3 Characterization of SMRT Unlike other nuclear receptors, unliganded RAR and TR possesε a εtrong εilencing domain which represses basal level promoter activity of their target genes (Damm et al., Nature 339:593-597 (1989) ; Brent et al., New Biol . 1:329-336 (1989) ; Baniahmad et al. , Cell 61:505-514 (1990); and Baniahmad et al., EMBO J . 11:1015-1023 (1992)) . The preferential interaction of SMRT with RAR and TR in the absence of hormone suggestε that SMRT may play a role in mediating the tranεcriptional silencing effect of the receptor. To further investigate the involvement of SMRT in silencing, the interaction of SMRT with mutant receptors which display diεtinct εilencing and/or tranεactivation activitieε waε teεted aε followε. S-methionine labeled receptors were used as probes to hybridize immobilized GST-SMRT in the presence (10 μM) or absence of all-trans retinoic acid (atRA) . The total bacteria extract expresεing GST-RXR waε included aε a control. When quantitated by phoεphoimager, RAR403 shows a 4-fold better interaction with SMRT than wild type RAR. Both full length RAR or a deletion mutant expreεεing only the ligand binding domain (LBD, referred to aε ΔΔR) aεεociate with SMRT; this association is blocked by ligand. Theεe reεultε confirm that the LBD alone is sufficient in the interaction. The carboxy-terminal deletion mutant RAR403 is a potent dominant negative suppressor of basal level promoter activity of RAR target genes (Damm et al., Proc . Natl . Acad . Sci . USA 90:2989-2993 (1993) ; Tsai and Collins, Proc . Natl . Acad . Sci . USA 90:7153-7157 (1993); and Tsai et al., Genes Dev 6:2258-2269 (1992)). Aε might be predicted from the above studies, RAR403 and its amino terminal deletion derivative, ΔΔR403, interact strongly with SMRT in either the presence or absence of ligand, consistent with SMRT mediating the repressor activity of this mutant. Example 4 Interaction of SMRT with TR Mutants The interaction of SMRT with two different classes of TR mutants was analyzed next. The first mutant employed is the naturally occurring oncogene, v-erbA, which has strong εilencing ability but no tranεactivation activity (Sap et al., (1989) supra; Sap et al., Nature 324:635-640 (1986); Weinberger et al., Nature 318:670-672 (1985); and Weinberger et al., Nature 324:641-646 (1986)). The second mutant employed is a single amino acid change (Pro 160 -> Arg) of the rTRa (TR160) which has previously been shown to loεe itε capacity in baεal level εuppression but retains hormone dependent transactivation (Thompson et al., Science 237:1610-1614 (1987) ; and Damm and Evans, Proc . Natl . Acad . Sci . USA 90:10668-10672 (1993)). If SMRT iε involved in silencing, it would be expected that SMRT εhould interact with the v-erbA, but εhow little or no association with the εilencing-defective TR160 mutant. Interaction of the oncogenic v-erbA and rTRα R160 mutant (TR160) with GST-SMRT was determined in a far-western asεay as described above (see Example 2) . When quantitated by phosphoimager, the v-erbA showε an 18-fold better interaction with SMRT than hTR?, and the TR160 mutant shows a 10-fold lower signal than the rTRα. Aε one might expect, v-erbA interactε εtrongly with SMRT both in preεence or absence of ligand. In contrast, full length TR160 mutant or LBD of TR160 (ΔΔTR160) does not interact εignificantly with SMRT when compared to the wild type receptor. Theεe data demonεtrate that SMRT plays an important role in mediating transcriptional silencing effects of both RAR and TR. These data also suggest that the release of SMRT from receptors could be a prerequisite step in ligand-dependent transactivation by nuclear receptors. Example 5 Formation of ternary complexes containing SMRT RAR and TR form heterodimers with RXR, resulting in a complex with high DNA binding ability (Bugge et al., EMBO J . 11:1409-1418 (1992) ; Yu et al., Cell 67:1251-1266 (1991); and Kliewer et al., Nature 355:446-449 (1992)). Since SMRT interactε with RAR and TR, teεtε were conducted to determine whether SMRT can alεo interact with the receptor-DNA complex. Thuε, the interaction of SMRT with RXR-RAR heterodimer on a DR5 element (i.e., an AGGTCA direct repeat spaced by five nucleotides) was determined in a gel retardation aεεay, which is carried out as follows. In vitro translated receptor or unprogram ed reticulocyte lyεate (URL) was incubated with 1 μg of poly dldC on ice for 15 minutes in a total volume of 20 μl containing 75 mM KCI, 7.5% glycerol, 20 mM Hepes (pH 7.5), 2 mM DTT and 0.1% NP-40, with or without ligand (in the range of about 10-100 32 nM employed) . A P labeled, double stranded oligonucleotide probe was added into the binding reaction (10,000 cpm per reaction) , and the reaction waε further incubated for 20 minuteε at room temperature. The protein-DNA complex waε separated on a 5% native polyacrylamide gel at 150 volts. SMRT is εeen to form a ternary complex with the RXR-RAR heterodimer on a DNA reponse element in the gel retardation asεay. Addition of ligand releases SMRT from this complex in a dose-dependent manner. Similarly, SMRT is seen to form a ternary complex with the RXR-TR heterodimer on a TR responεe element; addition of T3 disrupts the formation of thiε complex. Theεe data demonstrate that SMRT can be recruited to DNA responεe elements via protein-protein interaction with RAR or TR in the absence of hormone. Binding of hormone disrupts receptor-SMRT interaction and releases SMRT from the receptor-DNA complex. Example 6 Transient transfection assay CV-1 cells were plated in 24 well plates at a density of 50,000 cells per well. Expression plasmidε were tranεfected into cellε by lipofection uεing DOTAP. In each tranεfection, 5 ng of GAL-RAR and 15 ng of v-erbA or SMRT were uεed together with 150 ng of reporter conεtruct containing 4 copieε of GAL4 binding sites in front of a minimal thymidine kinase promoter and a CMX-/?