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WO2000008052A1 - Nouveau recepteur hormonal nucleaire retinien - Google Patents

Nouveau recepteur hormonal nucleaire retinien Download PDF

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Publication number
WO2000008052A1
WO2000008052A1 PCT/US1999/017885 US9917885W WO0008052A1 WO 2000008052 A1 WO2000008052 A1 WO 2000008052A1 US 9917885 W US9917885 W US 9917885W WO 0008052 A1 WO0008052 A1 WO 0008052A1
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Prior art keywords
polypeptide
receptor
retinor
binding domain
dna binding
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Fabienne C. De La Brousse-Elwood
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Tularik Inc
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Tularik Inc
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Priority to AU55500/99A priority Critical patent/AU5550099A/en
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Priority to IL13979800A priority patent/IL139798A0/xx
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention pertains to the field of nuclear receptors that modulate gene expression.
  • the receptors are found in human tissues, including the retina, and are thought to be involved in tissue-specific gene expression. Novel nuclear receptors and nucleic acids encoding them are provided. Also provided are assays for identifying ligands, response elements, and activity modulator compounds for the nuclear receptors of the invention.
  • hormones Development of and homeostasis in complex eukaryotes, including humans and other mammals, birds, and fish, is regulated by a wide variety of substances, including hormones.
  • the effects of these hormones is mediated through binding of the hormone to a specific, high affinity receptor.
  • hormone-mediated modulation is appropriate only for a specific cell or tissue type, the hormone receptor is generally expressed only in those cells or tissues.
  • Hormone receptors are a large family of ligand-activated transcription factors that modify the expression of target genes by binding to specific cis-acting sequences
  • Hormone receptors include those that remain sequestered in the cytoplasm in the absence of their cognate ligands (e.g., steroid hormone receptors). Upon binding of the ligand, the steroid hormone receptors are translocated to the nucleus where they bind to hormone response elements, typically as homodimers.
  • nuclear receptors Most of the nuclear receptors, conversely, are not sequestered in the cytoplasm in the absence of their ligands but rather remain in the nucleus. These receptors, which include the thyroid hormone, retinoid, fatty acid, and eicosanoid receptors, typically bind to their cognate response elements as heterodimers with a 9-cis-retinoic acid receptor (RXR). Often, binding of a nuclear receptor to a response element occurs in the absence of the cognate ligand. In some cases, binding of the nuclear receptor to the response element represses basal promoter activity.
  • RXR 9-cis-retinoic acid receptor
  • ligands for specific receptors include steroids, vitamin D, thyroid hormones, retinoic acid, and the like.
  • ligands are presently unknown for other hormone receptors. Such receptors are often termed "orphan receptors.”
  • the present invention provides novel RetinOR orphan nuclear receptor polypeptides.
  • the invention provides an isolated polypeptide which comprises at least one domain of a RetinOR polypeptide.
  • the domain can be a DNA binding domain that comprises an amino acid sequence which is substantially identical to a DNA binding domain of a RetinOR polypeptide, or a ligand binding domain that comprises an amino acid sequence which is substantially identical to a ligand binding domain of a RetinOR polypeptide.
  • RetinORl and RetinOR2 polypeptides include both the ligand binding domain and the DNA binding domain are native to the RetinOR polypeptides
  • nucleic acids that encode the RetinOR polypeptides.
  • the nucleic acids include a polynucleotide sequence that is substantially identical to a polynucleotide sequence that encodes at least one a RetinOR functional domain. These domains can be either or both of a DNA binding domain of a human RetinOR polypeptide and a ligand binding domain of a human RetinOR polypeptide.
  • the invention also provides chimeric receptors that have a DNA binding domain and a ligand binding domain. Generally, at least one of the DNA binding domain and the ligand binding domain is substantially identical to a corresponding domain of a RetinOR polypeptide. Assays are also provided by the invention.
  • the invention provides methods of identifying a response element that is modulated by a RetinOR polypeptide. These methods generally involve contacting a polynucleotide which comprises a candidate RetinOR response element with a RetinOR polypeptide; and determining whether the RetinOR polypeptide binds to the candidate RetinOR response element. Also provided by the invention are methods of identifying a ligand which modulates activity of a RetinOR polypeptide.
  • a cell that contains a polynucleotide that encodes a fusion polypeptide that has: 1) a DNA binding domain of a polypeptide; and 2) a ligand binding domain that is substantially identical to a ligand binding domain of a RetinORl polypeptide.
  • the cell will also contain a reporter gene construct that includes a response element to which the DNA binding domain can bind, with the response element being operably linked to a promoter that is operative in the cell and the promoter is operably linked to a reporter gene.
  • a candidate ligand for a RetinORl polypeptide is also provided.
  • the cell is incubated under conditions suitable for expression of the fusion polypeptide, after which it is determined whether the reporter gene is expressed at a higher or lower level in the presence of the candidate ligand compared to expression in the absence of the candidate ligand.
  • the invention provides methods of screening to identify a compound that modulates activity of a RetinOR polypeptide. These methods involve contacting a cell with a test compound.
  • the cell contains: a) a polynucleotide that encodes a fusion polypeptide that has 1) a DNA binding domain of a polypeptide which binds to DNA; and 2) a ligand binding domain that is substantially identical to a ligand binding domain of a RetinORl polypeptide.
  • a reporter gene construct that includes a response element to which the DNA binding domain can bind.
  • the response element is operably linked to a promoter that is operative in the cell and the promoter is operably linked to a reporter gene.
  • the assay involves determining whether the test compound modulates activity of a RetinOR polypeptide by detecting whether the reporter gene is expressed at a higher or lower level in the presence of the test compound compared to the reporter gene expression level in the absence of the test compound.
  • Figure 1 shows an alignment of polynucleotide sequences of clones encoding human RetinORl (FLRetinOR.2a.consensus; SEQ ID NO: 1) and human RetinOR2
  • FIG. 2 shows an alignment of the amino acid sequences of hRetinORl (SEQ ID NO: 4) and hRetinOR2 (SEQ ID NO: 5), along with the consensus sequence.
