WO2001016597A1 - In vitro transcription systems and uses - Google Patents

Abstract

The invention relates to methods of identifying agents that interact with retinoid X receptor dimers. The invention also relates to in vitro chromatin based DNA template transcription systems.

Description

In Vitro Transcription Systems and Uses

Background of the Invention

Field of the Invention

The invention relates to methods of identifying agents that interact with retinoid X receptor dimers. The invention also relates to in vitro chromatin based

DNA template transcription systems.

Related Art

Retinoic acids (RAs) exert their pleiotropic effects on vertebrate development and homeostasis by binding to nuclear receptors (NRs) (Kastner, P., et al, Cell 53:859-869 (1995), and references therein). These receptors belong to a gene superfamily that includes the receptors for steroid hormones, thyroid hormones, vitamin D3, and a growing number of so-called orphan receptors (for reviews, see Gronemeyer, H., & Laudet, V., Protein Profile 2: 1173-1236 (1995); Perlmann, T. & Evans, R.M., Cell 90:391-397 (1997)). Two families of receptors, the retinoic acid receptor isotypes (RARα, RARβ and RARγ) and the retinoid X receptors isotypes (RXRα, RXRβ and RXRγ) are implicated in the transduction of the RA signal (Chambon, P., FASEB J. 70:940-954 (1996), and references therein). RARs bind all-trans RA (tRA) and 9-cis RA (9cRA), whereas RXRs respond exclusively to 9cRA (Allenby, G., et al, Proc. Natl. Acad. Sci. USA 90:30-34 (1993), and references therein). The C-terminal region of RARs and

RXRs contains both the ligand binding domain (LBD), which functions as a ligand-dependent transactivation domain (activation function 2 (AF-2)), and surfaces for both homo- and hetero-dimerization as well as for interaction with other factors (see below). An additional ligand- independent activation function, AF-1, is present within the N-terminal region (see, Chambon, P., FASEB J.

70:940-954 (1996)).

RARs and RXRs can bind as dimers to RA response elements (RAREs) consisting of two hexameric motifs [PuG(G/A)(T/A)CA] usually arranged as direct repeats. However, RXRs readily heterodimerize with RARs, and RXR/RAR heterodimers bind to and transactivate from RAREs made up of direct repeat motifs separated by 5 (DR5) and 2 (DR2) bp much more efficiently than RXR homodimers on their own. This indicates that RXR/RAR heterodimers might be the functional units transducing the retinoic acid signals in vivo

(Chambon, P., FASEB J. 70:940-954 (1996); Leid, M., et al, Trends Biochem. Sci. 17:427-433 (1992); and references therein). Several lines of evidence support this possibility: (i) genetic studies have established the functionality of RXR/RAR heterodimers in the RA-responsive F9 embryonal carcinoma cell line (Clifford, J., etal, EMBOJ. 75:4142-4155 (1996); Chiba, H., etal, J. Cell Science 139:135-

747 (1997); Chiba, H., et al, Mol. Cell. Biol 77:3013-3020 (1997)), as well as in the mouse (Kastner, P., et al, Cell 55:859-869 (1995); Kastner, P., et al, Development 124:313-326 (1997); Kretzel, W., et al, Science 279:863-867 (1998); Mascrez, B., et al, Development 725:4691-4707 (1998); and references therein), and (ii) synergistic effects of RXR- and RAR-selective synthetic retinoid on target gene expression, proliferation, apoptosis and/or differentiation have been observed in a variety of cultured cell lines, including the embryonal carcinoma cell lines F9 and P19 (Clifford, J., et al, EMBOJ. 75:4142-4155 (1996); Chiba, H., et al, J. Cell. Science 759:735-747 (1997); Chiba, H., et al, Mol Cell. Biol. 77:3013-3020 (1997); Apfel, CM., et al, J. Biol. Chem. 270:30165-30112

(1995); Lotan, R., et al, Cancer Res. 55:232-236 (1995); Roy, B., et al, Mol. Cell. Biol 75:6481-6487 (1995); Chen, J-Y., et al, Nature 552:819-822 (1996); Horn, V., et al, FASEB J. 70:1071-1077 (1996); La Vista-Picard, N., et al, Mol. Cell. Biol. 76:4137-4146 (1996); Taneja, R., et al, Proc. Natl. Acad. Sci. USA 95:6197-6202 (1996); Taneja, R., etal, EMBOJ. 76:6452-6465 (1997); Botling,

J., et al, J. Biol. Chem. 272:9443-9449 (1997); Minucci, S., et al, Mol Cell. Biol 77:644-655 (1997); Joseph, B., et al, Blood 97:2423-2432 (1998)). However, in all cases, the liganded RXR was transcriptionally inactive, unless its RAR partner was itself liganded. This intra-heterodimeric subordination of the RXR AF-2 activity to the binding of a RAR agonistic ligand could be caused by an allosteric effect of the unliganded RAR on its liganded RXR partner (Vivat, V., et al, EMBO J. 76:5698-5709 (1997)).

Transfection studies have suggested that the AF-2 activation function of NRs is mediated through coactivators (intermediary factors) (Tasset, D., et al, Cell 62: 1177-1187 (1990)). Numerous proteins that interact directly with NRs in an agonistic ligand-dependent manner have been cloned and characterized, and several of them have been shown to enhance the activity of NR AF-2s when co- expressed in transiently transfected mammalian cells (Chambon, P., FASEB J. 70:940-954 (1996); Glass, C.K., etal, Curr. Opin. Cell. Biol. 9:222-232 (1997); and references therein). Some of these putative coactivators, SRC-I (Spencer,

T.E., et al, Nature 559:191-198 (1997)), CBP/p300 (Bannister, A.J. & Kouzarides, T., Nature 554:641-643 (1996); Ogryzko, V.V., et al, Cell 57:953- 959 (1996)) and ACTR (Chen, H., et al, Cell 90:569-580 (1997)) can interact with the histone acetyltransferase p/CAF (Yang, X-J., et al, Nature 382:319-324 (1996)) and also possess an intrinsic histone acetyltransferase activity. Moreover,

CBP and p300 also interact with RNA helicase A, which in turn binds RNA polymerase II (Nakajima, T., et al, Cell 90:1107-1112 (1997)).

It has been shown that RXRs form heterodimers in solution with either RARs, TRs or VDR and that the receptor domains required for heterodimeric interactions overlap with the LBD of each receptor. Ligand dependent transcription activation by the RXR/VDR heterodimer has been shown (Rachez, C. et al, Nature 595:824-828 (1999)). The formation of heterodimers between RXRs and PPARs was also demonstrated (Kliewer, S.A. et al. Nature 555:771- 774 (1992); Bardot, O. etal, Biochem. Biophys. Res. Comm. 192:31-45 (1993)). RXR also heterodimerizes with liver X receptors (LXRs; Apfel et al, Mol. Cell

Biol. 14:1025-1035 (1994), farnesoid X receptor (FXR; Forman et al, Cell 57:687-693 (1995)), benzoate X receptor (BXR; Blumberg et al, Genes & Dev. 72:1269-1277 (1998)), constitutively active receptor or constitutive androstane receptors (CARs; Choi et al, J. Biol. Chem. 272:23565-23571 (1997); Forman et al, Nature 595:612-615 (1998)), and steroid and xenobiotic receptor (SXR;

Blumberg et al, Genes & Dev. 72:3195-3205 (1998)). Summary of the Invention

The invention is directed to methods of identifying agents that interact with retinoid X receptor dimers. The invention is also directed to in vitro chromatin based DNA template transcription systems.

Brief Description of the Figures

FIG. 1. DNA templates and SI nuclease probe. The structures of the (DR5)5β2G, (17m)5β2G and internal control pGl reporter templates are schematically represented with the positioning of the response elements relative to the transcription start site. FIG.2. Analysis of RARcc/RXRoc heterodimers and chromatin structure.

FIG.2(A). Purification of RARα/RXRα heterodimers: FhRARα and HmRXRα were co-expressed in Sf9 cells and affinity -purified using aNi²⁺ column followed by anti-Flag agarose column that bind the HmRXR moiety and the FhRAR moiety of the heterodimer, respectively. Purified heterodimers (100 ng of protein) were separated on a 10% SDS-PAGE gel before staining with Coomassie Blue (lane 1 ) or Western blot analysis using monoclonal antibodies recognizing either human RARα (lane 2) or mouse RXRα (lane 3). FIG.2(B). Overall chromatin structure was not affected by RARα/RXRα heterodimers: chromatin or naked (DR5)5β2G templates (200 pM) incubated in the presence or absence of FhRARα/HmRXRα (1 nM) and tRA (10 ⁶ M) were digested with varying concentrations of micrococcal nuclease in a final volume of 80 μl, separated on a 1.5% agarose gel, and Southern blotted using a [³²P] probe corresponding to the -40 to +5 region of the (DR5)5β2G promoter. DNA supercoiling was estimated as described (Becker, P.B., et al, Methods Cell Biol. 44:201-223 (1994)) on DNA (200 ng) treated (or not treated) by topoisomerase 1 (10 units; final volume of 45 μl). DNA was separated on a 1% agarose gel in the presence or absence of 1.2 μM chloroquine. Migration of relaxed and supercoiled template DNA is indicated. FIG.3. RARα/RXRα heterodimers activate transcription from chromatin templates in a ligand- and template-specific manner. FIG. 3(A). tRA-induced derepression of transcription from chromatin templates by RARα/RXRα. In vitro transcription was performed on chromatin or naked (DR5)5β2G templates (200 pM) using a HeLa cell nuclear extract (100 μg) for 45 min in the presence or absence of FhRARα/HmRXRα (1 nM) and tRA (1 μM) in a final reaction volume of 50 μl as indicated. SI nuclease analysis was carried out after deproteinization. FIG. 3(B). Template specificity of activated transcription. Activation of transcription on chromatin (DR5)5β2G or (17M)5β2G templates was determined in the presence of 1 nM of activator (either Gal4(l-147), Gal4-

VP 16 or FhRARα/HmRXRα) with or without tRA ( 1 μM) as above. S 1 nuclease digestion of RNA transcripts originating from β2G and pGl templates generated 179- and 60-nt fragments, respectively (see FIG. 1).

