# Patent

## Title
Methods and compositions for secretion of heterologous polypeptides

## Abstract

This invention was made with Government support under Grant No. NIDDK 1 R55 DK45657-01, awarded by the National Institutes of Health. The Government has certain rights in the invention.

## Background
The present invention relates to intracellular receptors, and methods for the modulation thereof. In a particular aspect, the present invention relates to novel heterodimeric complexes. In another aspect, the present invention relates to methods for modulating processes mediated by retinoid X receptor and/or orphan receptor LXR.

All-trans retinoic acid (atRA) and 9-cis retinoic acid (9cRA) are metabolites of vitamin A that mediate tissue specific expression of target genes. This is accomplished through binding to two classes of nuclear hormone receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs; reviewed by Mangelsdorf et al., in The retinoids Sporn et al., eds. (New York, Raven Press) 319-349 (1994); and by Kastner et al., in Vitamin A in health and disease, R. Blomhoff, ed. (New York: Marcel Dekker, Inc.) 189-238 (1994)). 9cRA is a high affinity ligand for both RARs and RXRs, whereas atRA is a ligand for only RARs. Like other members of the nuclear receptor superfamily, the retinoid receptors transactivate their target genes by binding to specific sites called hormone response elements found within the 5', regulatory region of the target gene.

The highest affinity hormone response elements for the retinoid receptors, as well as the vitamin D receptor (VDR), thyroid hormone receptors (TRs), and peroxisome proliferator activated receptors (PPARs) have been characterized as direct repeats (DRs) of the canonical hexad, AGGTCA, separated by one to five nucleotides (reviewed by Mangelsdorf et al., 1994, supra and Giguere, V., in Endocr. Rev. 15:61-79 (1994)). RAR, VDR, TR and PPAR preferentially bind to their hormone response elements in vitro as heterodimers complexed with RXR. Reconstitution studies in yeast (see Heery et al., in Proc. Natl. Acad. Sci. USA 90:4281-4285 (1993); and Allegretto et al., in J. Biol. Chem. 268:26625-26633 (1993)) and RXR gene disruption experiments in mice (see Sucov et al., in Genes Dev. 8:1007-1018 (1994); and Kastner et al., in Cell 78:987-1003 (1994)) confirm the function of the RXR heterodimer and suggest that it has an obligatory role in vivo as well as in vitro. Thus, RXRs appear to be essential pleiotropic regulators of several signaling pathways.

In terms of retinoid signaling, two distinct pathways are known, the RXR/RAR heterodimer and RXR homodimer. The RXR/RAR heterodimer mediates atRA or 9cRA action through its high affinity binding to a direct repeat response element having a spacer of 5 nucleotides (i.e., a DR5 element), and to some extent DR2 elements. Recently, it has been shown that when the RXR/RAR heterodimer is bound to DNA, RXR occupies the 5- half-site and RAR occupies the 3' half-site of the DR5 element (see Perlmann et al., in Genes Dev. 7:1411-1422 (1993); and Kurokawa et al., in Genes Dev. 7:1423-1435 (1993)). In this configuration, RXR is unable to bind ligand and thus functions as a silent partner (Kurokawa et al., in Nature 371:528-531 (1994)). The role of RXR as a silent partner is consistent with the finding that other receptors that heterodimerize with RXR do not require 9cRA for their activation (see, for example, Yu et al., in Cell67:1251-1266 (1991); Kliewer et al., in Nature 355:446-449 (1992); Zhang et al., in Nature 355:441-446 (1992); Marks et al., in EMBO J. 11:1419-1435 (1992); Leid et al., in Cell 68:377-395 (1992); and Kliewer et al., in Nature 358:771-774 (1992)).

In the RXR homodimer, RXR acts as its own partner and mediates 9cRA action through binding to DR1 elements. Interestingly, the RXR/RAR heterodimer also binds the DR1 element, and does so with higher affinity than the RXR homodimer. The consequence of this binding is that the RXR/RAR heterodimer is a potent repressor of 9cRA activation through the RXR homodimer (Mangelsdorf et al., in Cell66:555-561 (1991); Kurokawa et al., 1994, supra). These findings suggest that in order for the RXR homodimer to be active (i.e., for RXR to be able to function in vivo as a 9cRA receptor), the ratio of RXR to RAR in a cell must be very high. This may explain why cells that endogenously express RXR and RAR (even at low levels) yield significant retinoid responses with DR5-containing reporter genes, but do not yield any response with DR1-containing reporter genes, unless RXRs are overexpressed in these cells (see Mangelsdorf et al., 1991, supra). Given the ubiquitous expression pattern of RARs and RXRs, it is of interest to determine whether 9cRA activation of RXR occurs only through DR1 elements, or whether any other pathways exist for mediating a 9cRA-RXR response.

In accordance with the present invention, we have discovered that an orphan member of the nuclear receptor superfamily, named LXR.alpha. (see SEQ ID NO:1), in the presence of RXR ligand (e.g., 9cRA), is a potent inducer of transactivation through a distinct retinoid response element. The LXR.alpha. response to retinoids is due to the unique interaction of LXR.alpha. with endogenous RXR in cells. This interaction permits RXR to work as an active, ligand-binding heterodimeric partner. These results demonstrate the ability of LXR.alpha. to function as a tissue-specific mediator of a novel retinoid-responsive pathway.

FIG. 1 presents an amino acid sequence comparison between human (h)LXR.alpha. and other members of the nuclear receptor superfamily. Sequence alignments were determined using the University of Wisconsin Genetics Computer Group programs (Devereux et al., in Nucleic Acids Res. 12:387-395 (1984)). Regions of significant similarity between hLXR.alpha. and other receptors are presented schematically as percent amino acid identify. dEcR, Drosophila ecdysone receptor (Koelle et al., in Cell67:59-77 (1991)); hVDR, human vitamin D receptor (Baker et al., in Proc. Natl. Acad. Sci. USA 85:3294-3298 1988); hRAR.alpha., human retinoic acid receptor .alpha. (Gigueere et al., in Nature 330:624-629 (1987)); hTR.mu., human thyroid hormone receptor .mu. (Weinberger et al., in Nature 324:641-646 (1986)); hRXR.alpha., human retinoid X receptor .alpha. (Mangelsdorf et al., in Nature 345:224-229 (1990)); hGR, human glucocorticoid receptor (Hollenberg et al., in Nature 318:635-641 (1985)).