  • the hRetinOR2 polypeptide is truncated compared to hRetinORl due to a 40 bp insertion in the hRetinOR2 nucleotide sequence, which insertion introduces a stop codon.
  • Nucleic acids are "homologous" when they are derived, naturally or artificially, from a common ancestor sequence. During natural evolution, this occurs when two or more descendent sequences diverge from a parent sequence over time, i.e., due to mutation and natural selection. Under artificial conditions, divergence occurs, e.g., in one of two ways. First, a given sequence can be artificially recombined with another sequence, as occurs, e.g., during typical cloning, to produce a descendent nucleic acid. Alternatively, a nucleic acid can be synthesized de novo, by synthesizing a nucleic acid which varies in sequence from a selected parental nucleic acid sequence.
  • homology is typically inferred by sequence comparison between two sequences. Where two nucleic acid sequences show sequence similarity it is inferred that the two nucleic acids share a common ancestor. The precise level of sequence similarity required to establish homology varies in the art depending on a variety of factors.
  • nucleic acids are considered homologous where they share sufficient sequence identity to allow direct recombination to occur between the two nucleic acid molecules.
  • nucleic acids utilize regions of close similarity spaced roughly the same distance apart to permit recombination to occur.
  • the recombination can be in vitro or in vivo.
  • one advantage of the invention is the ability to recombine more distantly related nucleic acids than standard recombination techniques permit.
  • sequences from two nucleic acids which are distantly related, or even not detectably related can be recombined using cross-over oligonucleotides which have subsequences from two or more different non-homologous target nucleic acids.
  • the two nucleic acids can only be indirectly recombined using oligonucleotide intermediates, they are not considered homologous for purposes of this disclosure.
  • Two nucleic acids “correspond" when they have the same sequence, or when one nucleic acid is a subsequence of the other, or when one sequence is derived, by natural or artificial manipulation from the other.
  • Nucleic acids are "elongated" when additional nucleotides (or other analogous molecules) are incorporated into the nucleic acid. Most commonly, this is performed with a polymerase (e.g., a DNA polymerase), e.g., a polymerase which adds sequences at the 3' terminus of the nucleic acid.
  • a polymerase e.g., a DNA polymerase
  • a polymerase e.g., a polymerase which adds sequences at the 3' terminus of the nucleic acid.
  • Two nucleic acids are “recombined” when sequences from each of the two nucleic acids are combined in a progeny nucleic acid.
  • Two sequences are “directly” recombined when both of the nucleic acids are substrates for recombination.
  • Two sequences are "indirectly recombined” when the sequences are recombined using an intermediate such as a cross-over oligonucleotide. For indirect recombination, no more than one of the sequences is a substrate for recombination.
  • a collection of "fragmented nucleic acids” is a collection of nucleic acids derived by cleaving one or more parental nucleic acids (e.g., with a nuclease, or via chemical cleavage).
  • a “full-length protein” is a protein having substantially the same domains as a corresponding protein encoded by a natural gene.
  • the protein can have modified sequences relative to the corresponding naturally encoded gene (e.g., due to recombination and selection), but is at least 95% as long as the naturally encoded gene.
  • isolated refers to material that is substantially or essentially free from components which normally accompany the enzyme as found in its native state. Thus, the polypeptides of the invention do not include materials normally associated with their in situ environment.
  • isolated proteins of the invention are at least about 80% pure, usually at least about 90%, and preferably at least about 95% pure as measured by band intensity on a silver stained gel or other method for determining purity.
  • Protein purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized.
  • recombinant when used with reference to a cell, or nucleic acid, or vector, indicates that the cell, or nucleic acid, or vector, has been modified by the introduction of a heterologous nucleic acid or the alteration of a native nucleic acid, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual alignment and inspection.
  • substantially identical in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 75%, preferably 85%, most preferably 90-95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues.
  • the sequences are substantially identical over the entire length of the coding regions and/or untranslated regions.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated if changes from the default parameters are desired.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol. Biol.
  • BLAST algorithm Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always > 0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • BLOSUM62 scoring matrix see Henikoff & Henikoff, Proc. Natl Acad. Sci. USA 89:10915 (1989)
  • the default settings are appropriate.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl Acad. Sci.
  • nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • a further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
  • Bind(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • stringent conditions refers to conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances.
  • Tm thermal melting point
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na + ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Consatively modified variations of a particular polynucleotide sequence refers to those polynucleotides that encode identical or essentially identical amino acid sequences, or where the polynucleotide does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of “conservatively modified variations.” Every polynucleotide sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine
  • each "silent variation" of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • G Glycine
  • A Alanine
  • V Valine
  • V Valine
  • L Leucine
  • I Isoleucine
  • Aromatic Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C);
  • Acidic Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q).
  • sequences are preferably optimized for expression in a particular host cell used to produce the proteins (e.g., yeast, human, and the like).
  • conservative amino acid substitutions in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties (see, the definitions section, supra), are also readily identified as being highly similar to a particular amino acid sequence, or to a particular nucleic acid sequence which encodes an amino acid. Such conservatively substituted variations of any particular sequence are a feature of the present invention. See also, Creighton (1984) Proteins, W.H. Freeman and Company. In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also “conservatively modified variations".
  • the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • antibodies raised to the one or more of the RetinOR polypeptides (or subsequences thereof) or to the polypeptides partially encoded by the hRetinORl or hRetionOR2 polynucleotide sequences of SEQ ID NO: 1 or SEQ ID NO: 2 can be selected to obtain antibodies specifically immunoreactive with the full length proteins and not with other proteins except perhaps to polymorphic variants.
  • a variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • the present invention pertains to the discovery of novel nuclear hormone receptors that are found in retinal cells and possibly other cells. Also provided are isolated nucleic acids that encode the retinal nuclear hormone receptors, as well as chimeric receptor polypeptides and nucleic acids encoding such polypeptides. The invention also provides methods for identifying ligands that can modulate activity of the retinal nuclear hormone receptors, and methods for identifying compounds that can affect the ability of such ligands to interact with the hormone receptors. Methods of identifying response elements that are responsive to the nuclear hormone receptors are also provided.