FIG.4. RARα/RXRα heterodimers bind all five RAREs in the promoter region of the (DR5)5β2G chromatin template, irrespective of the presence of tRA.

Chromatin or naked (DR5)5 β2G templates (250 ng) were incubated in the presence or absence of FhRARα/HmRXRα and tRA (10^~6 M) (under the conditions described above for transcription reactions) for 30 min, subjected to

DNase I digestion (5 units; final volume 50 μl), then analyzed by primer extension foot printing (see Materials and Methods). Sites of increased (closed triangle) or decreased (open triangle) sensitivity to DNase I are shown.

FIG. 5. Dose-dependent synergistic effects of specific retinoids on activation of transcription by RARα/RXRα heterodimers. FIG. 5(A). Dose- dependent activation by tRA and 9cRA. Transcription reactions were performed as described in FIG. 3 on (DR5)5β2G template by using FhRARα/HmRXRα in presence of varying concentrations (5x10^"'° to 10^"6 M) of tRA (open circles) or 9cRA (closed squares). FIG.5(B). Receptor-selective and synergistic activation of transcription. Transcription reactions were performed as described above using synthetic retinoid agonists and antagonists at the concentrations indicated. The receptor specificity of retinoids used are as follows: tRA (panRAR-specific ligand), 9cRA (panRAR- and panRXR-ligand), Compound I (RARα-specific agonist), Compound IV (RARγ -specific agonist), SRI 1237 (panRXR-specific agonist), and Compound II (RARα-specific antagonist). Transactivation by FhRARα/HmRXRα is expressed relative to that observed from the internal control template (pGl). Induction by tRA (10 ⁶ M) was arbitrarily set to 100%. All points are the average of at least two independent experiments run in duplicate.

FIG. 6. p300 enhances transactivation by RARα/RXRα heterodimers in vitro. FIG.6(A). Addition of exogenous acetylCoA (AcCoA) does not effect ligand-dependent transactivation by RARα/RXRα. Transcription reactions were performed in the presence or absence of acetylCoA (1 μM) on naked or chromatin (17M)5β2G or (DR5)5β2G templates plus or minus 1 nM activator (either

Gal4(l-147), Gal4-VP16 or FhRARα/HmRXRα) and/or tRA (1 μM), as described in FIG. 3. FIG.6(B). Addition of acetylCoA does not further enhance p300-activated transcription. Transcription was performed on (DR5)5β2G templates in the presence or absence of FhRARα/HmRXRα and/or tRA (5x10^"8 M). Where indicated, the co-activator p300 (0.5 nM) and acetyl CoA

(1 μM) were added.

Detailed Description of the Preferred Embodiments

It has been discovered that an in vitro chromatin based DNA template transcription system, in the presence of RXR/RAR heterodimers, mimics the effects of retinoids on gene transactivation as observed in vivo. Activation of transcription by RXR/RAR heterodimers depends on packaging of the template into a nucleosomal structure and that it is specific, in that it requires the heterodimer, a cognate ligand, and a cognate response element. Moreover, it has been discovered that the agonist-bound transcription activation function of RXR can act synergistically with that of RAR but that the binding of an agonist to RAR is a prerequisite for effective activation of transcription by agonist-bound RXR.

The invention is directed to a method of identifying an agent which interacts with a retinoid X receptor (RXR) dimer, the method comprising: (a) adding an agent to a chromatin based DNA template in the presence of the RXR dimer; and (b) measuring activation of transcription, thereby determining whether the agent interacts with the RXR dimer. In the method, activation of transcription can be compared to the method performed in the absence of the agent or in the presence of a known agent. Another embodiment of the invention is directed to a method of identifying a retinoic acid receptor (RAR) agonist, the method comprising: (a) adding an agent to a chromatin based DNA template in the presence of an RXR/RAR dimer; and (b) measuring activation of transcription, thereby determining whether the agent is an RAR agonist. In the method, activation of transcription can be compared to the method performed in the absence of the agent or in the presence of a known RAR agonist.

The invention is also directed to a method of identifying an RXR agonist, the method comprising: (a) adding an agent to a chromatin based DNA template in the presence of an RXR/RAR dimer and an RAR agonist; and (b) measuring activation of transcription, thereby determining whether the agent is an RXR agonist. In the method, activation of transcription can be compared to the method performed in the absence of the agent or in the presence of a known RXR agonist.

The invention is further directed to a method of identifying an RAR antagonist, the method comprising: (a) adding an agent to a chromatin based DNA template in the presence of an RXR/RAR dimer and an RAR agonist; and

(b) measuring activation of transcription, thereby determining whether the agent is an RAR antagonist. In the method, activation of transcription can be compared to the method performed in the absence of the agent or in the presence of a known RAR antagonist. The invention is directed to a method of identifying an RXR antagonist, the method comprising: (a) adding an agent to a chromatin based DNA template in the presence of a RXR/RAR dimer, an RAR agonist, and an RXR agonist; and (b) measuring activation of transcription, thereby determining whether the agent is an RXR antagonist. In the method, activation of transcription can be compared to the method performed in the absence of the agent or in the presence of a known

RXR antagonist. The invention is directed to a method of identifying a co-activator of an RXR dimer, the method comprising: (a) adding a first agent to a chromatin based DNA template in the presence of the RXR dimer and a second agent which is an agonist of the RXR dimer; and (b) measuring activation of transcription, thereby determining whether the first agent is a co-activator of the RXR dimer. In the method, activation of transcription can be compared to the method performed in the absence of the first agent or in the presence of a known co-activator. In another embodiment, this method can be used for identifying a co-repressor of the RXR dimer. In the method, activation of transcription can be compared to the method performed in the absence of the first agent or in the presence of a known co-repressor.

The invention is further directed to a method of identifying a modulator which modulates interactions between a RXR dimer and a co-activator of the RXR dimer, the method comprising: (a) adding an agent to a chromatin based DNA template in the presence of the RXR dimer, an agonist of the RXR dimer, and a co-activator of the RXR dimer; and (b) measuring activation of transcription, thereby determining whether the agent modulates interactions between the RXR dimer and the co-activator of the RXR dimer. In the method, activation of transcription can be compared to the method performed in the absence of the agent. In another embodiment, this method can be used for identifying a co-repressor of the RXR dimer. In the method, activation of transcription can be compared to the method performed in the absence of the agent.

The invention is directed to an in vitro chromatin based DNA template transcription system comprising: (a) a chromatin based DNA template; and (b) an RXR dimer. The invention is also directed to a kit comprising the in vitro chromatin based DNA template transcription system.

Each of the terms and elements of the invention as described in the above embodiments are detailed as follows. By a "nuclear receptor" or "nuclear receptor superfamily receptor" or

"steroid/thyroid hormone receptor superfamily" is intended a ligand-dependent transcription factor that regulates the expression of target genes involved in metabolism, development, and reproduction. Nuclear receptors include receptors for which specific ligands have not yet been identified (termed "orphan receptors"). These hormone binding proteins can bind to specific DNA sequences to modulate transcriptional activity of a target gene, upon binding of a ligand to the receptor. Exemplary nuclear receptors include, but are not limited to, retinoic acid receptors (RARs; α, β and γ), retinoid X receptors (RXRs; α, β and γ), vitamin D₃ receptor (VDR), thyroid receptors (TRs; α and β), peroxisome proliferator activated receptors (PPARs; α, β, δ and γ), liver X receptors (LXRs; α and β) (Willy, P.J. et al, Genes & Dev. 9:1033-1045 (1995); Willy, P.J. et al,

Genes & Dev. 77:289-298 (1997); Mukherjee, R. et al, Nature 556:407-410 (1997); Peet, D.J. et al, Curr. Opin. Genet. Dev. 5:571-575 (1998); Janowski, B.A. etal, Proc. Natl Acad. Sci. USA 96:266-271 (1999)), farnesoid X receptor (FXR) (Makishima, M. et al, Science 284:1362-1365 (1999)); Wang, H. et al, Mol. Cell3:543-553 (1999)), benzoateX receptor (BXR) (Blumberg etal, Genes

& Dev. 1 :1269-1277 (1998)), constitutively active receptor or constitutive androstane receptors (CARs; α and β) (Choi et al, J. Biol. Chem. 272:23565- 23571 (1997); Forman et al, Nature 595:612-615 (1998)), and steroid and xenobiotic receptor (SXR) (Blumberg et al, Genes & Dev. 72:3195-3205 (1998)), HNF4 (Sladek et al, Genes & Dev. 4:2353-2365 (1990)), the COUP family of receptors (Miyajima et al, Nucl Acids Res. 76:11057-11074 (1988)), nerve growth factor-induced receptor (NGFI-B) (Crawford, P.A. et al, Mol. Cell. Biol. 75:4331-4336 (1995)), ultraspiracle receptor (Oro et al, Nature 547:298- 301 (1990)), and the like. By an "RXR dimer" is intended a dimer formed by an RXR (α, β or γ) and a second nuclear receptor, and includes an RXR homodimer and RXR heterodimer. By an "RXR homodimer" is intended a dimer of an RXR (α, β or γ) and another RXR (α, β or γ). By an "RXR heterodimer" is intended a dimer of an RXR (α, β or γ) and a non-RXR nuclear receptor capable of dimerizing with an RXR, including, but not limited to, an RAR (α, β or γ), VDR, TR (α or β),