FIG. 2 collectively demonstrates that retinoids specifically induce LXR.alpha..

In FIG. 2a, CV1 cells were cotransfected with either a control plasmid (Control) or an expression plasmid for hLXR.alpha., as indicated, in combination with the luciferase reporter plasmid TK-LXRE3-LUC and then incubated with various ligands (see below) or an ethanol control (ETOH).

In FIG. 2b, LXR.alpha. and RXR.alpha. dose response to 9-cis retinoic acid is shown. CV1 cells were cotransfected with expression plasmids for hLXR.alpha. (squares) or hRXR.alpha. (circles) and the reporter plasmids TK-LXRE3-LUC or TK-CRBPII-LUC, respectively, and then incubated with increasing concentrations of 9-cis retinoic acid.

In FIGS. 2c-2e, LXR.alpha., RXR.alpha., and RAR.alpha. activation by retinoids is shown to be response element specific. Thus, CV1 cells were cotransfected with either a control plasmid (-) or expression pladmids for hLXR.alpha., hRXR.alpha., or hRAR.alpha. in combination with reporter plasmids TK-LXRE3-LUC (see FIG. 2c), TK-CRBPII-LUC (see FIG. 2d), or TK-.mu.RE-LUC (see FIG. 2e) and then incubated with various retinoid ligands (see below) or an ethanol control.

FIG. 3 collectively demonstrates that the putative ligand binding domain of LXR.alpha. contains a functional, RXR-specific heterodimerization domain, but not a functional retinoid binding domain.

FIG. 3A provides a schematic representation of GAL4 and VP16 hybrid proteins used in these experiments. The amino acid sequences from each parent protein used to construct the hybrid receptors are shown above each schematic.

FIG. 3B illustrates the fact that transactivation of the GAL4-LXR.alpha. construct requires the presence of VP16-RXR.alpha., but not VP16-RAR.alpha., in the mammalian two hybrid system. In negative control experiments, none of the VP16-receptors alone are able to confer transactivation.

FIG. 3C illustrates the ligand specificity of the GAL4-receptor chimeras. Thus, it is demonstrated that retinoid responsiveness can be conferred through the ligand binding domains of RXR and RAR, but not LXR. In both 3B and 3C, CV1 cells were cotransfected as described previously with CMX vectors driving expression of the indicated hybrid proteins and the reporter plasmid TK-MH100x4-LUC, which contains four copies of the GAL4 DNA upstream activation sequence. In FIG. 3C, ligands were added as described with reference to FIG. 2. Luciferase activity is expressed as RLUs (relative light units) corrected for transfection efficiency using an internal .mu.gal standard and represents the mean of triplicate assays (.+-.SEM).

FIG. 4 demonstrates that the LXRE is a functional, high affinity binding site for LXR/RXR heterodimers.

FIG. 5 illustrates the transactivation of LXR.alpha. TR.mu., or RXR.alpha. on an LXRE (TK-LXRE3-LUC) or a canonical DR4 response element (TK-DR4x2-LUC) reporter. Like the canonical DR4 element, other known thyroid hormone response elements such as the MHC-TRE (Izumo and Mahdavi, in Nature 334:539-542 (1988)) and the FAS-TRE (Amy et al., in Biochem. J. 271:675-679 (1990)) also demonstrate high affinity LXR.alpha. binding, but are not retinoid responsive. Cotransfections and luciferase assays were performed as described with reference to FIG. 2; ligands were added at 10.sup.-4 M methoprene acid (MA) or 10.sup.-6 M thyroid hormone (T.sub.3). Luciferase activity is expressed as (.+-.SEM of triplicate assays).

FIG. 6 summarizes the multiple RXR signaling pathways. As a dimer partner, RXR is a master regulator of multiple hormone response pathways, including at least three that are retinoid responsive: (1) The RXR homodimer binds and is responsive to 9cRA on DR1 target gene elements (RXREs). This response is strongly repressed by RAR, which preferentially heterodimerizes with RXR. The interaction with RAR prevents RXR from binding ligand and forms a high affinity complex on the RXRE that is transcriptionally inactive. Thus, in order for RXR to respond to ligand, the ratio of RXR to RAR in a cell must be very high. (2) As a heterodimer, RXR does not bind ligand, but functions as a silent partner to RAR in mediating atRA and 9cRA responses through RAREs composed of DR5 (and presumably DR2) sequences. (3) As described herein, the orphan receptor LXR.alpha. defines a third 9cRA response pathway in which RXR participates as the active ligand binding partner. The LXR/RXR heterodimer responds to retinoid through a specific DR4-like element (the LXRE). Unlike the RXR response on a DR1 element, the LXR/RXR response is uniquely sensitive to 9-cRA and can function potently in the presence of the low levels of endogenous RXR in the cell. (4) Finally, RXR subserves the role of heterodimeric partner to several receptor systems (designated by XR), including vitamin D, thyroid hormone, and peroxisome proliferators.

RXRs are unique in their ability to function as both homodimeric receptors and as obligate heterodimeric partners to receptors in multiple hormone response pathways. The central role of RXR in these pathways is summarized in FIG. 6. In the retinoic acid response pathway, where RXR is present as part of a heterodimer, RXR does not bind ligand, but rather serves as a cofactor to RAR in preferentially activating target genes containing DR5 response elements. As a homodimer, the ability of RXR to bind ligand and function exclusively as a 9cRA receptor is limited to DR1-containing target genes.