  • the present invention provides novel retinal nuclear receptor polypeptides.
  • the polypeptides are useful for several purposes. For example, one can use the RetinOR polypeptides of the invention to modulate expression of genes that are influenced by these receptors.
  • the polypeptides are also useful in screening assays to identify compounds that are capable of modulating, either increasing or decreasing, gene expression.
  • the human RetinOR receptor occurs in at least two forms, hRetinORl (SEQ ID NO: 4) and hRetinOR2 (SEQ ID NO: 5).
  • the hRetinOR2 variant is a truncated version of hRetinORl, arising from a 40 bp insertion in the coding region of an hRetinOR2 coding region.
  • the insertion introduces a premature stop codon into the coding region, thus resulting in a shorter polypeptide.
  • the full-length hRetinORl polypeptide is 410 amino acids in length, while the hRetinOR2 variant is 372 amino acids in length.
  • the truncated hRetinOR2 polypeptide lacks an AF-2 region (Durand et al. (1994) EMBOJ. 13: 5370).
  • the retinal nuclear receptors of the invention include isolated hRetinORl and hRetinOR2 retinal nuclear receptor polypeptides that are substantially free of other polypeptides and other cellular components with which the native receptors are naturally associated.
  • the invention also provides polypeptides that include an amino acid sequence that is substantially identical to an hRetinORl and/or hRetinOR2 polypeptide or a portion of these polypeptides.
  • the invention provides isolated polypeptides that include an amino acid sequence that is substantially identical to a functional domain of a human RetinOR (hRetinOR) retinal nuclear receptor.
  • Functional domains of nuclear receptors such as hRetinORl and hRetinOR2 include, for example, a DNA binding domain, a ligand binding domain, and an AF2 domain.
  • the invention provides polypeptides that are substantially identical to a DNA binding domain of the RetinOR polypeptides.
  • the polypeptides of the invention include amino acid sequences that comprise one or more of the zinc fingers which are part of the hRetinORl and hRetinOR2 DNA binding domains.
  • the DNA binding domains of the invention typically include the amino acid sequence CRVCGDSSSGKHYG IYACNGCSGFFKRSVRRRLIYRCQVGAGMCPVDKAHRNQCQACRLKKCLQAGM (SEQ ID NO: 4), which includes two "C4"-type zinc fingers.
  • conservative amino acid substitutions can be made without loss of DNA binding activity.
  • Polypeptides that contain substitutions, or that include less than the full- length DNA binding domain can easily be tested for DNA binding ability using assays that are well known to those of skill in the art.
  • polypeptides that include an amino acid sequence that is substantially identical to a ligand binding domain of an hRetinORl and/or hRetinOR2 polypeptide.
  • the ligand binding domain of the hRetinORl polypeptide includes twelve ⁇ - helical structures, while that of the hRetinOR2 polypeptide lacks one half of helix 10 through helix 12 of the ligand binding domain.
  • the ligand binding domain of hRetinORl and hRetinOR2 include the amino acids between about residue 227 and about residue 274 in SEQ ID NO: 4 and SEQ ID NO: 5, respectively.
  • the ligand binding domains of the RetinOR polypeptides generally include the amino acid sequence LLFMAVKWAKNLPVFSSLP
  • the ligand binding domains will include additional amino acids of RetinOR on either or both ends of this amino acid sequence.
  • the ligand binding domains also include amino acid residues beginning at about amino acid 212 in SEQ ID NO: 4 and SEQ ID NO: 5; in other embodiments the ligand binding domain includes amino acids of the AF2 domain as described below.
  • the AF-2 domain of RetinORl includes the amino acids LLCDMFKN (SEQ ID NO: 15 LLCDMFKN), which are found at the carboxyl terminus of the polypeptide, residues 403 to 410.
  • RetinOR2 which is truncated as a result of a stop codon introduced by a 40 bp insertion, lacks an AF-2 domain.
  • ligand binding domain generally includes, unless otherwise stated, not only the ligand binding domain but also the AF-2 domain of RetinORl polypeptides.
  • the RetinOR polypeptides of the invention can be made by methods known to those of skill in the art.
  • the RetinOR proteins or subsequences thereof are synthesized using recombinant DNA methodology. Generally this involves creating a DNA sequence that encodes the polypeptide, modified as desired, placing the DNA in an expression cassette under the control of a particular promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein.
  • polypeptides of the invention can be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeasts, filamentous fungi, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines.
  • E. coli E. coli
  • yeasts yeasts
  • filamentous fungi various higher eukaryotic cells
  • COS COS
  • CHO CHO and HeLa cells lines and myeloma cell lines.
  • myeloma cell lines myeloma cell lines.
  • bacteria examples include, but are not limited to, Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus.
  • Filamentous fungi that are useful as expression hosts include, for example, the following genera: Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Mucor, Cochliobolus, and Pyricularia. See, e.g., US Patent No.
  • a polynucleotide that encodes a RetinOR polypeptide of the invention can be operably linked to appropriate expression control sequences for a particular host cell in which the polypeptide is to be expressed.
  • appropriate control sequences include a promoter such as the T7, tip, or lambda promoters, a ribosome binding site and preferably a transcription termination signal.
  • the control sequences typically include a promoter which optionally includes an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc. , and a polyadenylation sequence, and may include splice donor and acceptor sequences.
  • yeast convenient promoters include GAL 1,10 (Johnson and Davies (1984) Mol. Cell. Biol. 4:1440-1448) ADH2 (Russell et al. (1983) J. Biol. Chem. 258:2674-2682), PHO5 (EMBOJ. (1982) 6:675-680), and MF ⁇ l (Herskowitz and Oshima (1982) in 77ze Molecular Biology of the Yeast Saccharomyces (eds. Strathern, Jones, and Broach) Cold Spring Harbor Lab., Cold Spring Harbor, N. Y., pp. 181 -209).
  • Expression cassettes are typically introduced into a vector which facilitates entry into a host cell, and maintenance of the expression cassette in the host cell.
  • Vectors that include a polynucleotide that encodes a RetinOR polypeptide are provided by the invention. Such vectors often include an expression cassette that can drive expression of the RetinOR polypeptide.