-gal construct aε an internal control. The relative luciferaεe activity waε calculated by normalizing to the β-qal activity. Example 7 Reversal of transcriptional εilencing Recently, it has been εhown that over expreεsion of RAR or TR could reverεe the tranεcriptional εilencing effect of the GAL4 DBD fuεion of TR (GAL-TR) or RAR (GAL-RAR) (Baniahmad et al., Mol Cell Biol 15:76-86 (1995); and Caεanova et al., Mol Cell Biol 14:5756-5765 (1994)), preεumably by competition for a limiting amount of a co-repressor. A similar effect is observed herein when over expresεion of v-erbA or RAR403 mutants are shown to reverse the silencing effect of GAL-RAR and GAL-TR on the basal activity of a luciferase reporter (see Figure 3A and 3B) . In principle, over expreεεion of SMRT εhould restore repreεεor activity when co-expressed with v-erbA or RAR403 competitors. Indeed, reεults presented in Figure 3C show that both the full length and the C-terminal domain of SMRT (C-SMRT) can titrate out v-erbA or RAR403 competitor activity and re-endow GAL-RAR and GAL-TR with silencing activity. In contraεt, neither v-erbA nor SMRT εhow any effect on the tranεactivation activity of GAL-VP16 fusion. Thus, SMRT iε able to block the titration effect of v-erbA and RAR403 and functionally replaceε the putative co-repressor in this system. Example 8 Direct recruitment of SMRT to a heterologous promoter If SMRT is the mediator of transcription εilencing of TR and RAR by interaction with template-bound unliganded receptorε, then direct recruitment of SMRT to a heterologouε promoter εhould reεult in repreεεion of basal level activity. Thiε waε tested by fusing full length SMRT to the GAL4 DBD (GAL-SMRT) . The effect of the reεulting fuεion protein on the activity of the thymidine kinaεe promoter containing four GAL4 binding εiteε was analyzed. Figure 3D εhowε that GAL-SMRT, like GAL-TR, can εilence baεal promoter activity in a doεe-dependent manner. In contraεt, GAL-RXR εhows no suppreεεion. Theεe data εuggeεt that SMRT, when recruited to a promoter by direct DNA binding or via aεεociation with an unliganded receptor, functions as a potent tranεcriptional repreεεor. While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variationε are within the εpirit and εcope of that which iε described and claimed. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Evans, Ronald M. Chen, J. Don (ii) TITLE OF INVENTION: TRANSCRIPTIONAL CO-REPRESSOR THAT INTERACTS WITH NUCLEAR HORMONE RECEPTORS AND USES THEREFOR (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Pretty, Schroeder, Brueggemann & Clark (B) STREET: 444 South Flower Street, Suite 2000 (C) CITY: Los Angeles (D) STATE: CA (E) COUNTRY: USA (F) ZIP: 90071 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US 08/522,726 (B) FILING DATE: 01-SEP-1995 (C) CLASSIFICATION: (viii) ATTORNE /AGENT INFORMATION: (A) NAME: Reiter, Stephen E. (B) REGISTRATION NUMBER: 31,192 (C) REFERENCE/DOCKET NUMBER: P41 90042 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 619-546-1995 (B) TELEFAX: 619-546-9392 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1495 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: both (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Met Glu Ala Trp Asp Ala His Pro Asp Lys Glu Ala Phe Ala Ala Glu 1 5 10 15 Ala Gin Lys Leu Pro Gly Asp Pro Pro Cys Trp Thr Ser Gly Leu Pro 20 25 30 Phe Pro Val Pro Pro Arg Glu Val lie Lys Ala Ser Pro His Ala Pro 35 40 45 Asp Pro Ser Ala Phe Ser Tyr Ala Pro Pro Gly His Pro Leu Pro Leu 50 55 60 Gly Leu His Asp Thr Ala Arg Pro Val Leu Pro Arg Pro Pro Thr lie 65 70 75 80 Ser Asn Pro Pro Pro Leu lie Ser Ser Ala Lys His Pro Ser Val Leu 85 90 95 Glu Arg Gin lie Gly Ala lie Ser Gin Gly Met Ser Val Gin Leu His 100 105 110 Val Pro Tyr Ser Glu His Ala Lys Ala Pro Val Gly Pro Val Thr Met 115 120 125 Gly Leu Pro Leu Pro Met Asp Pro Lys Lys Leu Ala Pro Phe Ser Gly 130 135 140 Val Lys Gin Glu Gin Leu Ser Pro Arg Gly Gin Ala Gly Pro Pro Glu 145 150 155 160 Ser Leu Gly Val Pro Thr Ala Gin Glu Ala Ser Val Leu Arg Gly Thr 165 170 175 Ala Leu Gly Ser Val Pro Gly Gly Ser lie Thr Lys Gly lie Pro Ser 180 185 190 Thr Arg Val Pro Ser Asp Ser Ala lie Thr Tyr Arg Gly Ser lie Thr 195 200 205 His Gly Thr Pro Ala Asp Val Leu Tyr Lys Gly Thr lie Thr Arg lie 210 215 220 lie Gly Glu Asp Ser Pro Ser Arg Leu Asp Arg Gly Arg Glu Asp Ser 225 230 235 240 Leu Pro Lys Gly His Val lie Tyr Glu Gly Lys Lys Gly His Val Leu 245 250 255 Ser Tyr Glu Gly Gly Met Ser Val Thr Gin Cys Ser Lys Glu Asp Gly 260 265 270 Arg Ser Ser Ser Gly Pro Pro His Glu Thr Ala Ala Pro Lys Arg Thr 275 280 285 Tyr Asp Met Met Glu Gly Arg Val Gly Arg Ala lie Ser Ser Ala