PPAR (α, β, δ or γ), LXR (α or β), BXR, CAR (α or β), SXR and FXR. Preferred non-RXR nuclear receptors capable of dimerizing with an RXR include RARs, TRs, PPARs, LXRs, BXR, CARs, SXR and FXR. More preferred non- RXR nuclear receptors capable of dimerizing with an RXR include RARs, TRs and PPARs. Generally, the nuclear receptor structure contains an amino-terminal activation function (AF-1; A/B domain), the DNA-binding domain (DBD; C domain), a hinge region (D domain), and a carboxy-terminal ligand-binding domain, LBD (E domain), which includes the activation function AF-2, required for ligand-dependent activation by nuclear receptors. By an "agent which interacts with an RXR dimer" is intended a compound which binds to an RXR dimer to mediate transcription of a target gene, i.e., "RXR dimer mediated transcription." The agent can activate or repress transcription of the target gene. Such agents can be, but are not limited to, peptides, carbohydrates, steroids and vitamin derivatives, which may each be natural or synthetic (prepared, for example, using methods of synthetic organic and inorganic chemistry that are well-known in the art).

For example, such an agent includes a "retinoid" which is a compound which binds to one or more of the retinoid receptors (RARα, RARβ, RARγ, RXRα, RXRβ and RXRγ). Compounds can be either "RAR retinoids" or "RXR retinoids" depending on their binding characteristics (RAR retinoids bind to one or more RARs; RXR retinoids bind to one or more RXRs (also referred to as "rexinoids")). Of course, some of such compounds can bind to both RARs and RXRs.

Many RAR and RXR agonists and antagonists are known in the art, such as, for example, 4-[[(2,3-Dihydro-l,l,3,3-tetramethyl-2-oxo-lH-inden-5-yl) carbonyl]amino]benzoic acid (Compound I; WO 98/47861), 4-[[[5,6-Dihydro-5,5- dimethyl-8-(3-quinolinyl)-2-naphthalenyl]carbonyl]amino]benzoic acid (Compound II; U.S. PatentNo. 5,559,248; U.S. PatentNo. 5,849,923), 3-Fluoro- 4[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)hydroxyacetyl] aminojbenzoic acid (Compound IV; U.S. Patent No. 5,624,957), 4-[2-(5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-l,3-dioxolan-2-yl]benzoic acid (SRI 1237; U.S. Patent No. 5,552,271).

Agents that interact with RXR dimers, including RAR and RXR agonists and antagonists, can be screened using the methods of the present invention. In the invention, agents that interact with only one partner or both partners of the

RXR dimer can be identified. In fact, because the invention mimics the effects of retinoids on gene transactivation in vivo, the invention provides more accurate methods of identifying RXR dimer agonists and antagonists. Moreover, the action of the identified agent can be further confirmed by binding assays known in the art to determine which partner of the RXR dimer is bound by the identified agent

(Rochel, N. et al, Biochem. Biophys, Res. Comm. 230:293-296 (1997)).

Generally, agents which cause transactivation via their receptors are examples of "agonists," while agents which do not cause transactivation, but instead block the transactivation caused by other agonists, are examples of "antagonists." However, because CARs are constitutively expressed, a reverse agonist is needed to deactivate transcription and a reverse antagonist is needed to activate transcription.

Agents can have the ability to bind to multiple receptors. By agents that are "specific" for a nuclear receptor are intended compounds that only bind to one, two or three particular nuclear receptor(s) and not to others. By agents that are

"selective" for a nuclear receptor are intended compounds that preferably bind to one, two or three particular nuclear receptor(s) over others by a magnitude of approximately five-fold or greater than to other retinoid receptors, preferably eight-fold or greater, more preferably, ten-fold or greater. By a "ligand for a member of the nuclear receptor" is intended an agent, compound or hormone that binds to a nuclear receptor, which in turn can activate an appropriate hormone response element. Thus, a ligand acts to modulate transcription of a gene maintained under the control of a hormone response element. Ligands include hormones, steroid or steroid-like compounds, retinoids, thyroid hormones, pharmaceutically active compounds, and the like. Exemplary ligands include ligands for retinoid receptors (e.g., al\-trans retinoic acid, 9-cis retinoic acid, etc.), ligands for thyroid hormone receptors (e.g., thyroid hormone), and ligands for vitamin D₃ receptor (e.g., 1,25 -dihydroxy vitamin D₃). Other ligands which bind to nuclear receptors can be identified by the present invention. By a "hormone" is intended a substance produced in a gland of an animal, human and nonhuman, which exerts specific effects on other parts of the body. By a "co-regulator" is intended a "co-activator" or a "co-repressor." By a "co-activator" is intended a molecule or factor, generally a protein or RNA, that interacts with nuclear receptors and enhance their transactivation. The co-regulator can complex with other molecules or factors to interact with nuclear receptors. Exemplary co-activators include, but are not limited to, ERAP-160 (GRIP- 170; p 160), ERAP- 140, RIP- 140, RIP- 160, TBP/TAF_ns, SRC- 1 (hSRC- 1 ; NCoA-l/mSRC-1), hSRC-3, Trip-1 (Sug-1), Trips, TIFl α, TIFlβ, γ, ARA-70, TRAPs (DRIPs), CBP, p300, CBP, PCAF (hGCN5), TIF2/hSRC-2 (GRIP- l/mSRC-2;NCoA-2,pl60), mSRC-3/hSRC-3, TRIP230, L7/SPA,p/CIP/mSRC-

3 (ACTR/hSRC-3; RAC3/hSRC-3; AIB-L/hSRC; TRAM-l/hSRC-3; pi 60; SRC- 3), E6-AP, RPF-1 (hRSP5), BRG-1 (SWI2/SNF2), Brahma, NSD-1, PGC-1, HMG-1, HMG-2, NCoA-62, BX42, TSC-2 (Tuberin), PBP (TRAP220; TRIP2; mPIP9), positive cofactors (PC2; PC4), ADA, SMCC, SRA, SNURF, ARIP3, Mizl, PIAS3 and GBP (reviewed in, McKenna, E.J. et al, Endocrine Reviews

20:321-344 (1999); Torchia, J. et al, Curr. Opin. Cell Biol. 70:373-383 (1998)).

By a "co-repressor" is intended a molecule or factor, generally a protein or RNA, that interacts with nuclear receptors and lowers the transcription rate at their target genes. Exemplary co-repressors include, but are not limited to, NCoR (RIP- 13), SMRT (silencing mediator for retinoic acid and thyroid receptors;

TRAC2), repressor domains of SMRT (e.g., SRD-1, SRD-2, amino acids 1-981 thereof, etc.), TRUP (SURF-3; PLA-X; L7a), SUNCoR, NURD, mSin3A, protein-protein interaction domains of mSin3A (e.g., PAH-1, PAH-2, PAH-3, PAH-4, combinations thereof, etc.), N-CoR, Mad/Mxi-1, mSin3B, Sin3, etc. (reviewed in, McKenna, E.J. et al, Endocrine Reviews 20:321-344 (1999);

Torchia, J. et al, Curr. Opin. Cell Biol. 70:373-383 (1998)). In the invention, methods are provided for identifying a "modulator" which promote dissociation of the co-activator or co-repressor complex from the nuclear receptors (e.g., retinoid and/or thyroid hormone receptors) or promote association of co-activator or co-repressor complexes with the nuclear receptors. As used herein, an "agent" is alternatively intended a molecule, factor, substance or compound which is screened for an intended function, such as co-activator, co-repressor, or modulator function, as it will be clear from the context in which the term is used.

The RXR dimers of the invention can be obtained by expressing the receptors proteins in eukaryotic or bacteria cells and purifying the receptors. In one embodiment, the receptor is purified from tissues or cells which naturally produce the receptor. Alternatively, the receptor can be expressed recombinantly, for example, by inserting the gene encoding the receptor into the baculovirus or vaccinia virus genome and infecting the baculovirus or vaccinia virus, respectively, into insect or human cells, respectively. The receptors can also be expressed in yeast. Exemplary constructs for production of the receptor can be obtained from, for example, human, mouse or chicken, and include, but are not limited to, human Flag-tagged RARα and mouse His-tagged RXRα (Dilworth, et al, Proc. Natl. Acad. Sci. USA 96:2000-2004 ( 1999)), human Flag-tagged VDR and human Flag- tagged RXRα (Rachez et al, Nature 595:824-828 (1999)), and human Flag- tagged TRα (Fondell et al, Proc. Natl Acad. Sci. USA 96:1959-1964 (1996)). Other constructs can be generated by subcloning the cDNA from existing DNA vectors of, for example, LXRs (Willy et al, Genes & Dev. 9:1033-1045 (1995)), PPARα (Isseman and Green, Nature 547:645-650, FXR (Forman et al, Cell 57:687-693 (1995)), LXRα (Apfel et al, Mol. Cell Biol. 74:7025-7035 (1994)),

BXR (Blumberg etal, Genes & Dev. 72:1269-1277 (1998)), CARα and β (Choi et al, J. Biol. Chem. 272:23565-23571 (1997); Forman et al, Nature 595:612-

615 (1998)), and SXR (Blumberg et al, Genes & Dev. 72:3195-3205 (1998)).