  • To easily obtain a vector of the invention one can clone a polynucleotide that encodes the RetinOR polypeptide into a commercially or commonly available vector.
  • a variety of common vectors suitable for this purpose are well known in the art.
  • common vectors include pBR322 derived vectors such as pBLUESCRIPTTM, and ⁇ -phage derived vectors.
  • vectors include Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp series plasmids) and pGPD- 2.
  • YIp5 Yeast Integrating plasmids
  • YRp series plasmids the YRp series plasmids
  • pGPD- 2 pGPD- 2.
  • a multicopy plasmid with selective markers such as Leu-2, URA-3, Trp-1, and His-3 is also commonly used.
  • a number of yeast expression plasmids such as YEp6, YEpl3, YEp4 can be used as expression vectors.
  • the above-mentioned plasmids have been fully described in the literature (Botstein et al. (1979) Gene 8:17-24; Broach et al. (1979) Gene, 8:121-133).
  • yeast expression plasmids see, e.g., Parents, B., 7E ⁇ ST(1985), and Ausubel, Sambrook, and Berger, all supra).
  • Expression in mammalian cells can be achieved using a variety of commonly available plasmids, including pSV2, pBC12BI, and p91023, as well as lytic virus vectors (e.g., vaccinia virus, adenovirus, and baculovirus), episomal virus vectors (e.g., bovine papillomavirus), and retroviral vectors (e.g., murine retro viruses).
  • lytic virus vectors e.g., vaccinia virus, adenovirus, and baculovirus
  • episomal virus vectors e.g., bovine papillomavirus
  • retroviral vectors e.g., murine retro viruses.
  • nucleic acids that encode the polypeptides of the invention can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment or electroporation for mammalian cells.
  • Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes, among others.
  • Techniques for transforming fungi are well known in the literature and have been described, for instance, by Beggs et al. ((1978) Proc. Natl. Acad. Sci. USA 75: 1929-1933), Yelton et al. ((1984) Proc. Natl. Acad. Sci.
  • the RetinOR proteins can be purified, either partially or substantially to homogeneity, according to standard procedures of the art, such as, for example, ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, Springer- Verlag, N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to Protein
  • polypeptides may then be used (e.g., in screening assays for modulators for gene expression or as immunogens for antibody production).
  • the RetinOR protein(s) may possess a conformation substantially different than the native conformations of the constituent polypeptides. In this case, it may be necessary to denature and reduce the polypeptide and then to cause the polypeptide to re-fold into the preferred conformation.
  • Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art (See, Debinski et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al, (1992) Anal.
  • RetinOR polypeptides without diminishing their biological activity.
  • Some modifications may be made to facilitate the cloning, expression, or incorporation of the polypeptide into a fusion protein.
  • Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.
  • the invention also provides antibodies that can specifically bind retinal nuclear receptors such as RetinORl and RetinOR2.
  • These antibodies can be prepared using as immunogens recombinant or synthetic polypeptides of 10 amino acids in length, or greater, selected from amino acid subsequences of the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5 are the preferred polypeptide immunogen (antigen) for the production of monoclonal or polyclonal antibodies.
  • an immunogenic peptide conjugate is also included as an immunogen.
  • Naturally occurring polypeptides are also used either in pure or impure form. Methods of producing polyclonal and monoclonal antibodies are well known to those of skill in the art.
  • Recombinant polypeptides are expressed in eukaryotic or prokaryotic cells (as described below) and purified using standard techniques. The polypeptide, or a synthetic version thereof, is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the polypeptide.
  • the invention provides isolated and/or recombinant nucleic acids that encode RetinOR polypeptides, and functional domains thereof.
  • the nucleic acids are useful for many purposes. For example, one can use the RetinOR nucleic acids of the invention to produce RetinOR polypeptides.
  • the nucleic acids of the invention are also useful as probes to identify RetinOR-encoding nucleic acids in human tissues, and also in those of other mammals.
  • the nucleic acids are also useful to study the expression of RetinOR, both in vitro and in vivo.
  • the nucleic acids also find use as antisense molecules that can alter expression of the RetinOR nuclear receptors.
  • Polynucleotide sequences that code for two variants of human retinal nuclear receptors, hRetinORl and hRetinOR2, are provided as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the protein coding regions of both hRetinORl and hRetinOR2 begin with an ATG codon at nucleotide position 88 of the respective polynucleotide sequences, relative to the beginning of the shaded portion of Figure 1.
  • the two polynucleotide sequences are identical except that hRetinOR2 includes a 40 bp insertion following nucleotide 1188.
  • This insertion which is due to a mRNA splicing variation, introduces a stop codon into the hRetinOR2 polynucleotide sequence, which results in the hRetinOR2 polypeptide being truncated compared to the hRetinOR2 polypeptide as discussed below.
  • a stop codon occurs beginning at nucleotide position 1204 of the hRetinOR2 polynucleotide sequence (SEQ ID NO: 2) and at nucleotide position 1318 of the hRetinORl polynucleotide sequence (SEQ ID NO: 1).
  • Untranslated regions both 5' and 3' of the retinal nuclear receptor coding regions are also provided in each of the hRetinOR polynucleotide sequences, and additional untranslated sequence information is provided in SEQ ID NO: 3.
  • the invention provides isolated nucleic acids that are at least substantially identical to a polynucleotide sequence that encodes all or part of an hRetinORl or hRetinOR2 polypeptide, or to an untranslated region that flanks one of such coding regions.
  • the percent similarity can be spread throughout for the entire hRetinORl or hRetinOR2 polynucleotide sequence, within the coding region, or within subsequences or regions of the polynucleotide sequences described herein.
  • the invention provides polynucleotide sequences that are at least about 90% identical to a subsequence of the hRetinORl or hRetinOR2 polynucleotide sequences that is at least about 52 nucleotides in length, more preferably at least about 75 nucleotides, and most preferably at least about 100 nucleotides in length.
  • the invention also provides isolated and/or recombinant nucleic acids that encode one or more functional domains of the retinal nuclear receptor.