Ser 290 295 300 lie Glu Gly Leu Met Gly Arg Ala lie Pro Pro Glu Arg His Ser Pro 305 310 315 320 His His Leu Lys Glu Gin His His lie Arg Gly Ser lie Thr Gin Gly 325 330 335 lie Pro Arg Ser Tyr Val Glu Ala Gin Glu Asp Tyr Leu Arg Arg Glu 340 345 350 Ala Lys Leu Leu Lys Arg Glu Gly Thr Pro Pro Pro Pro Pro Pro Ser 355 360 365 Arg Asp Leu Thr Glu Ala Tyr Lys Thr Gin Ala Leu Gly Pro Leu Lys 370 375 380 Leu Lys Pro Ala His Glu Gly Leu Val Ala Thr Val Lys Glu Ala Gly 385 390 395 400 Arg Ser lie His Glu lie Pro Arg Glu Glu Leu Arg His Thr Pro Glu 405 410 415 Leu Pro Leu Ala Pro Arg Pro Leu Lys Glu Gly Ser lie Thr Gin Gly 420 425 430 Thr Pro Leu Lys Tyr Asp Thr Gly Ala Ser Thr Thr Gly Ser Lys Lys 435 440 445 His Asp Val Arg Ser Leu lie Gly Ser Pro Gly Arg Thr Phe Pro Pro 450 455 460 Val His Pro Leu Asp Val Met Ala Asp Ala Arg Ala Leu Glu Arg Ala 465 470 475 480 Cys Tyr Glu Glu Ser Leu Lys Ser Arg Pro Gly Thr Ala Ser Ser Ser 485 490 495 Gly Gly Ser lie Ala Arg Gly Ala Pro Val lie Val Pro Glu Leu Gly 500 505 510 Lys Pro Arg Gin Ser Pro Leu Thr Tyr Glu Asp His Gly Ala Pro Phe 515 520 525 Ala Gly His Leu Pro Arg Gly Ser Pro Val Thr Met Arg Glu Pro Thr 530 535 540 Pro Arg Leu Gin Glu Gly Ser Leu Ser Ser Ser Lys Ala Ser Gin Asp 545 550 555 560 Arg Lys Leu Thr Ser Thr Pro Arg Glu lie Ala Lys Ser Pro His Ser 565 570 575 Thr Val Pro Glu His His Pro His Pro lie Ser Pro Tyr Glu His Leu 580 585 590 Leu Arg Gly Val Ser Gly Val Asp Leu Tyr Arg Ser His lie Pro Leu 595 600 605 Ala Phe Asp Pro Thr Ser lie Pro Arg Gly lie Pro Leu Asp Ala Ala 610 615 620 Ala Ala Tyr Tyr Leu Pro Arg His Leu Ala Pro Asn Pro Thr Tyr Pro 625 630 635 640 His Leu Tyr Pro Pro Tyr Leu lie Arg Gly Tyr Pro Asp Thr Ala Ala 645 650 655 Leu Glu Asn Arg Gin Thr lie lie Asn Asp Tyr lie Thr Ser Gin Gin 660 665 670 Met His His Asn Thr Ala Thr Ala Met Ala Gin Arg Ala Asp Met Leu 675 680 685 Arg Gly Leu Ser Pro Arg Glu Ser Ser Leu Ala Leu Asn Tyr Ala Ala 690 695 700 Gly Pro Arg Gly lie lie Asp Leu Ser Gin Val Pro His Leu Pro Val 705 710 715 720 Leu Val Pro Pro Thr Pro Gly Thr Pro Ala Thr Ala Met Asp Arg Leu 725 730 735 Ala Tyr Leu Pro Thr Ala Pro Gin Pro Phe Ser Ser Arg His Ser Ser 740 745 750 Ser Pro Leu Ser Pro Gly Gly Pro Thr His Leu Thr Lys Pro Thr Thr 755 760 765 Thr Ser Ser Ser Glu Arg Glu Arg Asp Arg Asp Arg Glu Arg Asp Arg 770 775 780 Asp Arg Glu Arg Glu Lys Ser lie Leu Thr Ser Thr Thr Thr Val Glu 785 790 795 800 His Ala Pro lie Trp Arg Pro Gly Thr Glu Gin Ser Ser Gly Ser Ser 805 810 815 Gly Ser Ser Gly Gly Gly Gly Gly Ser Ser Ser Arg Pro Ala Ser His 820 825 830 Ser His Ala His Gin His Ser Pro lie Ser Pro Arg Thr Gin Asp Ala 835 840 845 Leu Gin Gin Arg Pro Ser Val Leu His Asn Thr Gly Met Lys Gly lie 850 855 860 lie Thr Ala Val Glu Pro Ser Lys Pro Thr Val Leu Arg Ser Thr Ser 865 870 875 880 Thr Ser Ser Pro Val Arg Pro Ala Ala Thr Phe Pro Pro Ala Thr His 885 890 895 Cys Pro Leu Gly Gly Thr Leu Asp Gly Val Tyr Pro Thr Leu Met Glu 900 905 910 Pro Val Leu Leu Pro Lys Glu Ala Pro Arg Val Ala Arg Pro Glu Arg 915 920 925 Pro Arg Ala Asp Thr Gly His Ala Phe Leu Ala Lys Pro Pro Ala Arg 930 935 940 Ser Gly Leu Glu Pro Ala Ser Ser Pro Ser Lys Gly Ser Glu Pro Arg 945 950 955 960 Pro Leu Val Pro Pro Val Ser Gly His Ala Thr lie Ala Arg Thr Pro 965 970 975 Ala Lys Asn Leu Ala Pro His His Ala Ser Pro Asp Pro Pro Ala Pro 980 985 990 Pro Ala Ser Ala Ser Asp Pro His Arg Glu Lys Thr Gin Ser Lys Pro 995 1000 1005 Phe Ser lie Gin Glu Leu Glu Leu Arg Ser Leu Gly Tyr His Gly Ser 1010 1015 1020 Ser Tyr Ser Pro Glu Gly Val Glu Pro Val Ser Pro Val Ser Ser Pro 1025 1030 1035 1040 Ser Leu Thr His Asp Lys Gly Leu Pro Lys His Leu Glu Glu Leu Asp 1045 1050 1055 Lys Ser His Leu Glu Gly Glu Leu Arg Pro Lys Gin Pro Gly Pro Val 1060 1065 1070 Lys Leu Gly Gly Glu Ala Ala His Leu Pro His Leu Arg Pro Leu Pro 1075 1080 1085 Glu Ser Gin Pro Ser Ser Ser Pro Leu Leu Gin Thr Ala Pro Gly Val 1090 1095 1100 Lys Gly His Gin Arg Val Val Thr Leu Ala Gin His He Ser Glu Val 1105 1110 1115 1120 He Thr Gin Asp Tyr Thr Arg His His Pro Gin Gin Leu Ser Ala Pro 1125 1130 1135 Leu Pro Ala Pro Leu Tyr Ser Phe Pro Gly Ala Ser Cys Pro Val Leu 1140 1145 1150 Asp Leu Arg Arg Pro Pro Ser Asp Leu Tyr Leu Pro Pro Pro Asp His 1155 1160 1165 Gly Ala Pro Ala Arg Gly Ser Pro His Ser Glu Gly Gly Lys Arg Ser 1170 1175 1180 Pro Glu Pro Asn Lys Thr Ser Val Leu Gly Gly Gly Glu Asp Gly He 1185 1190 1195 1200 Glu Pro Val Ser Pro Pro Glu Gly Met Thr Glu Pro Gly His Ser Arg 1205 1210 1215 Ser Ala Val Tyr Pro Leu Leu Tyr Arg Asp Gly Glu Gin Thr Glu Pro 1220 1225 1230 Ser Arg Met Gly Ser Lys Ser Pro Gly Asn Thr Ser Gin Pro Pro Ala 1235 1240 1245 Phe Phe Ser Lys Leu Thr Glu Ser Asn Ser Ala Met Val Lys Ser Lys 1250 1255 1260 Lys Gin Glu He Asn Lys Lys Leu Asn Thr His Asn Arg Asn Glu Pro 1265 1270 1275 1280 Glu Tyr Asn He Ser Gin Pro Gly Thr Glu He Phe Asn Met Pro Ala 1285 1290 1295 He Thr Gly Thr Gly Leu Met Thr Tyr Arg Ser Gin Ala Val Gin Glu 1300 1305 1310 His Ala Ser Thr Asn Met Gly Leu Glu Ala He He Arg Lys Ala Leu 1315 1320 1325 Met Gly Lys Tyr Asp Gin Trp Glu Glu Ser Pro Pro Leu Ser Ala Asn 1330 1335 1340 Ala Phe Asn Pro Leu Asn Ala Ser Ala Ser Leu Pro Ala Ala Met Pro 1345 1350 1355 1360 He Thr Ala Ala Asp Gly Arg Ser Asp His Thr Leu Thr Ser Pro Gly 1365 1370 1375 Gly Gly Gly Lys Ala Lys Val Ser Gly Arg Pro Ser Ser Arg