Up until the present invention, activation through the RXR homodimer has been observed to be dependent on the artificial overexpression of RXR, since in all cell types tested the ubiquitous endogenous expression of RAR and RXR leads to the preferential formation of heterodimers that prevent RXR from binding ligand. This apparent lack of target cells in which the expression of RAR is sufficiently lower than RXR to allow formation of RXR homodimers raises the question of how RXR functions in vivo as a 9cRA receptor. In order for RXR to function in vivo as a 9cRA receptor, it was suspected that an alternative pathway exists that allows RXR to so function. Indeed, in accordance with the present invention, the orphan receptor LXR.alpha. has been identified as a cofactor that permits RXR to function as a potent 9cRA receptor with a distinct target gene specificity. The LXR effect is mediated through a unique heterodimeric interaction, which switches the role of RXR from a silent partner to an active, ligand-binding partner in the heterodimer complex. Significantly, this finding establishes a new pathway by which RXR can function as a bona fide receptor and further, it defines a third retinoid response system with a novel target gene specificity.

Unlike the RXR homodimer, the LXR/RXR heterodimer response to 9cRA in cells can occur in the presence of endogenous RXR and RAR. In the context of the LXR heterodimer, the mechanism of RXR activation by ligand appears similar to that of RAR: both receptors require a partner for specific DNA binding and ligand activation. The nature of the LXR heterodimer, however, is unique in several respects. The heterodimerization of LXR and RXR is specific, as no evidence of an LXR interaction with other receptors has been found. Furthermore, while the LXR/RXR heterodimer binds to a variety of DR4-like response elements, it is only ligand responsive on the LXRE. This finding indicates that DNA binding alone is not sufficient for ligand inducibility.

One fundamental question in the field of nuclear hormone receptors is the function of the ever increasing subclass of this protein family that have been referred to as orphan receptors. Although initially activators for these potential ligand-dependent transcription factors are unknown, their remarkable similarity to other nuclear receptors suggests that they may have ligands and operate in a fashion similar to the known receptors. Indeed, studies on both the known and orphan receptors have revealed that many of these proteins have transcription modulating activity that may be considered ligand-independent. As previously demonstrated for RXR and as shown here for LXR, these receptors can serve important ligand-independent functions as obligate heterodimer partners. Based on these findings, it can be predicted that other orphan receptors exist that serve similar roles, some of which are likely to intersect the retinoid response system.

In accordance with the present invention, there are provided retinoid-inducible response elements having the sequence:

5'-GGTTTA-NNNN-AGTTCA (SEQ ID NO:2), wherein each N is independently selected from A, G, T or C. Optionally, the above-described retinoid-inducible response element can have additional 3' residues, N.sub.1 N.sub.2, wherein N.sub.1 is selected from A or T, and N.sub.2 is selected from C or G (SEQ ID NO:3).

A presently preferred retinoid-inducible response element of the invention has the sequence:

5'-GGTTTA-AATA-AGTTCA (SEQ ID NO:4), optionally having additional 3' residues, N.sub.1 N.sub.2, as defined hereinabove (SEQ ID NO:5).

An especially preferred retinoid-inducible response element of the invention has the sequence:

5'-CTTGCGGTTCCCAG-GGTTTA-AATA-AGTTCA (SEQ ID NO:6), optionally having additional 3' residues, N.sub.1 N.sub.2, as defined hereinabove (SEQ ID NO:7).

In accordance with another embodiment of the present invention, there is provided a method to render RXR-containing cells responsive to RXR selective ligands, said method comprising contacting said cells with at least one isoform of LXR. This method is particularly effective when using cells which further contain members of the steroid/thyroid superfamily with which RXR forms heterodimers, e.g., RAR, VDR, TR, PPAR, and the like.

As employed herein, the phrase "isoform of LXR" refers to any orphan receptor that by sequence comparison is related to LXR.alpha. described herein. Thus, isoforms of LXR includes the receptor described by Apfel et al., in Mol. Cell. Biol. 14:7025-7035 (1994), the receptor described by Song et al., in Proc. Natl. Acad. Sci. USA 91:10809-10813 (1994), and the like.

In accordance with yet another embodiment of the present invention, there is provided a method to alter the target gene specificity of RXR-containing cells, said method comprising contacting said cells with at least one isoform of LXR.

In accordance with still another embodiment of the present invention, there is provided a method to activate, in RXR-containing cells, target gene(s) operatively associated with response element(s) having the sequence:

5'-GGTTTA-NNNN-AGTTCA (SEQ ID NO:2), wherein each N is independently selected from A, G, T or C, said method comprising contacting said cells with at least one isoform of LXR.

In accordance with a further embodiment of the present invention, there are provided methods of screening for RXR- and/or LXR-selective ligands, said method comprising: construct into a suitable host cell, wherein said reporter construct comprises an LXRE (or RXRE), operatively linked to a reporter gene, and RXR- (or LXR-)specific ligand(s), and

The coincident expression pattern of LXR.alpha. and RXR.alpha. suggests these genes may have a role in adult physiology as was originally predicted for RXR.alpha. (Mangelsdorf et al., in Nature 345:224-229 (1990)). Accordingly, invention methods can be applied to the treatment of a wide variety of disease states in which RXR mediated processes are involved.

The invention will now be described in greater detail by reference to the following non-limiting examples.

Sequential low-stringency screening (as described previously, see Mangelsdorf et al., (1990) supra) of a human liver .lambda.gtll cDNA library with a 503 bp cDNA probe encoding the human RAR.alpha. DNA-binding domain (i.e., the KpnI/SacI restriction fragment of hRAR.alpha., which includes the DNA-binding domain) led to the isolation of several potential nuclear receptor clones.