  • nucleic acids are provided that are at least substantially identical to a polynucleotide sequence that encodes a DNA binding domain or a ligand binding domain of a retinal nuclear receptor such as hRetinORl or hRetinOR2.
  • nucleic acids that are at least substantially identical to a polynucleotide sequence that encodes an AF-2 domain of a RetinORl polypeptide. DNA binding domains and ligand binding domains of the RetinOR polypeptides are described above.
  • the nucleic acids e.g., RetinORl and RetinOR2 nucleic acids, or subsequences (probes)
  • the nucleic acids can be isolated by cloning or amplification by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (SSR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • SSR self-sustained sequence replication system
  • a wide variety of cloning and in vitro amplification methodologies are well-known to persons of skill. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger, Sambrook, and Ausubel (all supra.); Cashion et al, U.S.
  • the hRetinOR cD ⁇ As can be isolated by routine cloning methods.
  • the cD ⁇ A sequence provided in S ⁇ Q ID NO: 1 and/or S ⁇ Q ID NO: 2 can be used to provide probes that specifically hybridize to a RetinOR gene, in a genomic DNA sample, to a RetinOR mRNA, in a total RNA sample, or to a RetinOR cDNA in a cDNA library (e.g., in a Southern or Northern blot).
  • the target RetinOR nucleic acid Once the target RetinOR nucleic acid is identified, it can be isolated according to standard methods known to those of skill in the art (see, e.g., Sambrook, Berger, and Ausubel, supra.).
  • the RetinORl nucleic acids of the invention can be isolated by amplification methods such as polymerase chain reaction (PCR).
  • the invention also provides nucleic acid constructs in which a RetinOR polynucleotide of the invention is operably linked to a promoter that is functional in a desired host cell.
  • Such constructs are often provided as an "expression cassette", which can also include other sequences involved in transcription, translation, and post-translational modification of the RetinOR polypeptide. Examples of suitable promoters and other control sequences are described herein.
  • the invention also provides expression vectors, and host cells that comprise the claimed recombinant nucleic acids.
  • the invention provides chimeric receptors that have a ligand binding domain and a DNA binding domain. At least one of the ligand binding domain and the DNA binding domain of the chimeric receptors of the invention is substantially identical to the corresponding domain of a RetinOR polypeptide.
  • These chimeric receptors are useful for many purposes. For example, one can use the chimeric receptors to identify ligands for the RetinOR nuclear receptors and to identify response elements that are responsive to RetinOR nuclear receptors.
  • the chimeric receptors are also useful in screening assays for identifying compounds that can modulate interactions between RetinOR nuclear receptors and their ligands and/or response elements.
  • the chimeric receptors of the invention include those having a ligand binding domain that is at least substantially identical to a ligand binding domain of an hRetinORl or an hRetinOR2 receptor, as well as those that have a DNA binding domain that is not substantially identical to a DNA binding domain of an hRetinORl or l ⁇ RetinOR2 receptor.
  • the DNA binding domain binding domain can be about 90% or less identical to that of an hRetinORl or hRetinOR2 polypeptide, more preferably about 75% or less, and most preferably about 60% or less identical.
  • the DNA binding domain is derived from a receptor other than a hRetinOR receptor.
  • the DNA binding domain is at least substantially identical to a DNA binding domain from a nuclear hormone receptor or a steroid hormone receptor.
  • DNA binding domains derived from steroid, thyroid, and retinoid hormone receptor are suitable for use in the chimeric receptors of the invention.
  • the DNA binding domains of receptors for steroid, thyroid, and retinoid hormones typically include two zinc finger units (Rhodes and Klug (Feb. 1993) Scientific American, pp. 56-65).
  • the DNA binding domains of these receptors, as are those of the hRetinOR receptors are generally cysteine-rich regions of about 65 amino acids that fold into two cysteine-rich "C4" type zinc fingers.
  • receptors from which one can derive DNA binding domains that are suitable for use in the chimeric receptors of the invention include, for example, androgen receptors, estrogen receptors, glucocorticoid receptors, mineralcorticoid receptors, progesterone receptors, retinoic acid receptors (including ⁇ , ⁇ (hap), and ⁇ ), thyroid hormone receptors (including ⁇ and ⁇ ), the gene product of the avian erythroblastosis virus oncogene v-erbA (which is derived from a cellular thyroid hormone receptor), vitamin D3 receptor, Drosophila ecdysone receptor (EcR), COUP transcription factor (also known as ear3) and its Drosophila homolog 7UP (svp), hepatocyte nuclear factor 4 (HNF-4), Ad4BP, apolipoprotein Al regulatory protein- 1 (ARP-1), peroxisome proliferator activated receptor (PPAR), Drosophila protein knirps (kni),
  • the chimeric receptors of the invention include a DNA binding domain from a DNA-binding polypeptide other than a nuclear receptor.
  • a DNA binding domain from a DNA-binding polypeptide other than a nuclear receptor For example, chimeric receptors that have the DNA binding domain of GAL4, which is a positive regulatory protein of yeast (Giniger et al. (1985) Cell 40: 767-774; Sadowski et al. (1992) Gene 118: 137-141) linked to a ligand binding domain of a RetinOR polypeptide are provided.
  • GAL4 is a positive regulatory protein of yeast (Giniger et al. (1985) Cell 40: 767-774; Sadowski et al. (1992) Gene 118: 137-141) linked to a ligand binding domain of a RetinOR polypeptide are provided.
  • GAL4 DNA binding domain-containing fusion proteins can be readily expressed by cloning a coding sequence for a RetinOR ligand binding domain into a commercially available expression vector that includes a GAL4 DNA binding domain coding sequence under the control of a promoter (e.g., pAS2-l (CLONTECH Laboratories, Inc.).
  • a promoter e.g., pAS2-l (CLONTECH Laboratories, Inc.
  • Another example of a well-characterized DNA binding domain for which expression vectors are commercially available is that of LexA (pLexA, CLONTECH).
  • the chimeric receptors can also include a nuclear localization sequence associated with the DNA binding domain (see, e.g., Silver et al. (1984) Proc. Nat 1 Acad. Sci. USA 8 ⁇ : 5951 -5955 for a GAL4 nuclear localization sequence).