A variety of methodologies are known in the art that can be used to obtain, isolate or purify the nuclear receptors, including, but not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and affinity chromatography.

Nuclear receptors bind to specific DNA sequences known as response elements (REs) or hormone response elements (HREs). Those of skill in the art can readily determine suitable hormone response elements (HREs) for use in the practice of the present invention, such as, for example, the response elements described in U.S. Patent No. 5,091,518 and WO 92/16546. The recognition of

REs by a given RXR dimer is dependent on the actual sequence, orientation and spacing of the repeated motifs. Naturally occurring HREs are composed of direct repeats (DRs; Umesono et al, Cell 65:1255-1266 (1991)), and inverted repeats (IRs; Umesono et al,

Nature 556:262-265 (1988)).

Direct repeats and inverted repeats can have a gap which separates the two core-binding sites. Thus, for example, spacers of 1, 3, 4 and 5 nucleotides serve as preferred DR response elements for heterodimers of RXR with PP AR, VDR,

T₃R and RAR, respectively (Naar et al, Cell 65: 1267- 1279 ( 1991 ); Kliewer et al,

Nature 358:111-114 (1992); and lssemann etα/., Biochimie 75:251-256 (1993)).

The optimal gap length for each heterodimer is determined by protein-protein contacts which appropriately position the DNA binding domains (DBDs) of RXR and its partner (Kurokawa et al, Genes & Dev. 7:1423-1435 (1993); Perlmann et al, Genes & Dev. 7:1411-1422 (1993); Towers et al, Proc. Natl Acad. Sci.

USA 90:6310-6314 (1993); and Zechel et al, EMBO J 75:1414-1424 (1994)).

Exemplary DRl is provided in Vivat et al, EMBO J 76:5697-5709 (1997).

Exemplary DR3 is provided in Rachez et al, Nature 595:824-828 (1999). Exemplary DR4 is provided in Fondell et al. , Proc. Natl Acad. Sci. USA 96: 1959-

1964 (1996). Exemplary DR5 is provided herein and in Dilworth et al, Proc.

Natl. Acad. Sci. USA 96:1995-2000 (1999).

The preferential RXR/RAR heterodimer binding repertoire in vitro to

DNA (DRl, DR2, and DR5, in order of increasing efficiency) is similar to the "natural" RARE repertoire, which suggest that RXR/RAR»heterodimers are the functional units that transduce the retinoid signal in vivo. Similarly, RXR/TR, RXR/VDR, and RXR/PPAR bind preferentially to DR4, DR3 , and DRl elements, respectively (Giguere, V. Endocr. Rev. 75:61-79(1994); Glass, C.K. Endocr. Rev. 75:391-407 (1994); and Mader, S. et al J. Biol. Chem. 265:591-600 (1993)). RXRs also bind as homodimers to a DRl element (Nakshatri, H., and Chambon, P. J. Biol Chem. 269:890-902 (1994)).

RXR/LXR binds to DR4, RXR/BXR binds to modified DR4, RXR/CAR binds to DR5, RXR SXR binds to DR4, and RXR/FXR binds to IR1 (inverted repeat with a 1 bp spacer).

Direct repeat hormone response elements (HREs) contemplated for use in the practice of the invention are composed of at least one direct repeat of two or more half sites, optionally separated by one or more spacer nucleotides (with spacers of 1 -5 preferred). The spacer nucleotides can be selected from any one of A, C, G or T. Each half site of direct repeat HREs contemplated for use in the practice of the invention comprises the sequence -RGBNNM- wherein R is selected from A or G; B is selected from G, C, or T; each N is independently selected from A, T, C, or G; and M is selected from A or C; with the proviso that at least 4 nucleotides of said -RGBNNM- sequence are identical with the nucleotides at corresponding positions of the sequence -AGGTCA-. Response elements employed in the practice of the invention can optionally be preceded by N_x, wherein x falls in the range of 0 up to 5. Exemplary hormone response elements include, but are not limited to, direct repeats of -PuG(G/A)(T/A)CA- (Mader, S. et al, J Biol Chem. 265:591-600 (1993)).

Response elements are operatively linked to a reporter or target gene, whereby expression of the reporter or target gene indicates the action of a ligand, RXR dimer and/or the response element. Exemplary reporter genes include, but are not limited to, chloramphenicol acetyl transferase (CAT), β-galactosidase (β- gal), luciferase (LUC), and β-globin.

In a steady state, eukaryotic chromosomes ("chromatin") are organized into a repeating protein DNA unit, the nucleosome. The basic protein unit of the nucleosome is the histone, a small, highly basic, globular moiety. A nucleosome core particle contains a histone octamer, made up of two copies of each of histones H2A, H2B, H3 and H4, around which is wrapped 1.7 turns of a left- handed DNA superhelix (-200 bp of DNA).

By a "chromatin based DNA template" or "chromatin template" or "chromatin assembled template" is intended a nucleosomal array generated by complexing an oligonucleotide sequence with histone octamers (H2A, H2B, H3 and H4) an or histone HI. The oligonucleotide sequence or DNA template comprises a hormone response element, at least a minimal promoter element (including a TATA box and a transcription start site, i.e., -35 to +80 of any natural eukaryotic or viral gene promoter), and a reporter gene, as described above. A "naked" oligonucleotide sequence or DNA template is not complexed with histone octamers.

A chromatin based DNA template is prepared by adding a chromatin assembly extract to the oligonucleotide sequence in the presence of histones. A chromatin assembly extract contains the proteins and factors necessary for assembly of the DNA template around the histones into nucleosomes and for movement of the nucleosome along the DNA template to allow transcriptionally repressive and permissive states.

Methods for preparing chromatin assembly extracts useful in the present invention are known in the art (Becker, P.B. et al, Meth. Cell Biol. 44:201-223 (1994); Pazin, M.J. etal, Science 266:2007-2011 (1994); Kamakaka, R.T. etal,

Genes & Dev. 7:1779-1795 (1993)).

Chromatin assembly extracts can be prepared, for example, from tissue culture cells (Banerjee, S. and Cantor, C.R., Mol Cell Biol. 70:2863-2873 (1990)), Xenopus eggs and oocytes (Almouzni, G. and Mechali, M., EMBO J. 9:573-582 (1988)); Shimamura, A. et al, Mol. Cell. Biol 5:4257-4269 (1988)),

Drosophila ISWI (Ito et al, Genes & Dev. 75:1529-1539 (1999); Carona et al. Mol. Cell 5:239-245 (1999)), human SNF2h (Leroy et al, Science 252:1900- 1904 (1998)), and preferably, Drosophila embryos (Becker, P.B. and Wu, C, Mol. Cell Biol 72:2241-2249 (1992); Becker, P.B. et al, Methods Cell Biol. 44:201-223 (1994)). For example, a method of preparation of S-190 Drosophila chromatin assembly extracts is provided in the "Materials and Methods" section, infra. A method for preparing S-150 chromatin assembly extracts is provided in Becker, P.B. et al, Meth. Cell Biol 44:201-223 (1994). Chromatin can be assembled on relaxed or supercoiled circular DNA by preincubating the extract with histones to assemble histone octamers and adding the template of interest. Core histones can be purified according to the method of Simon, R.H., & Felsenfeld, G., Nucl Acids. Res. 6:689-696 (1979), or calf thymus histones are commercially available (Boehringer Mannheim). The appropriate amount of histones can be determined empirically, using as a guide a stoichiometry of histones to DNA of -0.8:1 (w/w) (Albright, S.C. et al, J. Biol. Chem. 254:1065-1073 (1979)). Details of a method for chromatin assembly on a DNA template are provided in the "Example" section, infra.

DNA supercoiling assay is based on topological changes that accompany the wrapping of DNA around a nucleosome core (Becker, P.B. et al, Meth. Cell

Biol 44:201-223 (1994)). Winding of DNA around a nucleosome core introduces one positive superhelical turn in the plasmid DNA, which is relaxed by topoisomerase I activity present in the embryo extracts. When nucleosomes are removed by proteinase K digestion and DNA purification, one negative superhelical turn corresponding to each assembled nucleosome appears in the closed circular DNA. The superhelical density of a plasmid, i.e., the absolute number of superhelical turns, can be directly counted by visualization of the plasmid topoisomers on two-dimensional agarose gels or by resolving duplicate samples on multiple agarose gels containing different chloroquine concentrations. The introduction of supercoils into a plasmid can simply be visualized by agarose gel electrophoresis as a rapid indicator of nucleosome reconstitution.

Generally, supercoiling of the chromatin can be assayed by incubating the assembled chromatin with the heterodimer (e.g., Flag-tagged human RARα/His- tagged mouse RXRα) in the presence of ligand. Supercoiling can be determined by adding topoisomerase I and/or chloroquine and resolving the DNA on an agarose gel. Details of a supercoiling assay are provided in the "Materials and Methods" section.

DNA supercoiling measures the wrapping of DNA around a particle but does not necessarily indicate the reconstitution of a full octamer of core histones (Becker, P.B. et al, Meth. Cell Biol 44:207-223 (1994)). The winding of DNA around a complete histone octamer or a tetramer of histones H3 and H4 cannot be distinguished by the supercoiling assay. Therefore, nuclease digestion assay is used to provide information on the nature of the nucleosome core particle as well as on the average distance between particles. Generally, no more than 20% of the genome is organized as active chromatin in a given cell type. Active chromatin is less compact than bulk chromatin, and is more accessible to enzymes. Nuclease digestions can be used to investigate changes in nucleosome organization and positioning around a given gene in different cell types and stages. Such nucleases include, for example, DNase I, DNase II, micrococcal nuclease, SI nuclease, copper/phenanthroline, and restriction enzymes.