Positive clones were isolated, subcloned into pGEM vectors (Promega) restriction mapped, and sequenced by the dideoxy method with sequenase (US Biochemical) using both single-stranded and double-stranded DNA templates. Sequences were aligned and analyzed by the University of Wisconsin Genetics Computer Group programs (Devereux et al., supra). Several LXR clones were isolated; the longest, designated .lambda.HL1-1, contained a 2.8 kb insert that was subsequently subcloned into the EcoR1 site of pBluescript-KS (Stratagene). Because the size of this insert is identical to that of the vector and contains .about.1.2 kb of 5' sequence that appears to be unrelated to the LXR cDNA, the first 1151 nucleotides of this clone were excised by EcoRV digestion at two restriction sites, one within the insert and the other in the multiple cloning site of the vector. The resultant DNA was religated and named pXR2.DELTA.RV. The putative cDNA insert of pXR2.DELTA.RV is 1680 bp (see SEQ ID NO:1) and contains the entire coding region of hLXR.alpha. as well as a potential polyadenylation signal sequence and 33 bases of the poly(A+) tail. Eighteen bp downstream from an in-frame termination codon at nucleotide 127 is the predicted translation start codon for LXR.alpha. at nucleotide 148. Translation from this ATG predicts an LXR.alpha. protein of 447 amino acids (M.sub.r 49,000) that has been confirmed by in vitro transcription/translation of the LXR.alpha. cDNA depicted in SEQ ID NO:1. This clone also contains 188 bp of 3' UTR which includes a canonical polyadenylation signal and a tract of 33 adenosines of the presumptive poly(A) tail. From these results, it can be concluded that this cDNA encodes the full-length LXR.alpha. protein.

Based on its initial isolation from the liver and its liver-rich expression pattern, this orphan receptor was designated LXR.alpha..

A comparison of the human LXR.alpha. orphan receptor with a number of other members of the nuclear receptor superfamily is depicted in FIG. 1. Human (h)LXR.alpha. shares closest identity with the Drosophila ecdysone receptor (dEcR), having a 71% similar DNA binding domain and 39% similar putative ligand binding domain. The degree of sequence identity of hLXR.alpha. with the known human receptors, however, is much less, but overall it is greatest with the human vitamin D receptor (hVDR). Interestingly, of the non-steroid subclass of receptors with known ligands, hLXR.alpha. has the lowest similarity to hRXR.alpha. (52% in the DNA binding domain, 22% in the ligand binding domain). From genomic Southern analysis, and preliminary evaluation of other LXR.alpha.-related cDNAs, it is noted that LXR likely constitutes a gene family with at least one other member.

Construction of wild-type receptor inserted into the expression vectors RSV and CMX, have been detailed elsewhere (Gigueere et al., in Cell46:645-652 (1986); Umesono et al., in Cell65:1255-1266 (1991); Mangelsdorf et al., (1990) supra). The hLXR.alpha. cDNA insert was excised from the plasmid pXR2.DELTA.RV with Kpn1 and BamHI and ligated into the expression vectors RSV and CMX. Chimeric Gal4-receptor expression plasmids (e.g. CMX-GAL4-LXR.alpha.) were constructed by first ligating the GAL4 portion of pSG424 (Sadowski and Ptashne, in Nucleic Acids Res. 17:7539-7530 (1989)) into the HindIII/SacI sites of pCMX (Umesono et al., in Cell 65:1255-1266 (1991)) to create pCMX-GAL4. This vector contains the CMV promoter fused to the coding sequence for GAL4 (1-147), followed by inframe polylinker cloning sites and translational stop codons. The cDNAs encoding the ligand-binding domain (LBD) of each of the receptors were then ligated into the polylinker to create GAL4-LBD fusions.

The following receptor amino acid sequences were used for these constructions and their corresponding DNA sequences ligated into pCMX-GAL4:hLXR.alpha. 166-447, hRXR.alpha. 203-462 and hRAR.alpha. 185-462.

Likewise, chimeric VP16-receptor expression plasmids were constructed by fusing the cDNA fragment encoding the 78 amino acid transactivation domain of VP16 (Triezenberg et al., in Genes Dev. 2:718-729 (1988)) in-frame to the cDNA encoding the desired nuclear receptor. The VP16-receptor fusions were engineered into CMX expression plasmids. Each chimera contains a 6-12 amino acid linker at the VP16-receptor fusion point to facilitate an in-frame junction. Kozak translational start sites (CACCATGG) were engineered into the 5-prime end of the VP16-receptor chimeras to provide strong initiator methionines. All fusion points and cloning sites were sequenced and the full-length proteins in vitro synthesized to insure sequence fidelity. Reporter plasmids for these studies were constructed by ligating the appropriate oligonucleotides into the HindIII site of the TK-LUC vector to create TK-LXRE-LUC, TK-MH100x4-LUC (Kang et al., in J. Biol. Chem. 268:9629-9635 (1993)), TK-DR4x2-LUC (Umesono et al., (1991) supra), TK-CRBPII-LUC (Mangelsdorf et al., (1991) supra), TK-.beta.SRE-LUC (Sucov et al., in Proc. Natl. Acad. Sci. USA 87:5392-5396 (1990)). All constructs were verified by sequencing.

CV-1 cells were maintained at 37.degree. C., 5% CO.sub.2 in DMEM containing 5% calf bovine serum (CBS). Transfections were performed in 8-well plates in media containing 5% dextran-charcoal stripped CBS by the calcium phosphate coprecipitation technique as described previously (Mangelsdorf et al., (1990) supra). Eight hours after transfections ligands were delivered to cells at 10.sup.3 -fold dilutions (0.1% vol/vol of solvent in media). Retinoids (gifts from Marcus Boehm at Ligand Pharmaceuticals, Inc.) were manipulated under gold light and stored dark in ethanol or methanol under nitrogen gas at -80.degree. C. Cells were harvested 36 hours after addition of ligand and analyzed for luciferase and .beta.-galactosidase activity using a Dynatech microtiter plate Model ML3000 luminometer and a Model MR5000 spectrophotometer, respectively.

For most experiments, cotransfection of DNA into mammalian cells was accomplished with 50 ng reporter plasmid, 50 ng RSV- or CMX-.beta.gal, 25 ng of each receptor expression plasmid and pGEM carrier to give 375 ng DNA/well. For controls, the empty CMX vector was used in place of the receptor cDNA. For the GAL4-receptor chimera experiments, 30 ng receptor and 80 ng of reporter plasmid were used. For GAL4 and VP16 chimera interaction assays, 30 ng of GAL4-LXR.alpha. and 15 ng of the VP16-RAR or -RXR were used along with 80 ng of the reporter plasmid. All transfection data points were normalized using an internal .beta.-galactosidase marker (Mangelsdorf et al., (1990) supra) and represent the mean of duplicate or triplicate assays.