  • the chimeric receptors of the invention can use an entire molecule as a DNA binding domain, or can use portions of molecules that are capable of binding to nucleic acids, directly or indirectly.
  • an electrophoretic mobility shift assay (EMSA) (Scott et al. (1994) J. Biol. Chem. 269: 19848-19858), in which a nucleic acid of interest is allowed to associate with various fragments of a polypeptide to identify those fragments that are capable of binding to the nucleic acid. Association of a portion of the protein with the nucleic acid will result in a retardation of the electrophoretic mobility of the nucleic acid.
  • DNase I footprinting is well known to those of skill in the art.
  • the DNA binding domain can be either a polypeptide or a nucleic acid.
  • the nucleic acid will be capable of specifically hybridizing to a target nucleic acid site, such as a response element. Hybridization of the nucleic acid to the target site will place the chimeric receptor in a position suitable for activating or repressing expression of a gene that is linked to the target site.
  • a target nucleic acid site such as a response element.
  • Hybridization of the nucleic acid to the target site will place the chimeric receptor in a position suitable for activating or repressing expression of a gene that is linked to the target site.
  • An example of an oligonucleotide being chemically linked to a protein by chemical coupling is found in Corey et al. (1989) Biochemistry 28: 8277-8286.
  • chimeric receptors are useful, for example, in assays to identify modulators of RetinOR transcriptional regulation activity as described below.
  • Ligand binding domains are useful, for example, in assays to identify modulators of RetinOR transcriptional regulation activity as described below.
  • the invention also provides chimeric RetinOR receptors in which the DNA binding domain is at least substantially identical to a DNA binding domain of a RetinOR polypeptide, such as an hRetinORl or hRetinOR2 polypeptide and the ligand binding domain is not substantially identical to a ligand binding domain of an hRetinORl or hRetinOR2 polypeptide.
  • the ligand binding domain binding domain can be about 90% or less identical to that of an hRetinORl or hRetinOR2 polypeptide, more preferably about 75% or less, and most preferably about 60% or less identical.
  • the ligand binding domains used in the chimeric receptors are typically derived from ligand binding domains of other receptors, including the receptors listed above. Ligand binding domains for many receptors are known to those of skill in the art.
  • the ligand binding domain and the DNA binding domain are linked together. Suitable methods of forming such linkages are known to those of skill in the art. For a review of methods for constructing fusion proteins between receptor ligand binding domains and DNA binding domains, see, e.g. , Mattioni et al. (1994) Methods in Cell Biology 43(Pt A): 335-352.
  • the linkage can be done using either recombinant or chemical methods. For example, a cysteine residue can be placed at either end of a domain so that the domain can be linked to another domain by, for example, a sulfide linkage.
  • linkers are typically polypeptide sequences, such as poly glycine sequences of between about 5 and 200 amino acids, with between about 10-100 amino acids being typical.
  • proline residues are incorporated into the linker to prevent the formation of significant secondary structural elements by the linker.
  • Preferred linkers are often flexible amino acid subsequences which are synthesized as part of a recombinant fusion protein.
  • the flexible linker is an amino acid subsequence comprising a proline such as Gly(x)-Pro-Gly(x) where x is a number between about 3 and about 100.
  • a linker can also be a single peptide bond, or one or more amino acid residues.
  • a chemical linker is used to connect synthetically or recombinantly produced ligand binding domain and DNA binding domain subsequences.
  • Such flexible linkers are known to persons of skill in the art. For example, poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
  • the chimeric receptors are conveniently produced by recombinant expression in a host cell.
  • the invention provides chimeric nucleic acids that encode a fusion protein that includes a DNA binding domain and a ligand binding domain, at least one of which is at least substantially identical to the corresponding domain of a RetinOR nuclear receptor of the invention.
  • the chimeric nucleic acid will also encode a linker region that provides a link between the two domains.
  • Techniques for making such chimeric nucleic acids are known to those of skilled in the art. For example, recombinant methods can be used (see, e.g., Berger and Sambrook, both supra.). Alternatively, the nucleic acid encoding the chimeric receptors can be synthesized chemically.
  • a nucleic acid that encodes the chimeric receptor is generally placed under the control of a promoter and other control elements that can drive expression of the chimeric gene in a desired host cell.
  • the invention also provides expression cassettes in which a promoter and/or other control elements are operably linked to a polynucleotide that encodes a chimeric receptor. Suitable promoters, other control sequences, and expression vectors are described above.
  • Also provided by the invention are methods for identifying corepressors, coactivators, and ligands for RetinOR receptors, response elements that are responsive to RetinOR receptors, and methods for screening to identify compounds that upregulate or downregulate expression of genes that are regulated by RetinOR receptors.
  • the RetinORl activation domain contains the amino acid sequence LLCDMFKN, which is consistent with the consensus AD-2 sequence for nuclear receptors (Durand et al. (1994) EMBOJ. 13: 5370-5382).
  • Nuclear receptors that include a glutamate (E) at the fourth amino acid position of the consensus sequence are typically transcriptional activators, while those receptors that have an aspartate (D) at the fourth amino acid position are generally transcription repressors.
  • RetinORl AD-2 sequence includes an aspartate (D) instead of a glutamate is indicative that RetinORl is a repressor when unbound by a ligand.
  • Screening assays for identifying ligands for the RetinOR receptors are provided by the invention.
  • the methods can involve the use of a chimeric polypeptide that includes a RetinORl ligand binding domain that is linked to a DNA binding domain for which a known response element is available. Suitable chimeric polypeptides and methods for their production are described above.
  • the response element that is bound by the DNA binding domain used in the chimeric polypeptide is generally used in a reporter gene construct.
  • Response elements including glucocorticoid response elements (GRE) and estrogen response elements (ERE), are described in, for example, Jantzen et al. (1987) Cell 49: 29; Martinez et al. (1987) EMBO J 6: 3719 and Burch et al. (1988) Mol. Cell. Biol. 8: 1123.