Micrococcal nuclease (MNase) assay relies on the ability of MNase to preferentially cleave the linker DNA between nucleosome core particles. After the initial endonucleolytic attack of linker DNA, the trimming activity associated with the enzyme progressively removes the linker DNA. Extensive digestion of chromatin with MNase can bring the size of the mononucleosome from 160-220 bp to the 147 bp DNA fragment protected by the nucleosome core particle whereas a partial digest results in a ladder of fragments representing oligonucleosomal DNAs. Details of a MNase assay is provided in the "Materials and Methods" section, infra.

The invention lends itself readily to the preparation of kits containing the elements necessary to carry out the methods disclosed herein. Such a kit can comprise a carrier being compartmentalized to receive in close confinement therein one or more contain means, such as tubes or vials. One of the container means can contain the DNA template. One of the container means can contain the chromatin extract. One or more of the container means can contain the histones. One or more of the containers can contain known agonists, antagonists, co-activators, co-repressors or modulators which can be used as controls. In addition, the kit can also include a "catalog" defined broadly as a booklet, book pamphlet, computer disk or the like, which can assist in carrying out the invention. The kit can contain all of the additional elements necessary to carry out the method of the invention, such as buffers, enzymes, pipettes, tubes, nucleic acids, nucleoside triphosphates, and the like.

As described herein, by a "compound" is intended a protein, nucleic acid, carbohydrate, lipid or a small molecule. It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of the invention or any embodiment thereof.

The following Example serve only to illustrate the invention, and are not to be construed as in any way to limit the invention.

Example

The supercoiled plasmid (DR5)5β2G that contains the RARβ2 core promoter (- 35 to +85) and five copies of the RA response element (RARE) of the RARβ2 gene was used to study activation of transcription by RAR/RXR heterodimers (see Materials and Methods; see FIGs. 1 and 2B). To determine whether a chromatin-assembled template was important, the transcriptional activity of purified RARα/RXRα heterodimers was analyzed (see, "Materials and Methods" section and FIG. 2A), using both naked and chromatin based DNA templates. Periodic nucleosomal arrays (FIG. 2B) were generated using supercoiled (DR5)5β2G plasmid and a chromatin-assembly extract (see,

"Materials and Methods" section). Note that the nucleosomal organization of the chromatin template was not grossly affected by the addition of RARα/RXRα heterodimers and tRA (FIG. 2B). When expressed relative to basal transcription from the internal control naked pGl template (FIG. 1), "constitutive" transcription on the naked (DR5)5β2G template was not affected by the presence of RA ligand and/or receptor heterodimers (FIG. 3 A). In marked contrast, very little transcription was observed from the corresponding chromatin template in the absence of

RARα/RXRα heterodimers, irrespective of the presence of RA. However, the addition of both heterodimers and tRA resulted in a potent activation of transcription (between 30- to 100-fold; FIG. 3A). Little ligand-dependent activation of transcription by RARα/RXRα heterodimers was observed when exogenous histones were not added to the Drosophila extract during chromatin assembly on the (DR5)5β2G plasmid. Optimal activation of transcription from the chromatin template was achieved using 1 nM RARα/RXRα heterodimer that corresponds to approximately five heterodimers per (DR5)5β2G template molecule (200 pM), i.e., one heterodimer per DR5 response element (FIG. 3A). Consistent with this observation, DNase I foot printing analysis showed that all five DR5 RAREs were bound by RARα/RXRα heterodimers at these concentrations, with no RARE being particularly favored (FIG. 4). Note that in contrast to activation of transcription, the binding of the heterodimers to the chromatin template was not dependent on the presence of tRA (FIG. 4). Binding of unliganded RAR/RXR heterodimers to chromatin is therefore clearly not sufficient for transcriptional activation, thus suggesting that the critical step in transactivation is a ligand-dependent transconformation of DNA-bound heterodimers.

The response element specificity of transcriptional activation by RARα/RXR heterodimers was examined by comparing transcription from the cognate (DR5)5β2G template and the (17M)5β2G template, in which the five DR5 RAREs have been replaced by five copies of the 17-mer binding site for the DNA binding domain [GAL(1-147)] of the yeast transactivator Gal4 (FIG. 1). RARα/RXRα heterodimers did not activate transcription from the chromatin- assembled (17M)5β2G template, whereas under similar conditions, the chimeric acidic transactivator GAL-VP16 (Sadowski, I., Nature 555:563-564 (1988)) efficiently activated transcription from that template, but not from the chromatin- assembled (DR5)5β2G template (FIG. 3B).

The above results demonstrate that activation of transcription by RAR/RXR heterodimers is dependent on packaging of the template into a nucleosomal structure and that it is specific, in that it requires the heterodimer, a cognate ligand and a cognate response element. As tRA binds RARs, but not RXRs, the effect of ligands that bind to RXRs was then investigated. Of interest, 9cRA that binds both RARs and RXRs was more efficient than tRA at limiting concentrations, with ED₅₀ of approximately 9x10^{" 10} M and 4x10^"9 M for 9cRA and tRA, respectively (FIG. 5 A). Because these differential effects of 9cRA and tRA suggested that synergistic activation of transcription might occur when both RARα and RXRα are liganded, transcriptional activation by RARα/RXRα heterodimers upon addition of receptor-specific synthetic retinoids (FIG.5B) was investigated. As expected, a stimulation was observed in the presence of the RARα-specific agonist Compound I, but not on addition of either the RARγ- specific agonist Compound IV or the RARα antagonist Compound II (Chen, J-Y., et al, Nature 552:819-822 (1996)) (FIG. 5B). Of interest, the RXR-specific pan-agonist SRI 1237 (Chen, J-Y., et al, Nature 552:819-822 (1996)) did not activate transcription on its own (FIG. 5B). However, a synergistic stimulation was observed upon concomitant addition of SR 11237 and limiting concentrations of RAR agonists (FIG. 5B; compare 10 ⁸ and 5x10^'8 M tRA in the presence and absence of SRI 1237, and also Compound I in the presence and absence of SRI 1237). In contrast, no stimulation resulted from the simultaneous addition of the RARα antagonist Compound II and the RXR agonist SRI 1237. It appears therefore that the AF-2 activation function of RXRα can act synergistically with that of RARα, but that the binding of an agonist to RARα is a prerequisite for effective activation of transcription by agonist-bound RXRα. This conclusion was further supported by the observation that the RARα-specific antagonist Compound II abrogated the synergistic effect of the RARα-specific agonist Compound I and RXR agonist SRI 1237 (FIG. 5B). Similarly, Compound II abrogated the 9cRA-induced transcriptional activation by RARα/RXRα heterodimers (FIG. 5B), even though 9cRA binds to both RARs and RXRs.

Acetylation and deacetylation of nucleosomal histones in transcriptionally active (euchromatin) and inactive (heterochromatin) chromatin, respectively, is well documented (reviewed in Kuo. M-H. & Allis, CD., BioEssays 20:615-626

(1998)). The facilitating role of histone acetylation in transcriptional activation has also been recently demonstrated in vitro (Nightingale, K.P., et al, EMBO J. 17:2865-2876 (1998); \lt\ey, R.Υ., etal, Nature 594:498-502 (1998)). The effect of the addition of acetylCoA to the in vitro transcription system was examined. No effect could be evidenced using either RARα/RXRα heterodimers or the Gal-

VP16 activator, in the presence of either naked or chromatin-assembled cognate templates (FIG. 6A). Because certain coactivators are thought to mediate transactivation by nuclear receptors at least in part through their intrinsic histone acetyltransferase activities (e.g. SRC-1, ACTR, CBP and p300, etc.), it was investigated whether addition of purified baculovirus-expressed p300 could stimulate transcriptional activation by RARα/RXRα in the in vitro system. p300 enhanced the activation of transcription by the heterodimers ~4-fold in the presence of tRA, while transcription of the chromatin template remained repressed in the absence of the agonistic ligand, irrespective of the presence of the heterodimers (FIG. 6B). No p300 effect was seen on naked DNA templates. The further addition of acetylCoA had no effect on the extent of transcriptional enhancement, even though the purified p300 coactivator exhibited histone acetyltransferase activity. However, the in vitro system contains some endogenous histone acetyltransferase activity that was not further enhanced by the addition of p300 to the transcription reaction.

Materials and Methods

DNA and Chromatin Templates. The plasmids (DR5)5β2G and (17M)5β2G (-5.2 kb) were constructed by inserting five copies of the DR5 RA response element from the mouse RARβ2 promoter or the 17-mer GAL4 binding site, respectively, upstream of the mouse RARβ2 core promoter [-35 to +85] which had been previously linked to the -9 to +1516 chicken β-globin gene sequence (FIG. 1). The most 3 ' DR5 element is positioned at approximately the same distance from the TATA box as the DR5 RARE found in the natural RARβ2 promoter (Zelent, A., et al, EMBOJ. 9:71-81 (1991)).