Lys Ala 1380 1385 1390 Lys Ser Pro Ala Pro Gly Leu Ala Ser Gly Asp Arg Pro Pro Ser Val 1395 1400 1405 Ser Ser Val His Ser Glu Gly Asp Cys Asn Arg Arg Thr Pro Leu Thr 1410 1415 1420 Asn Arg Val Trp Glu Asp Arg Pro Ser Ser Ala Gly Ser Thr Pro Phe 1425 1430 1435 1440 Pro Tyr Asn Pro Leu He Met Arg Leu Gin Ala Gly Tyr Met Ala Ser 1445 1450 1455 Pro Pro Pro Pro Gly Leu Pro Ala Gly Ser Gly Pro Leu Ala Gly Pro 1460 1465 1470 His His Ala Trp Asp Glu Glu Pro Lys Pro Leu Leu Cys Ser Gin Tyr 1475 1480 1485 Glu Thr Leu Ser Asp Ser Glu 1490 1495 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: both (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Gly Lys Tyr Asp Gin Trp Glu Glu Ser Pro Pro Leu Ser Ala Asn Ala 1 5 10 15 Phe Asn Pro Leu Asn Ala Ser Ala Ser Leu Pro Ala Ala Met Pro He 20 25 30 Thr Ala Ala Asp Gly Arg Ser Asp His Thr Leu Thr Ser Pro 35 40 45 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CGGAGGACTG TCCTCCG 17

Claims: That which is claimed is: 1. A co-suppreεsor of steroid/thyroid hormone receptor activity, said co-suppressor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptors. 2. A co-suppressor according to claim 1 having substantially the same εequence aε reεidues 1-1329 plus 1376-1495, as εet forth in SEQ ID NO:l. 3. A co-εuppreεεor according to claim 2 further compriεing the amino acid residues set forth in SEQ ID NO:2, i. e., residueε 1330-1375 of SEQ ID NO:l. 4. An antibody raised against the co-suppressor of claim 1. 5. A method to block the represεing effect of co-suppreεεors having a εtructure and function characteristic of the silencing mediator for retinoic acid and thyroid receptorε, εaid method compriεing administering an effective amount of an antibody according to claim 4. 6. Isolated polynucleic acid encoding the co- suppreεsor of claim 1. 7. A vector containing polynucleic acid acording to claim 6. 8. A complex comprising the co-suppressor of claim 1 and a homodimeric or heterodimeric member of the steroid/thyroid hormone superfamily of receptors, wherein said member contains a silencing domain which represses basal level promoter activity of target genes. 9. A complex according to claim 8 wherein εaid homodimeric or heterodimeric member of the steroid/thyroid hormone superfamily of receptorε iε selected from thyroid hormone receptor homodimer, thyroid hormone receptor- retinoid X receptor heterodimer, retinoic acid receptor homodimer, retinoic acid receptor-retinoid X receptor heterodimer or retinoid X receptor homodimer. 10. A complex according to claim 8 further comprising a responεe element for said member of the steroid/thyroid hormone superfamily of receptors. 11. A method to disεociate the complex of claim 8, said method compriεing contacting said complex with ligand for said member of the steroid/thyroid hormone superfamily of receptors. 12. A method to repress the activity of a member of the εteroid/thyroid hormone εuperfamily of receptorε containing a εilencing domain which repreεεeε basal level promoter activity of target genes, εaid method compriεing contacting said member of the steroid/thyroid hormone superfamily of receptors with a sufficient quantity of a co-εuppreεεor having a εtructure and function characteristic of the silencing mediator for retinoic acid and thyroid receptorε so aε to repress the activity of said member. 13. A method according to claim 12 wherein said member of the εteroid/thyroid hormone εuperfamily of receptors is selected from thyroid hormone receptor, retinoic acid receptor, vitamin D receptor or peroxisome proliferator activated receptor. 14. A method to identify compounds which relieve the suppression of steroid/thyroid hormone receptor activity caused by a co-suppresεor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptors, said method comprising comparing the size of the complex of claim 8 upon exposure to test compound, relative to the size of said complex in the abεence of teεt compound. 15. A method according to claim 14 wherein said complex further comprises a response element for said member of the steroid/thyroid hormone εuperfamily of receptors. 16. A method to identify compounds which relieve the suppression of steroid/thyroid hormone receptor activity caused by a co-suppreεsor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptors, without substantially activating said receptor, said method comprising: comparing the reporter signal produced by two different expresεion εyεtemε in the abεence and preεence of teεt compound, wherein εaid first expreεεion εystem compriεeε a complex compriεing: a homodimeric or heterodimeric member of the εteroid/thyroid hormone εuperfamily of receptorε selected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer, or retinoic acid receptor-retinoid X receptor heterodimer, a responεe element for said member of the steroid/thyroid hormone superfamily of receptorε, wherein εaid reεponse element is operatively linked to a reporter, and optionally, the co-εuppreεsor of claim 1, and wherein εaid second expression system comprises a complex comprising: a homodimeric or heterodimeric form of the εame member of the steroid/thyroid hormone superfamily of receptors as employed in said firεt expression εystem, wherein said member is mutated such that it retains hormone