  • GRE glucocorticoid response elements
  • ERP estrogen response elements
  • Many other response elements are known; a commonly used response element is the GAL4 upstream activating sequence (UAS G ) (Keegan et al. (1986) Science 14: 699-704), which is responsive to binding by chimeric receptors that include the GAL4 DNA binding domain.
  • UAS G GAL4 upstream activating sequence
  • the response element is operably linked to a promoter that is active in the particular host cell. Suitable promoters include those described above. In presently preferred embodiments, the promoter is operably linked to a reporter gene that, when expressed, produces a readily detectable product.
  • reporter gene plasmid systems are known, such as the chloramphenicol acetyltransferase (CAT) and ⁇ -galactosidase (e.g., bacterial LacZ gene) reporter systems, the firefly luciferase gene (See, e.g., Cara et al, (1996) J Biol. Chem., 271 : 5393-5397), the green fluorescence protein (see, e.g., Chalfie et al.
  • CAT chloramphenicol acetyltransferase
  • ⁇ -galactosidase e.g., bacterial LacZ gene
  • the firefly luciferase gene See, e.g.,
  • reporter plasmids are also described in US Patent No. 5,071,773.
  • Selectable markers which facilitate cloning of the vectors of the invention are optionally included. Sambrook and Ausubel, both supra, provide an overview of selectable markers.
  • the reporter plasmid and an expression plasmid that directs expression of the chimeric receptor are introduced into a suitable host cell. Standard transfection methods can be used to introduce the vectors into the host cells.
  • transfection methods include, for example, calcium phosphate precipitation (Chen and Okayama (1988) BioTechniques 6: 632), DEAE-dextran, and cationic lipid-mediated transfection (e.g., Lipofectin) (see, e.g., Ausubel, supra.).
  • the host cell prior to introduction of the expression plasmid, should not contain a RetinOR receptor. See, e.g., USPN 5,071,773 for suitable host cells for use in the assays.
  • the chimeric receptor is contacted with a candidate ligand.
  • a cell that contains a reporter gene construct and the chimeric peptide can be grown in the presence and absence of putative ligands. Due to the predicted repressive effect of RetinORl on reporter gene expression in the absence of ligand, cells grown in the absence of RetinORl ligand will exhibit a level of reporter gene expression that is below the basal level observed in the absence of the DNA-bound RetinORl ligand binding domain. Reporter gene expression is increased when cells are grown in the presence of a ligand for the RetinORl receptor if ligand binding to the RetinOR ligand binding domain causes relief of RetinORl - mediated repression. Conversely, a decrease in reporter gene expression will result if the binding of the ligand to a RetinOR polypeptide is associated with inhibition of gene expression.
  • Candidate ligands can include hormones and other molecules that, through binding to their cognate receptor, are capable of influencing gene expression.
  • potential ligands for the RetinOR receptors of the invention can include other receptor polypeptides, coactivators, and the like, which comprise the cellular machinery for regulation of gene expression.
  • nuclear hormone receptors often interact with transcriptional coactivators.
  • the invention also provides methods of identifying coactivators, corepressors and other molecules that interact with RetinOR receptors. These assay methods can involve introducing a coactivator or a corepressor that is a candidate ligand for RetinOR receptors into a host cell that contains a chimeric RetinOR receptor and reporter plasmid.
  • the coactivator can be introduced by means of an expression construct; this expression construct can be present on the same or a different vector than the expression construct for the chimeric receptor. Additional methods of identifying ligands for nuclear receptors such as the
  • RetinOR receptors can be adapted from assays described in commonly assigned US patent application Ser. No. 08/975,614, filed November 21, 1997. Briefly, these assays use a peptide sensor to which is attached a detectable label.
  • the peptides are based on corepressor or coactivator protein motif sequences, either naturally occurring or derived from mutational analysis. Alternatively, the peptides can be obtained through randomizing residues and selecting for binding to the RetinOR receptor polypeptide. In some embodiments, panels of predetermined or randomized candidate sensors are screened for receptor binding.
  • the sensor peptides are labeled with a detectable label.
  • the detectable labels can be primary labels (where the label comprises an element that is detected directly or that produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, as is common in immunological labeling).
  • Primary and secondary labels can include undetected elements as well as detected elements.
  • Useful primary and secondary labels in the present invention can include spectral labels such as fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon Green , rhodamine and derivatives (e.g., Texas red, tetrarhodimine isothiocynate (TRITC), etc.), digoxigenin, biotin, phycoerythrin, AMCA, CyDyes TM , and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, 32 P, 33 P, etc.), enzymes (e.g., horse radish peroxidase, alkaline phosphatase etc.), spectral colorimetric labels such as colloidal gold or colored glass or plastic (e.g.
  • fluorescent dyes e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon Green
  • the label may be coupled directly or indirectly to a component of the detection assay (e.g., the detection reagent) according to methods well known in the art.
  • a component of the detection assay e.g., the detection reagent
  • the detectable label is a fluorescent label, in which case fluorescence polarization detection provides a sensitive and efficient means of detecting whether the peptide sensor is bound to the RetinOR receptor polypeptide. See, e.g. , Schindler et al. (1995) Immunity 2: 689-697).
  • the sensor polypeptide and the RetinOR polypeptide are incubated under conditions that are suitable for sensor binding to the receptor polypeptide.
  • a candidate modulator of RetinOR binding to a corepressor or coactivator is included in the reaction mixture. If a candidate modulator decreases binding of the sensor peptide to the RetinOR polypeptide, the candidate modulator is a potential lead compound for blocking the RetinOR-mediated effect on transcription.
  • the present invention provides methods for obtaining response elements that are responsive to RetinOR receptors. Through use of such methods, one can identify genes for which expression is modulated by the RetinOR receptors.
  • the methods typically involve contacting a putative response element with a polypeptide that includes a RetinOR DNA binding domain (see, e.g., Ausubel et ah, supra.). Both cell-based and biochemical methods are provided.
  • a RetinOR receptor, or RetinOR DNA binding domain is used.
  • a chimeric polypeptide that has a RetinOR DNA binding domain and a ligand binding domain from a non-orphan receptor.
  • the ligand binding domain is preferably one for which an appropriate ligand is available. Suitable chimeric receptors are described above.