Chromatin assembly extracts were prepared from Drosophila embryos (0-6 hr) as described in Kamakaka, R.T. et al, Genes & Dev. 7:1779-1795 (1993)). Canton-S wild-type flies were grown at 25 °C at 70-80% humidity in population cages. The embryos were collected on apple juice-agar plates covered with yeast. Four batches of embryos [typically, 30-50 grams, collected (every 6 hr over a 24-hr period) between 0 and 6 hr after fertilization and then stored for < 18 hr at 4 °C], were harvested in nylon mesh with water and then dechorionated by immersion for 90 sec in 1 :1 (vol/vol) bleach [5.25% (wt/vol) sodium hypochlorite]/water [final concentration of sodium hypochlorite is 2.63% (wt/vol)] at room temperature. The embryos were quickly rinsed with embryo wash buffer

I [1 liter; 0.7% (wt/vol) NaCl and 0.04% (vol/vol) Triton X-100; buffer at room temperature], washed with water (at room temperature), and transferred to a 800- ml beaker in an ice bath. Embryo wash buffer II [500 ml, buffer at 4°C; 0.7% (wt/vol) NaCl and 0.05% (vol/vol) Triton X-100] was added to the beaker. The embryos were suspended with a glass rod and allowed to settle to the bottom of the beaker (for -2 min). The cloudy suspension above the embryos, which contained chorion particles and other debris, was removed by aspiration. This wash/settling/aspiration procedure was repeated once with embryo wash buffer II (500 ml; at 4°C), twice with 0.7% (wt/vol) NaCl solution (500 ml for each wash; buffer at 4°C), and once with buffer R [500 ml; buffer at 4°C; 10 mM HEPES

(K⁺) (pH 7.5), 10 mM KC1, 1.5 mM MgCl₂, 0.5 mM EGTA, 10% (vol/vol) glycerol, 10 mM β-glycerophosphate, 1 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride (PMSF)]. In the final wash, the embryos take longer to settle (-10 min), and the final volume of the embryo suspension before homogenization is roughly twice of that of the loosely packed volume of dechorionated embryos. The embryos were then transferred to a Wheaton Dounce homogenizer (40 ml) and disrupted by 15 strokes with the B pestle followed by 40 strokes with the A pestle. The homogenate was subjected to centrifugation in a Falcon 2059 tube in a Sorvall SS-34 rotor at 8000 rpm for 5 min. at 4°C The cloudy, yellow cytoplasmic fraction was collected with a syringe (the white layer at the top and the pellet at the bottom of the tube were avoided). MgCl₂ (from a 1 M stock solution) was added to increase the Mg(II) concentration from 1.5 mM to a final concentration of 7 mM. The extract was then subjected to centrifugation in a Beckman SW55 rotor at 45,000 rpm (192,000g) for 2 hr at 4°C After centrifugation, the white upper layer was removed with a spatula and the yellow- brown liquid was collected. This supernatant fraction was frozen in liquid nitrogen, thawed in water (at room temperature), and then subjected to a second centrifugation in the Beckman SW55 rotor at 45,000 rpm for 2 hr at 4°C The resulting chromatin reconstitution extract (also referred to as the Drosophila S-190 extract) was frozen in liquid nitrogen and stored at - 80°C The extracts remain active for >1 year at - 80° C

Chromatin was assembled on supercoiled circular DNA (see FIG. 2B) as follows. The chromatin assembly extract was preincubated with 3 μg of calf thymus core histones (Boehringer Mannheim) at room temperature to assemble histone octamers. After 30 minutes, 1 μg of(DR5)5β2G (or (17m)5β2G) and an ATP regeneration solution (3 mM MgCl₂, 1 mM DTT, 30 mM creatine phosphate,

3 mM ATP, 1 μg/mL creatine kinase) were added and allowed to incubate for 4 hr at 27°C (Becker, P.B., et al, Methods Cell Biology 44:201-223 (1994)).

Supercoiling assays were performed essentially as previously described (Pazin, M. J., et al , Science 266:2007-2011 (1994)). Two hundred nanograms of assembled chromatin (or naked control template) was incubated with 1 nM Flag- tagged human RARα/His-tagged mouse RXRα (FhRARα/HmRXRα) heterodimer in the presence of ligand or vehicle for 30 min at 27 °C The concentration of MgCl₂ was increased to 11.5 mM immediately before the addition of 10 units of topoisomerase I (Life Technologies, Cergy Pontoise, France), and allowed to incubate at 37 °C for 30 min. DNA was resolved on a 1% agarose gel in the presence or absence of 1.2 μM chloroquine for 18 hr at 2 volts/cm. Determination of supercoiling within (DR5)5β2G chromatin template using topoisomerase I and/or chloroquine (Becker, P.B., et al, Methods Cell Biology 44:201-223 (1994)) indicated the presence of at least 25 nucleosomes.

Micrococcal digestion analysis of reconstituted chromatin was performed essentially as previously described (Bellard, M., et al, in Methods Enzymol

770:317-346 (1989)). Assembled chromatin was incubated with 1 nM FhRARα/HmRXRα heterodimer or vehicle in the presence of 10^"6 M tRA for 30 min at 27 °C Chromatin was then digested with varying concentrations of micrococcal nucleate (1-20 units) for 1 min at 27 °C Proteins were removed by proteinase K treatment followed by an extraction with phenol-chloroform.

Digested DNA was separated on a 1.5% agarose gel for 4 hr at 4 volts/cm, transferred to a nitrocellulose membrane and analyzed by southern blotting using a probe corresponding to the promoter region of the (DR5)5βG plasmid. Micrococcal nuclease digestion (Bellard, M., et al, in Methods Enzymol. 770:317-346 (1989)) showed that they had a periodicity of approximately 160 bp

(FIG. 2B).

DNase I footprinting was performed essentially as previously described (Pazin, M.J., et al, Science 266:2007-2011 (1994)). Chromatin was assembled on 250 ng of (DR5)5β2G plasmid then incubated alone or with 1 nM FhRARα/HmRXRα heterodimer in the presence or absence of 10-6 M RA for

30 min at 27 °C CaCl₂ was added to a final concentration of 3 mM along with 5 U of DNase I (Boehringer Mannheim) for 90 sec. DNA fragments were amplified with VentR (exo-) [New England Biolabs, Beverley, MA] using a 30 bp primer complementary to a sequence located between -280 and -250 upstream of the RARβ2 promoter start site.

Protein Expression and Purification. The Spodoptera frugipenda cell line S/ was co-infected with baculoviruses expressing His-tagged mouse RXRα (HmRXRα) and Flag-tagged human RARα (FhRARα) for 48 hr. Sβ cells expressing the heterodimeric proteins were lysed by homogenization in a low salt buffer (20 mM Hepes pH 7.6, 100 mM KC1, 10 mM imidazole, lx PIC

[2.5 μg/mL leupeptin, 2.5 μg/mL pepstatin, 2.5 μg/mL aprotinin, 2.5 μg/mL antipain, 2.5 μg/mL chymostatin], and 1 mM PMSF). The FhRARα/HmRXRα heterodimer was partially purified by chromatography using a Ni²⁺ column (Amersham Pharmacia) and eluted with a low salt buffer containing 300 mM imidazole. The heterodimer was then further purified from the Ni²⁺ column eluate by affinity purification using agarose-coupled M2 anti-Flag antibodies (Sigma), as specified in the manufacturer's instructions. The purified heterodimer was eluted from the resin in a buffer consisting of 20 mM Hepes pH 7.6, 100 mM KCl, 1.5 mM MgCl₂, 0.5 mM EGTA, 50 μM ZnCl₂, 15% glycerol, 500 μg/mL competitor peptide (DYKDDDDK) (SEQ ID NO:l), 1 mM DTT, 1 mM PMSF and lx PIC.

Western blot analysis was performed by using the monoclonal antibodies anti-RARα 9α-9A6 (Gaub, M.P., et al, Exp. Cell Res. 201:335-346 (1992)) and anti-RXRα 1RX-6G12 (Rochette-Egly, C, et al, Biochem. Biophys. Res. Commun. 204:525-536 (1994)) (FIG. 2A). The DNA binding properties of the heterodimer were examined by electrophoretic mobility shift analysis (Kumar, V. & Chambon, P., Cell 55:145- 156 (1988)). Briefly, the purified heterodimer was incubated with 10^"6 M tRA on ice. After 15 min., the [³²P]-labeled DR5 oligonucleotide 5'-TCGGGAGGGTTCACCGAAAGTTCACTCGCC-3' (SEQ ID NO:2) hybridized to its unlabeled compliment were added to the reaction in the presence of 20 mM Hepes pH 7.6, 10 mM KCl, 10% Glycerol, 1.5 mM MgCl₂, 0.5 mM EGTA, 1 mg/mL BSA, and 2 μg poly dldC The reaction was then allowed to proceed for another 15 min at 22 °C Reaction mixtures were resolved by polyacrylamide gel electrophoresis (5% in 0.5x TBE) for 4 hr at 150 volts, dried and visualized by autoradiography.

The integrity of the ligand binding domain of RARα was examined using a ligand binding assay (Rochel, N., et al, Biochem. Biophys. Res. Comm. 230:293-296 (1997)). The RARα/RXRα heterodimer (10 fmol) was diluted to 200 μl in a buffer of 10 mM Tris-HCl (pH 8.0), 150 mM KCl then incubated in the presence of varying concentrations of diluted [³H]tRA (5xl0^"10 - 5xl0^"8 M) on ice.