dependent activation activity but has lost its ability to repress baεal level promoter activity of target geneε, the εame reεponεe element-reporter combination as employed in said first expreεεion system, and optionally, the co-suppreεsor of claim 1, and thereafter selecting those compounds which provide: a higher reporter signal upon exposure of said compound to said first expression syεtem, relative to reporter εignal in the absence of said compound, and substantially the same reporter signal upon exposure of εaid compound to said second expreεεion syεtem, relative to reporter signal in the absence of εaid compound, wherein εaid selected compounds are capable of relieving the suppression of steroid/thyroid hormone receptor activity cauεed by a co-εuppreεεor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptors, but substantially lacking the ability to activate steroid/thyroid hormone receptor activity. 17. A method according to claim 16 wherein said mutant receptor is εelected from RAR403 homodimers, RAR403- containing heterodimers, TR160 homodimers or TR160- containing heterodimers. 18. A method to identify compoundε which activate εteroid/thyroid hormone receptor activity, but εubεtantially lack the ability to relieve the εuppression caused by a co-suppressor having a structure and function characteriεtic of the silencing mediator for retinoic acid and thyroid receptors, said method comprising: comparing the reporter εignal produced by two different expreεεion εyεtemε in the abεence and preεence of test compound, wherein said firεt expreεεion εyεtem compriεeε a complex compriεing: a homodimeric or heterodimeric member of the εteroid/thyroid hormone superfamily of receptors εelected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer, or retinoic acid receptor-retinoid X receptor heterodimer, a reεponεe element for εaid member of the εteroid/thyroid hormone εuperfamily of receptors, wherein εaid reεponεe element is operatively linked to a reporter, and optionally, the co-suppreεsor of claim 1, and wherein said second expreεεion εyεtem compriεes a complex comprising: a homodimeric or heterodimeric form of the same member of the steroid/thyroid hormone superfamily of receptorε as employed in said first expression syεtem, wherein said member iε mutated εuch that it retains hormone dependent activation activity but has lost its ability to repress basal level promoter activity of target geneε, the εame reεponεe element-reporter combination aε employed in said first expression system, and optionally, the co-suppressor of claim 1, and thereafter selecting those compoundε which provide: a higher reporter εignal upon expoεure of said compound to said second expression system, relative to reporter signal in the absence of compound, and substantially the same reporter signal upon expoεure of εaid compound to εaid first expression system, relative to reporter signal in the abεence of εaid compound, wherein εaid εelected compounds are capable of activating steroid/thyroid hormone receptor activity, but substantially lacking the ability to relieve the suppreεεion cauεed by a co-εuppreεεor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptors. 19. A method to identify compounds which relieve the εuppreεεion of steroid/thyroid hormone receptor activity caused by a co-suppressor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptorε, and activate εaid receptor, said method comprising: comparing the reporter signal produced by two different expresεion εystems in the absence and presence of test compound, wherein said first expression system compriεeε a complex compriεing: a homodimeric or heterodimeric member of the steroid/thyroid hormone superfamily of receptors selected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer, or retinoic acid receptor-retinoid X receptor heterodimer, a response element for said member of the steroid/thyroid hormone superfamily of receptors, wherein said responεe element is operatively linked to a reporter, and optionally, the co-suppressor of claim 1, and wherein said εecond expreεεion εyεtem compriεeε a complex compriεing: a homodimeric or heterodimeric form of the same member of the steroid/thyroid hormone εuperfamily of receptors as employed in said first expresεion system, wherein said member is mutated εuch that it retainε hormone dependent activation activity but haε loεt itε ability to repreεε baεal level promoter activity of target genes, the same response element-reporter combination aε employed in εaid firεt expreεεion εyεtem, and optionally, the co-εuppreεsor of claim 1, and thereafter selecting those compounds which provide: increased reporter signal upon exposure of said compound to said second expreεsion system, relative to reporter signal in the absence of said compound, and substantially increased reporter signal upon exposure of said compound to said first expression syεtem, relative to reporter signal in the absence of said compound, wherein said selected compounds are capable of relieving the suppression of steroid/thyroid hormone receptor activity caused by a co-suppressor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptors, and activating said receptor. 