Murine poly(A)+ RNA used in these studies was prepared and assayed by Northern Analysis as described previously (Mangelsdorf et al., in Genes Dev. 6:329-344 (1992)). Equal amounts (10 .mu.g) of poly(A)+RNA were loaded in each lane and verified by ethidium bromide staining. The DNA hybridization probe used on each blot was the 534 bp EcoR1 fragment of the pXR2.DELTA.RV cDNA insert. This probe includes nucleotides 1147-1680 of the hLXR.alpha. cDNA and encodes part of the ligand binding domain and 3'-untranslated region of the clone. Autoradiography was for two days at -70.degree. C. with intensifying screens.

Northern blot analyses were performed to determine the tissue distribution and developmental pattern of LXR.alpha. gene expression. Poly(A+) RNA isolated from a variety of adult rat tissues or from staged whole mouse embryos (gestation day 10.5 to 18.5) was size-fractionated, transferred to nylon filters, and hybridized with the hLXR.alpha. cDNA. The distribution of LXR.alpha. mRNA reveals an expression pattern similar to that of RXR.alpha. (Mangelsdorf et al., (1990) supra), with strong expression in metabolic organs such as liver, kidney, and intestine. The 1.9 kb LXR.alpha. message is also notably present in spleen and to a lesser extent in the adrenals. Analysis of expression during mouse development indicates low levels of LXR.alpha. are detected at embryonic day 13.5 and continue to increase in abundance through parturition.

For EMSA experiments, receptor proteins were generated using a coupled in vitro transcription/translation system according to the manufacturer's instructions (Promega). Sequences of double-stranded oligonucleotides were as shown in the figures and text and were synthesized with HindIII overhangs. Binding reactions were performed in a total volume of 20 .mu.l consisting of 75 mM KC1, 20 mM Hepes, pH 7.4, 2 mM DTT, 7.5% glycerol, 0.1% NP-40, 2 MG poly (dI-dC) (Pharmacia), 40 pmol of a non-specific single-stranded oligonucleotide (for removal of non-specific binding in the lysates), and 2 .mu.l of receptor lysates or unprogrammed (control) lysates. Reactions containing lysates and competing oligonucleotides were incubated for 30 minutes on ice followed by the addition of 40 fmol of .sup.32 P-labeled oligonucleotide probe (labeled by end-filling) and further incubated for 20 minutes at room temperature. Samples were then analyzed on 5% polyacrylamide gels run in 0.5X TBE buffer. For antibody supershifting experiments, 1 .mu.l of anti-hRXR.alpha. antiserum (a gift from Jacki Dyck), or non-specific antiserum was added to the reactions after addition of the radiolabeled probe and incubated at room temperature for 10 minutes, followed by gel electrophoresis.

To identify a potential ligand for the orphan receptor LXR.alpha., a cell-based cotransfection screening assay similar to that used to successfully identify the RXR ligand (see Mangelsdorf et al., (1990) supra; Heyman et al., in Cell68:397-406 (1992)) was employed. Initially an LXR responsive gene was unknown, therefore the preliminary experiments were designed to first identify an LXR-specific DNA binding sequence that could serve as an LXR responsive element (referred to as an LXRE). During the course of these experiments, a specific, high affinity DNA binding site for LXR.alpha. was discovered in the promoter sequence of .DELTA.MTV, a promoter derived from the mouse mammary tumor virus LTR (see SEQ ID NO:6). This discovery suggested that the .DELTA.MTV LXRE sequence could be transferred to a heterologous promoter, such as thymidine kinase (TK), and used with a luciferase (LUC) reporter gene for screening potential LXR.alpha. ligands. Thus, an assay system was established by transfecting CV-1 cells with an expression plasmid harboring the cDNA for LXR.alpha. and the luciferase reporter plasmid TK-LXRE3-LUC (see FIG. 2a). TK-LXRE3-LUC contains three copies of the LXRE defined herein; TK-CRBPII-LUC contains the RXRE from the rat CRBPII promoter (Mangelsdorf et al., (1991) supra)); TK-.beta.RE-LUC contains the RAR response element from the mouse RAR.beta.2 promoter (Sucov et al., (1990) supra). Luciferase activity is expressed in relative light units (RLU) and represents the mean of triplicate assays (.+-.SEM) normalized to .beta.gal as an internal standard. For the experiments summarized in panels 2a and 2c-2e), three different retinoid ligands that have previously been used to distinguish between retinoid receptor systems were used: 10.sup.-5 M 9-cis retinoic acid (9cRA), which binds to both RXR and RAR (Heyman et al., (1992) supra; Levin et al., in Nature 355:359-361 (1992)); 10.sup.-5 M TTNPB, which is RAR-specific (Mangelsdorf et al., (1990) supra); and 10.sup.-4 M methoprene acid (MA), which is RXR-specific. The concentrations of other ligands used in (a) were 10.sup.-6 M thyroid hormone (T.sub.3), 10.sup.-6 M 1,25-dihydroxyvitamin D.sub.3 (VD), and 1 mM clofibric acid (CA).

In a comprehensive screen of several classes of compounds, it was found that LXR.alpha. activation is only induced by the presence of certain retinoid ligands. Remarkably, LXR.alpha. is strongly activated by both 9-cis retinoic acid (9cRA) and methoprene acid (MA), which have previously been shown to be RXR-specific ligands. The presence of both LXR.alpha. and retinoid is required to confer transactivation of the luciferase reporter gene containing the LXRE (compare "Control" to "LXRa" in FIG. 2a).