After 4 hr, unbound RA was separated from RARα-bound RA by washing through GF/C glass fibre filters (Whatman, Maidstone, England) using a buffer consisting of 50 mM Tris-HCl pH 7.5, 154 mMNaCl, 0.01% TritonX-100. The amount of [³H]tRA bound to the heterodimer was determined by liquid scintillation counting. Full-length p300 was prepared from Sβ cells infected with a p300- expressing baculovirus (Kraus, W.L. & Kadonaga, J.T., Genes Dev. 72:331-342 (1998)), its purification was monitored using arabbit polyclonal anti-p300 (C-20) antibody (Santa Cruz Biotechnology), and the histone acetyltransferase activity of the purified protein was confirmed as described (Ogryzko, V.V., Cell 87:953- 959 (1996)). His-tagged Gal(l-147) and GAL-VP16 (Tora, L., et al, Cell

59:477-487 (1989)) were expressed from pET3 expression vectors in the BL-21 pLysS bacterial strain and purified by Ni²⁺ column chromatography.

In vitro Transcription. Transcription was performed using a HeLa cell nuclear extract (Dignam, J.D., etal, Nucleic Acids Res 77:1475-1489 (1983)) as described (Pazin, M.J., et al, Science 266:2007-2011 (1994)). Chromatin or naked templates were incubated with 1 nM FhRARα/HmRXRα heterodimers (in the presence of ligand or vehicle) for 30 min at 27 °C prior to transcription initiation. Transcription reactions were initiated by the addition of the 100 μg HeLa nuclear extract (Dignam, J.D. et al, Nucl Acids Res. 77:1475-1489 (1983)), pGl (internal control plasmid) (Sassone-Corsi, P., et al, Cold Spring

Harbor Symp. Quant. Biol 50:141-152 (1985); see FIG. 1), and rNTPs (0.5 mM) and incubated at 30 °C for 45 min. Transcription was quantitated by SI nuclease analysis (Tora, L., et al. , Cell 59:477-487 (1989)) using a [³²P]-labeled probe (S 1 probe) that hybridizes with transcripts from the (DR5)5β2G, (17 m)5β2G and pG 1 plasmids through their transcription start sites to yield fragments of 179, 179 and 60 nt, respectively (FIG. 1).

Retinoids. 4-[[(2,3-Dihydro-l,l,3,3-tetramethyl-2-oxo-lH-inden-5-yl) carbonyl]amino]benzoic acid (Compound I; WO 98/47861), 4-[[[5,6-Dihydro-5,5- dimethyl-8-(3-quinolinyl)-2-naphthalenyl]carbonyl]amino]benzoic acid (Compound II; U.S. PatentNo. 5,559,248; U.S. PatentNo. 5,849,923), 3-Fluoro-

4[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)hydroxyacetyl] amino]benzoic acid (Compound IV; U.S. PatentNo. 5,624,957), SRI 1237 (U.S. Patent No. 5,552,271) were obtained from Bristol-Myers Squibb Company.

Discussion

Numerous studies aimed at reproducing transactivation by nuclear receptors in vitro have been reported over the last years. Most of these studies were performed with naked DNA templates and the reported transcriptional activations were either ligand-independent (Shemshedini, L., etal, J. Biol. Chem. 267:1834-1839 (1992); Sch itt, J. & Stunnenberg, H.G., Nucleic Acids Res 27:2673-2681 (1993); De Vos, P., et al, Nucleic Acids Res. 22:1161-1166 (1994)) or only modestly dependent on addition of agonistic ligands (Valcarcel,

R., etal, Genes Dev 5:3068-3079 (1994); Fondell, J.D., etal, Proc. Natl. Acad. Sci. USA 95:8329-8333 (1996); Lemon, B.D., et al, Mol. Cell. Biol 77:1923- 1937 (1997)). Moreover, these studies did not or only poorly reproduced the effects of agonistic and antagonistic ligands, as observed on responsive genes in vivo. In one case (Schild, C, et al, EMBO J 72:423-433 (1993)), a greater stimulation was observed with salt dialysis-reconstituted chromatin templates than with naked DNA templates, but the ligand-dependency of transcriptional activation was not established. With the recent finding that several putative transcriptional coactivators that interact in an agonist-ligand-dependent manner with NRs possess histone acetyltransferase activity, it became evident that more physiologically relevant templates might be required to faithfully reproduce in vitro the essential features of ligand-dependent transcriptional activations as observed in vivo. Estrogen- and anti-estrogen-regulated transcriptional activation by the estrogen receptor α (ERα), resembling the natural mechanism of action of ERα in vivo, was in fact recently achieved in vitro with chromatin, but not with naked DNA templates (Kraus, W.L. & Kadonaga, J.T., Genes Dev. 72:331-342 (1998)). Using a similar approach, it is shown by the present invention that "constitutive" transcription from a naked DNA template containing a RA- responsive promoter [(DR5)5β2G] is not affected by RARα/RXRα heterodimers, irrespective of the presence of the tRA agonist. In contrast, there is very little transcription when the same promoter is present in chromatin-assembled templates, unless tRA is bound to the RAR/RXR heterodimers, which results in activation of transcription to a level similar to that achieved with naked DNA templates. These observations clearly establish that the tRA-induced transcriptional activation mediated by RXR/RAR heterodimers corresponds to the relief of a repression generated by the chromatin organization of the template. Moreover, these results show that this relief does not correspond to the binding of RXR/RAR heterodimers to the chromatin template, because it is clear from DNase I footprinting data (FIG.4) that unliganded heterodimers specifically bind to the RA response elements. Thus, the critical events underlying ligand-induced transcriptional activation by RXR/RAR heterodimers must occur subsequent to their binding to the chromatin template.

Previous studies of the effects of retinoids on transcription of RA- responsive genes, differentiation and apoptosis of mouse embryonal carcinoma cells (Clifford, J., etal, EMBOJ. 75:4142-4155 (1996); Chiba, H., etal, J. Cell Science 759:735-747 (1997); Chiba, H., et al, Mol. Cell. Biol. 77:3013-3020 (1997); Roy, B., et al, Mol. Cell. Biol. 75:6481-6487 (1995); Horn, V., et al, FASEB J. 70: 1071-1077 (1996); Taneja, R., et al, Proc. Natl. Acad. Sci. USA 95:6197-6202 (1996); Taneja, R.,etα/.,E 5OJ. 76:6452-6465 (1997);Minucci,

S., et al, Mol Cell. Biol. 77:644-655 (1997)) and human acute promyelocytic leukemia cells (Chen, J-Y., etal, Nature 552:819-822 (1996)), as well as genetic studies in the mouse (Kastner, P., et al, Development 724:313-326 (1997); Mascrez, B., etal, Development 725:4691-4707 (1998); and references therein), have established that RAR/RXR heterodimers are the main functional units mediating the effect of retinoids in vivo (see "Related Art" section for additional references). Furthermore, these studies have shown that the ligand-dependent activation function AF-2 of both RAR and RXR partners are instrumental in this mediation. However, in all cases there is a subordination of the activity of RXR AF-2 to the binding of an agonistic ligand to the RAR partner. Most remarkably, the present in vitro system reproduces these in vivo features of retinoid action. Although ligands binding to RARα but not to RXRα (tRA, Compound I) can activate transcription on their own, a ligand binding to RXRα but not to RARα (SR 11237) is inactive on its own. Consistent with the subordination of RXR AF-2 activity to that of RAR AF-2, the transcriptional activation brought about by 9-cis RA that induces both RAR and RXR AF-2s, is abrogated by the addition of the

RARα antagonist Compound II. Furthermore, in agreement with previous in vivo observations (Clifford, J., et al, EMBOJ. 75:4142-4155 (1996); Roy, B., et l, Mol. Cell. Biol. 75:6481-6487 (1995); Taneja, R., et al, Proc. Natl. Acad. Sci. USA 95:6197-6202 (1996); Taneja, R., et al, EMBO J. 76:6452-6465 (1997); Botling, J., et al, J. Biol. Chem. 272:9443-9449 (1997); Minucci, S., et al, Mol.

Cell. Biol. 77:644-655 (1997); Durand, B., et al, EMBO J 75:5370-5382 ( 1994)), synergistic effects between limiting amounts of RAR ligands and a RXR- specific ligand were observed.

Structural studies (Wurtz, J-M., et al, Nature Struct. Biol. 5:87-94 (1996); Moras, D. & Gronemeyer, H., Curr. Opin. Cell. Biol. 70:384-391 (1998);

Nolte, R.T., etal, Nature 595:137-144 (1998); Darimont, B.D., et al, Genes & Dev. 72:3343-3356 (1998); and references therein) have demonstrated that binding of an agonistic ligand triggers a transconformation of the ligand binding domain. This generates an interaction surface for coactivators of the activation function AF-2, that are thought to recruit factors of the general transcription machinery and/or act on chromatin remodeling through histone acetyltransferase activities. Interestingly, Kraus and Kadonaga (Kraus, W.L. & Kadonaga, J.T., Genes & Dev. 72:331-342 (1998)) have recently reported that the p300 coactivator (Hanstein, B., et al, Proc. Natl. Acad. Sci. USA 95:11540-11545 (1996); Chakravarti, D., et al, Nature 555:99-103 (1996)) acts synergistically with ligand-activated ERα to stimulate transcription in vitro from a cognate chromatin template. Similarly, upon addition of exogenous p300, a further 4-fold enhancement was observed in ligand-dependent transcription from chromatin templates in the presence of RXRα/RARα heterodimers. Note that the actual level of enhancement by p300 is likely to be higher, because endogenous p300/CBP is already present in the HeLa cell extract used in the transcription system. However, though the purified p300 exhibits intrinsic histone acetyltransferase activity, the addition of acetylCoA to the present transcription system has no further effect on p300-activated transcription. A similar observation was made by Naar et al. (Naar, A.M., et al, Genes & Dev. 12:3020- 3031 (1998)) in a study of Spl/SREBP- 1 -activated transcription on a chromatin template in the presence of CBP. Thus, p300 may further enhance ligand-induced activation of transcription on chromatin templates by bridging RXRα/RARα heterodimers to RNA polymerase II through its interaction with RNA helicase A (Nakajima, T., et al, Cell 90:1107-1112 (1997)), rather than by locally remodelling the chromatin structure through histone acetylation.