20. A modified form of the co-εuppreεsor of claim 1 selected from: full length silencing mediator for retinoic acid and thyroid receptors plus GAL4 DNA binding domain, full length silencing mediator for retinoic acid and thyroid receptorε plus GAL4 activation domain, or full length silencing mediator for retinoic acid and thyroid receptors plus giutathione S-transferaεe (GST) tag. 21. A modified co-εuppressor according to claim 20, wherein said modified co-suppressor comprises full length silencing mediator for retinoic acid and thyroid receptors plus GAL4 activation domain. 22. A method to identify compounds which disrupt the ability of a co-suppresεor having a εtructure and function characteristic of the silencing mediator for retinoic acid and thyroid receptorε to complex with steroid/thyroid hormone receptors, without substantially activating said receptor, said method comprising: comparing the reporter signal produced by two different expression systems in the absence and preεence of test compound, wherein said firεt expreεεion εyεtem compriεes a complex compriεing: a modified co-εuppressor according to claim 21, a homodimeric or heterodimeric member of the steroid/thyroid hormone superfamily of receptors εelected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer or retinoic acid receptor-retinoid X receptor heterodimer, and a reεponεe element for εaid member of the εteroid/thyroid hormone superfamily of receptorε, wherein said response element is operatively linked to a reporter, and wherein said second expreεεion εystem compriεeε a complex comprising: εaid modified co-εuppreεεor, a homodimeric or heterodimeric form of the εame member of the εteroid/thyroid hormone εuperfamily of receptorε aε employed in said firεt expreεεion εystem, wherein εaid member is mutated such that it retainε hormone dependent activation activity but has lost its ability to repress basal level promoter activity of target genes, and the same response element-reporter combination as employed in εaid first expression system, and thereafter selecting those compounds which provide: a lower reporter εignal upon expoεure of said compound to said first expreεεion system, relative to reporter signal in the absence of said compound, and substantially the same reporter signal upon exposure of said compound to said second expresεion system, relative to reporter signal in the absence of εaid compound, wherein said εelected compoundε are capable of diεrupting the ability of a co-εuppressor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptors to complex with steroid/thyroid hormone receptorε, without εubεtantially activating εaid receptor. 23. A method according to claim 22 wherein said mutant receptor is selected from RAR403 homodimers, RAR403- containing heterodimerε, TR160 homodimerε or TR160- containing heterodimerε. 24. A method according to claim 22, wherein the hoεt iε a mammalian or yeast cell. 25. A method to identify compounds which activate steroid/thyroid hormone receptor activity, but substantially lack the ability to disrupt a complex comprising a steroid/thyroid hormone receptor and a co- suppresεor having a εtructure and function characteriεtic of the silencing mediator for retinoic acid and thyroid receptors, said method comprising: comparing the reporter signal produced by two different expreεεion εyεtemε in the abεence and preεence of test compound, wherein said first expreεεion εyεtem compriεeε a complex comprising: a modified co-suppreεsor according to claim 21, a homodimeric or heterodimeric member of the steroid/thyroid hormone superfamily of receptors selected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer or retinoic acid receptor-retinoid X receptor heterodimer, and a responεe element for εaid member of the steroid/thyroid hormone superfamily of receptors, wherein said response element is operatively linked to a reporter, and wherein εaid εecond expreεεion system compriseε: said modified co-suppressor, a homodimeric or heterodimeric form of the same member of the steroid/thyroid hormone εuperfamily of receptors as employed in said first expression εyεtem, wherein εaid member is mutated εuch that it retainε hormone dependent activation activity but haε lost its ability to repreεs basal level promoter activity of target genes, and the same response element-reporter combination as employed in said first expression syεtem, and thereafter εelecting those compounds which provide: a higher reporter εignal upon expoεure of said compound to said εecond expression syεtem, relative to reporter εignal in the abεence of compound, and substantially the same reporter signal upon exposure of εaid compound to said first expreεsion εystem, relative to reporter signal in the abεence of compound, wherein said selected comoundε are capable of activating steroid/thyroid hormone receptor activity, but subεtantially lack the ability to diεrupt the complex of a co-suppresεor having a εtructure and function characteriεtic of the εilencing mediator for retinoic acid and thyroid receptorε and a εteroid/thyroid hormone receptor. 