Since these results are similar to the activation specificity of RXR.alpha., a side-by-side comparison of dose responses to 9cRA was performed with LXR.alpha. and RXR.alpha. and their cognate response elements. As shown in FIG. 2b, both LXR.alpha. and RXR.alpha. show nearly identical concentration-dependent activation profiles in response to 9cRA. Similar dose responses were observed with several other RXR-specific ligands, including methoprene acid and LG69 (Kurokawa et al., in Nature 371:528-531 (1994)). These results suggest the existence of a distinct pathway of gene regulation mediated by LXR.alpha. and 9cRA.

Three Distinct Retinoid Response Pathways To further demonstrate that the LXR retinoid response is authentic and distinguishable from the pathways previously described by the RXR and RAR receptor systems, cotransfection experiments were performed to directly compare the three receptor systems by using receptor-specific ligands and response elements (see FIGS. 2c-e). For these experiments, the two retinoid ligands, methoprene acid (MA) and (E)-4-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl) -1-propenyl!benzoic acid (TTNPB), which have previously been shown to be uniquely specific for RXR and RAR, respectively (Mangelsdorf et al., (1990) supra; Kurokawa et al., (1994) supra) were used. Expression plasmids for the three receptors (LXR.alpha., RXR.alpha., and RAR.alpha.) were cotransfected into CV1 cells along with luciferase reporters containing their respective response elements and tested for ligand-dependent activation. In the absence of any response element, the empty reporter plasmid TK-LUC conferred no receptor or ligand dependent transactivation. However, as shown above, in the presence of the RXR-specific ligand, methoprene acid, LXR.alpha. strongly activates transcription from the LXRE-containing reporter (see FIG. 2c). In contrast, the RAR-specific ligand TTNPB had no effect on the activation of LXR.alpha.. Significantly, neither RXR.alpha. nor RAR.alpha. were able to substitute for LXR.alpha. in mediating LXRE dependent transactivation, even in the presence of the cognate ligands.

FIG. 2d and 2e demonstrate that the retinoid responsiveness of LXR.alpha. is specific to the LXRE, and cannot also be mediated through other retinoid response elements. Previously, a retinoid response element (that is specific for the RXR homodimer) has been identified from the rat CRBPII promoter (Mangelsdorf et al., (1991) supra). When this RXR response element (RXRE) is placed into the promoter of the luciferase reporter plasmid (TK-CRBPII-LUC), there is a marked response to the RXR ligand, methoprene acid (see FIG. 2d). This response occurs in the presence of RXR.alpha., but not RAR.alpha. or LXR.alpha.. It is noteworthy that even though these cells contain low levels of endogenous RXR, the RXR homodimer response requires the transfection of additional RXR.

In similar experiments, when a reporter plasmid containing an RAR response element (TK-SRE-LUC) is used, the retinoid response is limited to RAR and RXR and their cognate ligands (FIG. 4e). The response to TTNPB occurs through an RXR/RAR heterodimer, whereas the response to methoprene acid occurs through an RXR homodimer which can also weakly bind to this element (zhang et al., in Nature 358:587-591 (1992); Kurokawa et al., in Nature 371:528-531 (1994)). This activation occurs even in the absence of transfected RAR or RXR, confirming the existence of endogenous retinoid receptors. As has been previously reported, the addition of exogenous RAR.alpha. or RXR.alpha. accordingly increases the respective retinoid response. Importantly, there is no further increase in activation when LXR.alpha. is transfected into these cells. These data clearly demonstrate that LXR.alpha. has a retinoid response cis element specificity that is distinctly unique from RXR and RAR. Furthermore, the LXR response occurs in the absence of transfected RXR or RAR. Taken together, these results distinguish the human LXR.alpha. as a novel mediator of retinoid action.

LXR.alpha. Forms a Functional Heterodimer with RXR.alpha.

The data presented above suggest two possible explanations to account for the specific activation of LXR.alpha. by retinoids. One possibility is that LXR.alpha. is functioning in a fashion similar to other receptors. In this scenario, LXR.alpha. transactivation through an LXRE might be mediated by an LXR homodimer of an RXR/LXR heterodimer in which LXR binds the ligand. However, based on the RXR-like specificity of the retinoid response, another plausible explanation is that LXR.alpha. is not directly bound and activated by these retinoids, but instead is an obligatory heterodimeric partner of endogenous RXR, and that retinoid binding (and thus, transactivation) requires the presence of RXR. In either case, it is clear that receptor binding and transactivation through the LXRE is uniquely dependent on the presence of LXR.alpha., since both the RXR homodimer and RXR/RAR heterodimer fail to activate on the LXRE (see FIG. 2c).

To test the feasibility of these two hypotheses, the possibility of a heterodimeric interaction between LXR.alpha. and one of the other receptors was examined. For these studies, a mammalian version of the two hybrid system originally described for the detection of protein-protein interactions in yeast (Fields and Song, in Nature 340:245-246 (1989)) was employed. In this strategy, the ligand binding domain of one receptor (i.e., LXR.alpha.) is fused to the DNA binding domain of GAL4, creating a hybrid protein (GAL4-LXR.alpha., FIG. 3a) that can bind to specific upstream activation sequences (UAS.sub.G), but which lacks a constitutive transactivation domain and therefore cannot by itself activate transcription of a luciferase reporter (FIG. 3b). The use of the LXR.alpha. ligand binding domain as the "bait" in these experiments is based on previous studies which have localized a strong dimerization interface in the ligand binding domain of other nuclear receptors (Perlmann et al., (1993) supra; and Kurokawa et al., Genes Dev. 7:1423-1435 (1993)).

To screen for receptors that may interact with LXR.alpha., the candidate receptors were fused to the 78 amino acid transactivation domain of VP16. Each VP16-receptor chimera (FIG. 3a) was cotransfected along with GAL4-LXR.alpha. into CV-1 cells (FIG. 3b). In the presence of two receptors capable of dimerization, the VP16 transactivation domain is brought into functional proximity with the GAL4 DNA binding sequences, which in turn permits activation of the promoter driving luciferase expression. As is clearly demonstrated in FIG. 3b, only the VP16 hybrid protein containing RXR.alpha., but not RAR.alpha., is able to induce transcription in the presence of GAL4-LXR.alpha.. Similar VP16 hybrids of the vitamin D and thyroid hormone receptors are unable to confer transactivation. These experiments demonstrate a specific interaction between LXR.alpha. and RXR.alpha. and are strong evidence that these two receptors form a functional heterodimer in vivo.