In conclusion, an in vitro chromatin based DNA template transcription system to study the molecular events underlying activation of transcription by RXR/RAR heterodimers, that essentially mimics the in vivo synergistic effect of RAR- and RXR-selective retinoids and the subordination of RXR AF-2 activity to binding of an agonist to RAR, has been established. This presently crude system can now be dissected biochemically to ultimately provide a thorough molecular view of the events that occur during ligand-dependent transcriptional activation by retinoid receptors.

All documents, e.g., scientific publications, patents and patent publications recited herein are hereby incorporated by reference in their entirety to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference in its entirety. Where the document cited only provides the first page of the document, the entire document is intended, including the remaining pages of the document.

Claims

What Is Claimed Is:

1. A method of identifying an agent which interacts with a retinoid X receptor (RXR) dimer, said method comprising:

(a) adding an agent to a chromatin based DNA template in the presence of said RXR dimer; and

(b) measuring activation of transcription, thereby determining whether said agent interacts with said RXR dimer.

2. The method of claim 1 , wherein the second receptor of said RXR dimer is selected from the group consisting of retinoic acid receptors (RARs), RXRs, thyroid receptors (TRs), vitamin D₃ receptor (VDR), peroxisome proliferator activated receptors (PPARs), liver X receptors (LXRs), famesoid X receptor (FXR), benzoate X receptor (BXR), constitutive androstane receptors (CARs) and steroid and xenobiotic receptor (SXR).

3. The method of claim 2, wherein said RXR dimer is an RXRα/RARα dimer.

4. The method of claim 1, wherein said chromatin based DNA template comprises a hormone response element (HRE) which is capable of binding by said RXR dimer.

5. The method of claim 1 , further comprising adding a co-activator.

6. The method of claim 5, wherein said co-activator is selected from the group consisting of p300, CBP, SRC-1, ACTR, TIF2, PCAF, SWI/SNF, GCN5, PC2, RSF, NURF, CHRAC and ACF.

7. The method of claim 6, wherein PC2 is selected from the group consisting of ARC, TRAP, DRIP, and SMCC

8. The method of claim 5, wherein said co-activator is p300.

9. A method of identifying a retinoic acid receptor (RAR) agonist, said method comprising:

(a) adding an agent to a chromatin based DNA template in the presence of a retinoid X receptor (RXR)/RAR dimer; and

(b) measuring activation of transcription, thereby determining whether said agent is an RAR agonist.

10. The method of claim 9, wherein said chromatin based DNA template comprises a retinoic acid response element (RARE).

11. The method of claim 9, wherein said RXR/RAR dimer is an

RXRα/RARα dimer.

12. The method of claim 9, further comprising adding a co-activator.

13. The method of claim 12, wherein said co-activator is selected from the group consisting of p300, CBP, SRC-1, ACTR, TIF2, PCAF, SWI/SNF, GCN5, PC2, RSF, NURF, CHRAC and ACF.

14. The method of claim 13 , wherein PC2 is selected from the group consisting of ARC, TRAP, DRIP, and SMCC

15. The method of claims 12, wherein said co-activator is p300.

16. A method of identifying a retinoid X receptor (RXR) agonist, said method comprising:

(a) adding an agent to a chromatin based DNA template in the presence of an RXR/retinoic acid receptor (RAR) dimer and an RAR agonist; and (b) measuring activation of transcription, thereby determining whether said agent is an RXR agonist.

17. The method of claim 16, wherein said chromatin based DNA template comprises a retinoic acid response element (RARE).

18. The method of claim 16, wherein said RXR/RAR dimer is an

RXRα/RARα dimer.

19. The method of claim 16, further comprising adding a co-activator.

20. The method of claim 19, wherein said co-activator is selected from the group consisting of p300, CBP, SRC-1, ACTR, TIF2, PCAF, SWI/SNF, GCN5, PC2, RSF, NURF, CHRAC and ACF.

21. The method of claim 20, wherein PC2 is selected from the group consisting of ARC, TRAP, DRIP, and SMCC

22. The method claim 20, wherein said co-activator is p300.

23. A method of identify ing a retinoic acid receptor (RAR) antagonist, said method comprising:

(a) adding an agent to a chromatin based DNA template in the presence of a retinoid X receptor (RXR)/RAR dimer and an RAR agonist; and

(b) measuring activation of transcription, thereby determining whether said agent is an RAR antagonist.

24. The method of claim 23, further comprising adding said agent in the presence of an RXR agonist.

25. The method of claim 23, wherein said chromatin based DNA template comprises a retinoic acid response element (RARE).

26. The method of claim 23, wherein said RXR/RAR dimer is an RXRα/RARα dimer.

27. The method of claim 23, further comprising adding a co-repressor.

28. The method of claim 27, wherein said co-repressor is selected from the group consisting of SMRT, N-COR and NURD.

29. A method of identifying a retinoid X receptor (RXR) antagonist, said method comprising: (a) adding an agent to a chromatin based DNA template in the presence of a RXR/retinoic acid receptor (RAR) dimer, an RAR agonist, and an RXR agonist; and

(b) measuring activation of transcription, thereby determining whether said agent is an RXR antagonist.

30. The method of claim 29, wherein said chromatin based DNA template comprises a retinoic acid response element (RARE).

31. The method of claim 29, wherein said RXR dimer is an RXRα/RARα dimer.

32. The method of claim 29, further comprising adding a co-repressor.

33. The method of claim 32, wherein said co-repressor is selected from the group consisting of SMRT, N-COR and NURD.

34. A method of identifying a co-activator or co-repressor of a retinoid X receptor (RXR) dimer, said method comprising:

(a) adding a first agent to a chromatin based DNA template in the presence of said RXR dimer and a second agent which is an agonist of said RXR dimer; and

(b) measuring activation of transcription, thereby determining whether said first agent is a co-activator or co-repressor of said RXR dimer.

35. The method of claim 34, wherein the second receptor of said RXR dimer is selected from the group consisting of retinoic acid receptors (RARs), RXRs, thyroid receptors (TRs), vitamin D₃ receptor (VDR), peroxisome proliferator activated receptors (PPARs), liver X receptors (LXRs), famesoid X receptor (FXR), benzoate X receptor (BXR), constitutive androstane receptors (CARs) and steroid and xenobiotic receptor (SXR).

36. The method of claim 34, wherein said RXR dimer is an RXRα/RARα dimer.

37. The method of claim 34, wherein said chromatin based DNA template comprises a hormone response element (HRE) which is capable of binding by said RXR dimer.

38. A method of identifying a modulator which modulates interactions between a retinoid X receptor (RXR) dimer and a co-activator or co-repressor of said RXR dimer, said method comprising:

(a) adding an agent to a chromatin based DNA template in the presence of said RXR dimer, an agonist of said RXR dimer, and a co-activator or co-repressor of said RXR dimer; and (b) measuring activation of transcription, thereby determining whether said agent modulates interactions between an RXR dimer and said co-activator or co-repressor.

39. The method of claim 38, wherein said chromatin based DNA template comprises a hormone response element (HRE) which is capable of binding by said RXR dimer.

40. The method of claim 38, wherein the second receptor of said RXR dimer is selected from the group consisting of retinoic acid receptors (RARs),

RXRs, thyroid receptors (TRs), vitamin D₃ receptor (VDR), peroxisome proliferator activated receptors (PPARs), liver X receptors (LXR), famesoid X receptor (FXR), benzoate X receptor (BXR), constitutive androstane receptors (CARs) and steroid and xenobiotic receptor (SXR).

41. The method claim 40, wherein said RXR dimer is an RXRα/RARα dimer.

42. The method of claim 38, wherein said co-activator is selected from the group consisting of p300, CBP, SRC-1, ACTR, TIF2, PCAF, SWI/SNF, GCN5, PC2, RSF, NURF, CHRAC and ACF.

43. The method of claim 42, wherein PC2 is selected from the group consisting of ARC, TRAP, DRIP, and SMCC

44. The method of claim 38, wherein said co-repressor is selected from the group consisting of SMRT, N-COR and NURD.

45. An in vitro chromatin based DNA template transcription system comprising:

(a) a chromatin based DNA template; and

(b) a retinoid X receptor (RXR) dimer.

46. The system of claim 45, wherein the second receptor of said RXR dimer is selected from the group consisting of retinoic acid receptors (RARs), RXRs, thyroid receptors (TRs), vitamin D₃ receptor (VDR), peroxisome proliferator activated receptors (PPARs), liver X receptors (LXR), famesoid X receptor (FXR), benzoate X receptor (BXR), constitutive androstane receptors (CARs) and steroid and xenobiotic receptor (SXR).

47. The system of claim 46, wherein said RXR dimer is an

RXRα/RARα dimer.

48. The system of claim 46, wherein said DNA template further comprises a hormone response element (HRE) which is capable of binding by said RXR dimer.

49. The system of claim 46 which is contained in a kit.

50. The system of claim 49, wherein each of (a) and (b) are contained in separate compartments.