26. A method according to claim 25, wherein the hoεt is a mammalian or yeast cell. 27. A method to identify compounds which activate a steroid/thyroid hormone receptor, and diεrupt the ability of a co-suppressor having a εtructure and function characteriεtic of the silencing mediator for retinoic acid and thyroid receptors to complex with εaid receptor, εaid method comprising: comparing the reporter signal produced by two different expresεion εyεtemε in the absence and presence of teεt compound, 10 wherein εaid firεt expreεεion εyεtem compriεeε a complex compriεing: a modified co-suppressor according to claim 21, a homodimeric or heterodimeric member 15 of the steroid/thyroid hormone superfamily of receptors selected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer or retinoic 20 acid receptor-retinoid X receptor heterodimer, and a response element for said member of the steroid/thyroid hormone superfamily of receptors, wherein said responεe element is 25 operatively linked to a reporter, and wherein said second expression syεtem compriεes a complex comprising: said modified co-εuppreεεor, the εame homodimeric or heterodimeric 30 member of the steroid/thyroid hormone superfamily of receptors as employed in said firεt expreεεion εyεtem, wherein εaid member iε mutated such that it retainε hormone dependent activation activity but haε lost 35 its ability to repreεε baεal level promoter activity of target geneε, and the εame reεponεe element-reporter combination as employed in said first expression system, and thereafter 40 selecting those compoundε which provide: a reduction in reporter εignal upon exposure of compound to said first expression εyεtem, relative to reporter signal in the absence of εaid compound, and increased reporter signal upon exposure of compound to said second expression system, relative to reporter signal in the absence of said compound, wherein said selected compounds are capable of activating a steroid/thyroid hormone receptor and diεrupting a complex compriεing εteroid/thyroid hormone receptor and a co-εuppreεsor having a εtructure and function characteristic of the silencing mediator for retinoic acid and thyroid receptorε. 28. A method according to claim 27, wherein the hoεt is a mammalian or yeast cell. 29. A method to identify compoundε which activate a steroid/thyroid hormone receptor and/or disrupt the ability of a co-suppresεor having a εtructure and function characteriεtic of the εilencing mediator for retinoic acid and thyroid receptors to complex with said receptor, said method compriεing: comparing the reporter εignalε produced by a combination expreεεion εystem in the absence and preεence of teεt compound, wherein εaid combination expression syεtem compriεes: a first homodimeric or heterodimeric member of the steroid/thyroid hormone εuperfamily of receptors εelected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer, or retinoic acid receptor- retinoid X receptor heterodimer, a εecond homodimeric or heterodimeric form of the εame member of the εteroid/thyroid hormone εuperfamily of receptors aε employed in εaid first homodimer or heterodimer, wherein said member iε mutated εuch that it retains hormone dependent activation activity but has loεt itε ability to repreεs basal level promoter activity of target geneε, wherein either εaid first homodimer (or heterodimer) or said εecond homodimer (or heterodimer) iε operatively linked to a GAL4 DNA binding domain, a reεponεe element for εaid member of the εteroid/thyroid hormone εuperfamily of receptors, wherein said response element is operatively linked to a firεt reporter, a GAL4 reεponεe element, wherein εaid reεponεe element is operatively linked to a second reporter, and thereafter identifying as capable of relieving the suppreεεion of εteroid/thyroid hormone receptor activity cauεed by a co-εuppreεsor having a εtructure and function characteriεtic of the εilencing mediator for retinoic acid and thyroid receptorε, but substantially lacking the ability to activate steroid/thyroid hormone receptor activity those compounds which provide: a higher reporter signal from the reporter responsive to the first member upon exposure of εaid compound to said first member, relative to reporter εignal in the absence of said compound, and substantially the εame reporter εignal from the reporter reεponεive to the εecond member upon expoεure of εaid compound to εaid εecond member, relative to reporter εignal in the abεence of said compound, or identifying as capable of activating steroid/thyroid hormone receptor activity, but substantially lacking the ability to relieve the suppresεion cauεed by a co-εuppressor having a εtructure and function characteriεtic of the εilencing mediator for retinoic acid and thyroid receptorε thoεe compounds which provide: a higher reporter signal from the reporter responεive to the εecond member upon exposure of said compound to said second member, relative to reporter signal in the absence of compound, and substantially the same reporter signal from the reporter responsive to the first member upon exposure of said compound to εaid firεt member, relative to reporter εignal in the abεence of εaid compound, or identifying aε capable of relieving the εuppreεsion of εteroid/thyroid hormone receptor activity cauεed by a co-εuppreεεor having a εtructure and function characteristic of the silencing mediator for retinoic acid and thyroid receptors, and activating said receptor those compoundε which provide: increaεed reporter εignal from the reporter reεponεive to the εecond member upon exposure of said compound to εaid εecond member, relative to reporter εignal in the absence of said compound, and substantially increased reporter signal from the reporter reεponεive to the firεt member upon expoεure of said compound to said firεt member, relative to reporter signal in the absence of εaid compound. 