To address the possibility that the retinoid response elicited by LXR.alpha. was through the direct interaction of ligand with the putative ligand binding domain of LXR.alpha., ligand activation experiments were performed with the GAL4-receptor hybrids. Previous studies have shown that the ligand binding domains of nuclear receptors contain ligand-dependent activation domains (Nagpal et al., in Cell70:1007-1019 (1992)). Thus, when a nuclear receptor ligand binding domain is fused to the DNA binding domain of GAL4, this hybrid transcription factor alone can confer ligand-dependent transactivation of a UAS.sub.G -containing reporter (Nagpal et al., in EMBO J. 12:2349-2360 (1993)). Such heterologous system provides an effective means for directly assaying receptor/ligand interactions, even in the presence of endogenous wild-type receptors.

As shown in FIG. 3c, the chimeric constructs GAL4-RXR and GAL4-RAR confer ligand inducible transactivation that accurately mimics the cognate ligand specificity of wild-type receptor proteins: 9cRA activates both RXR and RAR, methoprene acid only activates RXR, and TTNPB specifically activates RAR. In contrast, no activation is seen with the GAL4-LXR chimera in the presence of these same ligands. These results indicate that retinoid receptor ligands cannot directly interact with the LXR.alpha. ligand binding domain when evaluated under similar conditions that permit their interaction with RXR and RAR. Furthermore, the specificity of 9cRA and methoprene acid for RXR, taken together with the heterodimerization studies, are strong evidence that the LXR.alpha. retinoid response requires the presence of RXR.alpha..

The previous experiments suggest the existence of an LXR/RXR heterodimer as the mediator of retinoid signaling through a unique retinoid response element, referred to as the LXRE. This element was originally identified in the 5' regulatory region of .DELTA.MTV, a promoter derived from the mouse mammary tumor virus LTR that is routinely used in conjunction with heterologous response elements to drive receptor-specific expression of reporter genes (Hollenberg and Evans, in Cell55:899-906 (1988)). In the context of the reporter gene, .DELTA.MTV promoter identified a region of 30 nucleotides (see SEQ ID N): ) that when removed from .DELTA.MTV-LUC abolished retinoid activation by LXR.alpha.. As demonstrated in FIG. 2, this sequence can be transferred to the TK-LUC vector and confer full LXR.alpha. activation by retinoids. As has been shown to be the case with all other nuclear receptor response elements, increasing the number of LXREs in the reporter plasmid coincidentally increases hormone responsiveness.

To test whether this sequence was specifically bound by an LXR/RXR heterodimer, electrophoretic mobility shift assays were performed using in vitro synthesized receptor proteins and .sup.32 P-labelled LXRE probe. LXR.alpha. and RXR.alpha. do not bind to the LXRE probe individually, but when mixed together a strong protein-DNA complex is formed. This complex is supershifted in the presence of RXR.alpha.-specific antisera, but not in the presence of a non-specific antisera. These results are consistent with the conclusion that only the LXR/RXR heterodimer is capable of high affinity binding to the LXRE.

To demonstrate the functionality of the LXR/RXR heterodimer, an experiment was performed by cotransfecting both LXR.alpha. and RXR.alpha. with the LXRE reporter in the presence of the RXR-specific ligand, methoprene acid (see FIG. 4). Thus, expression plasmids for hLXR.alpha., hRXR.alpha., or no receptor (-) were cotransfected into CV1 cells in combination with the reporter plasmids TK-LUC or TK-LXRE2-LUC as indicated. Cells were then incubated with ethanol control (-) or 10.sup.4 M methoprene acid (+) and assayed for luciferase activity as described above. TK-LXRE2-LUC contains two copies of the LXRE. In experiments in which LXR and RXR plasmids were cotransfected together the ratio of the two expression plasmids was 1:0.1 (25 ng hLXR.alpha.:2.5 ng hRXR.alpha.). In all other experiments were LXR and RXR were added alone, the amount of each receptor expression plasmid was 25 ng.

As demonstrated above, the addition of LXR.alpha. alone gives a marked ligand response, whereas RXR.alpha. alone gives no response. However, when both LXR.alpha. and RXR.alpha. are added together, there is a dramatic synergistic increase in both basal and retinoid responsive activity. The LXR/RXR heterodimer must be exquisitely sensitive to retinoid, because under the conditions these experiments were performed, even the addition of one-tenth the amount of exogenous RXR markedly increases the LXR-dependent response. This response has been observed in several mammalian cell types. From these experiments, it can be concluded that the presence of endogenous RXR in these cells (Titcomb et al., in Mol. Endocrinol 8:870-877 (1994) ) is responsible for the ability of LXR.alpha., when added alone, to confer retinoid responsiveness. This conclusion is consistent with the ability of endogenous RXR to also function as a heterodimeric partner for RAR and other nuclear hormone receptors. The important difference in this case is that RXR does not play the role of a silent partner, but rather takes on the role of the active receptor partner.

Previously it has been shown that the heterodimers formed between RXR and other nuclear receptors can bind and activate through DNA response elements that are direct repeats of the core sequence AGGTCA spaced by 1 to 5 nucleotides (designated as DR1 to DR5; reviewed by (Mangelsdorf et al., (1994) supra; Kastner et al., in Vitamin A in health and disease, R. Blomhoff, ed. (New York: Marcel Dekker, Inc.) 189-238 (1994); Gigueere, V., Endocr. Rev. 15:61-79 (1994)). Inspection of the LXRE sequence in SEQ ID NO:6 reveals the presence of two degenerate hexad sequences spaced by four nucleotides that resemble the canonical direct repeat of AGGTCA half-sites.

The configuration of the LXRE into a putative direct repeat spaced by four nucleotides (i.e., DR4) was examined by performing a competition analysis with ten different mutant LXRE oligos in which individual pairs of nucleotides were systematically changed to cytosine (C) or guanine (G) in the region within and flanking the DR4 motif. The ability of mutant LXREs to compete with the labelled native LXRE probe was analyzed using the gel mobility shift assay. Mutant oligos with substitutions within either half-site of the DR4 motif fail to compete with native LXRE for binding to the LXR/RXR heterodimer, even at 50-fold molar excess. In contrast, LXRE oligos with similar substitution mutations in the spacer and flanking regions are able to compete well for binding to LXR/RXR. Furthermore, the introduction or substraction of a single nucleotide into the spacer region (thereby creating a DR5 or DR3 motif, respectively) resulted in oligos that fail to competitively bind the LXRE.

The data presented above suggest that the minimal LXRE is a DR4-type response element. To further investigate this possibility, a series of perfect AGGTCA direct repeat oligos, spaced by 1 to 5 nucleotides (DR1-5), were used to compete with labelled LXRE for binding the LXR/RXR heterodimer. Only the DR4 oligo is observed to compete as well as native LXRE for binding to LXR/RXR, even at a 10-fold molar excess. Since DR4 sequences are known to bind RXR/thyroid hormone receptor heterodimers and function as potent thyroid hormone response elements, it was of interest to know if LXR.alpha. could also activate through these DR4 elements. Surprisingly, LXR.alpha. activation is specific only to the LXRE, and is not functional on a canonical DR4 thyroid hormone response element.

In independent experiments, it has also been demonstrated that the LXR/RXR can bind, but is completely inactive, on several other DR4 elements that are known to be thyroid hormone responsive as well. Interestingly, the LXRE is reciprocally a very poor thyroid hormone response element, in spite of the fact that the RXR/thyroid hormone receptor heterodimer binds to this sequence with high affinity. These experiments suggest that DNA binding alone is insufficient to permit transactivation. Thus, the LXRE represents a novel DR4 motif that can distinguish between two hormonal pathways.

While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed. SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 7 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 447 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: MetSerLeuTrpLeuGlyAlaProValProAspIleProProAspSer 151015 AlaValGluLeuTrpLysProGlyAlaGlnAspAlaSerSerGlnAla 202530 GlnGlyGlySerSerCysIleLeuArgGluGluAlaArgMetProHis 354045 SerAlaGlyGlyThrAlaGlyValGlyLeuGluAlaAlaGluProThr 505560 AlaLeuLeuThrArgAlaGluProProSerGluProThrGluIleArg 65707580 ProGlnLysArgLysLysGlyProAlaProLysMetLeuGlyAsnGlu 859095 LeuCysSerValCysGlyAspLysAlaSerGlyPheHisTyrAsnVal 100105110 LeuSerCysGluGlyCysLysGlyPhePheArgArgSerValIleLys 115120125 GlyAlaHisTyrIleCysHisSerGlyGlyHisCysProMetAspThr 130135140 TyrMetArgArgLysCysGlnGluCysArgLeuArgLysCysArgGln 145150155160 AlaGlyMetArgGluGluCysValLeuSerGluGluGlnIleArgLeu 165170175 LysLysLeuLysArgGlnGluGluGluGlnAlaHisAlaThrSerLeu 180185190 ProProArgArgSerSerProProGlnIleLeuProGlnLeuSerPro 195200205 GluGlnLeuGlyMetIleGluLysLeuValAlaAlaGlnGlnGlnCys 210215220 AsnArgArgSerPheSerAspArgLeuArgValThrProTrpProMet 225230235240 AlaProAspProHisSerArgGluAlaArgGlnGlnArgPheAlaHis 245250255 PheThrGluLeuAlaIleValSerValGlnGluIleValAspPheAla 260265270 LysGlnLeuProGlyPheLeuGlnLeuSerArgGluAspGlnIleAla 275280285 LeuLeuLysThrSerAlaIleGluValMetLeuLeuGluThrSerArg 290295300 ArgTyrAsnProGlySerGluSerIleThrPheLeuLysAspPheSer 305310315320 TyrAsnArgGluAspPheAlaLysAlaGlyLeuGlnValGluPheIle 325330335 AsnProIlePheGluPheSerArgAlaMetAsnGluLeuGlnLeuAsn 340345350 AspAlaGluPheAlaLeuLeuIleAlaIleSerIlePheSerAlaAsp 355360365 ArgProAsnValGlnAspGlnLeuGlnValGluArgLeuGlnHisThr 370375380 TyrValGluAlaLeuHisAlaTyrValSerIleHisHisProHisAsp 385390395400 ArgLeuMetPheProArgMetLeuMetLysLeuValSerLeuArgThr 405410415 LeuSerSerValHisSerGluGlnValPheAlaLeuArgLeuGlnAsp 420425430 LysLysLeuProProLeuLeuSerGluIleTrpAspValHisGlu 435440445 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGTTTANNNNAGTTCA16 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGTTTANNNNAGTTCANN18 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GGTTTAAATAAGTTCA16 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GGTTTAAATAAGTTCANN18 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CTTGCGGTTCCCAGGGTTTAAATAAGTTCA30 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; (A) DESCRIPTION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTTGCGGTTCCCAGGGTTTAAATAAGTTCANN32 __________________________________________________________________________

## Claims

1. An isolated nucleic acid comprising a retinoid-inducible response element wherein said response element has the sequence:

2. An isolated nucleic acid comprising a retinoid-inducible response element wherein said response element has the sequence: or T, and N.sub.2 is C or G.

3. An isolated nucleic acid comprising a retinoid-inducible response element wherein said response element has the sequence:

4. An isolated nucleic acid comprising a retinoid-inducible response element wherein said response element has the sequence: N.sub.1 is A or T, and N.sub.2 is C or G.

5. A retinoid-inducible response element according to any one of claims 1-4, wherein said response element selectively binds LXR-RXR heterodimer(s).