30. A method according to claim 29 wherein said combination expression system further comprises a co- suppresεor of steroid/thyroid hormone receptor activity, said co-suppressor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptors. 31. A method to identify compoundε which activate a εteroid/thyroid hormone receptor and/or diεrupt the ability of a co-εuppreεsor having a εtructure and function characteristic of the silencing mediator for retinoic acid and thyroid receptors to complex with said receptor, said method comprising: comparing the reporter signals produced by a combination expresεion εystem in the abεence and preεence of test compound, wherein said combination expreεεion system compriseε: a modified co-εuppreεεor according to claim 21, a first homodimeric or heterodimeric member of the steroid/thyroid hormone superfamily of receptors εelected from thyroid hormone receptor homodimer, thyroid hormone receptor-retinoid X receptor heterodimer, retinoic acid receptor homodimer, or retinoic acid receptor- retinoid X receptor heterodimer, a εecond homodimeric or heterodimeric form of the εame member of the εteroid/thyroid hormone superfamily of receptors as employed in said first homodimer or heterodimer, wherein εaid member iε mutated εuch that it retainε hormone dependent activation activity but haε loεt itε ability to repreεε baεal level promoter activity of target genes, wherein either said first homodimer (or heterodimer) or said second homodimer (or heterodimer) is operatively linked to a GAL4 DNA binding domain, a responεe element for εaid member of the steroid/thyroid hormone superfamily of receptors, wherein said response element is operatively linked to a first reporter, a GAL4 responεe element, wherein said response element iε operatively linked to a εecond reporter, and thereafter identifying aε capable of diεrupting the ability of a co-suppressor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptorε to complex with εteroid/thyroid hormone receptor, without εubεtantially activating εteroid/thyroid hormone receptor, thoεe compoundε which provide: a lower reporter εignal from the reporter reεponεive to the firεt member upon exposure of εaid compound to εaid firεt member, relative to reporter signal in the absence of said compound, and εubstantially the same reporter signal from the reporter responεive to the second member upon exposure of εaid compound to εaid second member, relative to reporter εignal in the abεence of εaid compound, or identifying as capable of activating steroid/thyroid hormone receptor activity, but substantially lacking the ability to disrupt a complex comprising a steroid/thyroid hormone receptor and a co- suppresεor having a εtructure and function characteriεtic of the silencing mediator for retinoic acid and thyroid receptors those compounds which provide: a higher reporter signal from the reporter reεponεive to the εecond member upon expoεure of εaid compound to said second member, relative to reporter εignal in the abεence of compound, and εubεtantially the εame reporter εignal from the reporter reεponεive to the firεt member upon expoεure of εaid compound to εaid firεt member, relative to reporter signal in the absence of said compound, or identifying aε capable of diεrupting a complex compriεing a εteroid/thyroid hormone receptor and a co- εuppreεεor having a εtructure and function characteristic of the silencing mediator for retinoic acid and thyroid receptors, and activating said receptor those compoundε which provide: a reduction in reporter εignal from the reporter reεponεive to the firεt member upon expoεure of said compound to εaid firεt member, relative to reporter εignal in the absence of said compound, and increaεed reporter εignal from the reporter reεponsive to the second member upon exposure of said compound to said second member, relative to reporter εignal in the abεence of εaid compound. 32. A method to identify compounds which relieve the suppreεεion of εteroid/thyroid hormone receptor activity cauεed by a co-εuppressor having a structure and function characteristic of the silencing mediator for retinoic acid and thyroid receptorε, εaid method compriεing determining the effect of adding teεt compound to an expreεεion εystem comprising: a modified member of the εteroid/thyroid hormone εuperfamily of receptorε, wherein εaid modified member containε an activation domain which renderε εaid receptor constitutively active, a fusion protein comprising the receptor interaction domain of SMRT operatively linked to the GAL4 DNA binding domain, and a GAL4 responεe element operatively linked to a reporter.

Inventors: CHEN J DON, EVANS RONALD M, CHEN, J., DON, EVANS, RONALD, M.
Applicants: SALK INST FOR BIOLOGICAL STUDI, THE SALK INSTITUTE FOR BIOLOGICAL STUDIES
Application year: 1996
Main classification: C12N1
IPC codes: A61K39, C07K14, C07K16, C07K19, C12N1, C12N5, C12N15, C12R1, G01N33