  • standard gel shift assays are performed to identify polynucleotides that can bind to a RetinOR DNA binding domain. These assays are performed by incubating a polypeptide that includes a RetinOR DNA binding domain, either as a purified protein or a complex mixture of proteins) with a labeled DNA fragment that contains the putative RetinOR binding site. Reaction products are analyzed on a nondenaturing polyacrylamide gel. To determine the specificity of the binding, one can perform competition experiments using polynucleotides that include a RetinOR binding site, or unrelated DNA sequences. Kits for performing gel shift assays are described in, for example, Gel Shift Assay Systems manual (Promega, Madison WI, Part No. TB110).
  • Another in vitro assay for identifying RetinOR response elements is the binding site selection method (see, e.g., US Patent No. 5,582,981).
  • a library of oligonucleotides having a randomized nucleotide sequence of about 18 nucleotides flanked by two known nucleotide sequences of sufficient length to allow hybridization to PCR primers that are complementary to these regions.
  • the oligonucleotides are end-labeled (e.g., with ⁇ - 32 P) and contacted with a RetinOR DNA binding domain polypeptide.
  • a low stringency gel shift experiment is performed.
  • PCR amplification is then carried out on those oligonucleotides to which the RetinOR DNA binding domain bound, as evidenced by retardation in the gel shift electrophoresis.
  • the selection and amplification process is repeated at least twice more using the amplified fragments.
  • In vivo assays for RetinOR response elements are also provided.
  • the in vivo assays are particularly suitable for confirming results obtained in an in vitro assay.
  • Cells are provided which contain a reporter construct that contains the putative response element in a position relative to a promoter at which binding of a RetinOR polypeptide can increase or decrease expression of an operably linked gene.
  • the putative response element can be, for example, a member of a library of polynucleotide fragments.
  • the chimeric receptor and the reporter constructs are introduced into a host cell. Suitable host cells are described in, for example, USPN 5,071,773.
  • the host cells that contain the reporter plasmid construct and the chimeric receptor are grown in the presence of the ligand for the ligand binding domain used in the chimeric receptor.
  • Those cells in which expression of the reporter gene in the presence of the ligand is greater or less than the expression in the absence of the ligand contain a reporter construct that includes a putative response element for a RetinOR receptor.
  • the response elements can be isolated from these cells by, for example, plasmid recovery, PCR amplification, or other methods known to those of skill in the art. Upon isolation, the response elements can be characterized (e.g., by sequencing) and used to identify genes for which expression is influenced by RetinOR receptors.
  • Screening assays to identify compounds that modulate RetinOR activity The invention also provides screening assays for identifying compounds that can modulate RetinOR biological activity. These compounds can function by, for example, altering the interaction between RetinOR receptors and their ligands (e.g., corepressors and/or coactivators) or between the RetinOR receptors and their response elements.
  • ligands e.g., corepressors and/or coactivators
  • the screening methods of the invention find use in studies of gene regulation in retinal tissue, and also find therapeutic use in situations in which it is desirable to increase or decrease expression of genes that are under the control of RetinOR nuclear receptors. Other uses will also be apparent those of ordinary skill in the art.
  • the screening methods of the invention use a cell that contains a polypeptide that has a ligand binding domain which is at least substantially identical to that of a RetinOR receptor.
  • the polypeptide will also include a DNA binding domain, which can be substantially identical to that of a RetinOR receptor, or to that of a receptor other than a RetinOR receptor, or to any DNA binding domain for which the response element is known (e.g., GAL4, nuclear hormone receptors, and the like).
  • a DNA binding domain for which the response element is known (e.g., GAL4, nuclear hormone receptors, and the like).
  • suitable chimeric polypeptides are described in more detail above.
  • the chimeric receptor polypeptide is introduced into the cell by expression of a polynucleotide that encodes the receptor polypeptide.
  • an expression vector that encodes the chimeric receptor can be introduced into the cell that is to be used in the assay.
  • the cells will also contain a response element that can be bound by the DNA binding domain.
  • the response element is operably linked to a promoter that is active in the cell.
  • the promoter is operably linked to a reporter gene that, when expressed, produces a readily detectable product.
  • the response element/reporter gene construct is conveniently introduced into cells as part of a "reporter plasmid.”
  • Host cells that contain the reporter plasmid, the chimeric receptor polypeptide, and the ligand are incubated in the presence of a test compound.
  • any chemical compound can be used as a potential RetinOR activity modulator in the assays of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used.
  • the assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma- Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.
  • high throughput screening methods involve providing a combinatorial library containing a large number of potential therapeutic compounds (potential modulator compounds). Such "combinatorial chemical libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487- 493 (1991) and Houghton et al, Nature 354:84-88 (1991)).
  • Other chemistries for generating chemical diversity libraries can also be used.
  • Such chemistries include, but are not limited to: peptoids (PCT Publication No. WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio-oligomers (PCT Publication No.
  • WO 92/00091 benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al, J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with ⁇ -D-glucose scaffolding (Hirschmann et al, J. Amer. Chem. Soc.
  • carbohydrate libraries see, e.g., Liang et al, Science, 274:1520-1522 (1996) and U.S. Patent 5,593,853
  • small organic molecule libraries see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like).
  • compositions, Kits and Integrated Systems The invention provides compositions, kits and integrated systems for practicing the assays described herein.
  • an assay composition having an expression vector for a chimeric RetinOR polypeptide, an expression vector that contains an appropriate reporter gene, and a suitable host cell is provided by the present invention.
  • Ligands that bind to the ligand binding domain of the chimeric receptor can also be included in the assay compositions, as can modulators of RetinOR activity.
  • kits for practicing the RetinOR assay methods noted above can include any of the compositions noted above, and optionally further include additional components such as instructions to practice a high-throughput method of assaying for RetinOR activity, or screening for an inhibitor or activator of RetinOR activity, one or more containers or compartments (e.g., to hold reagents, nucleic acids, or the like), and a control RetinOR activity modulator.
  • the invention also provides integrated systems for high-throughput screening of potential RetinOR modulators for an effect on expression of genes under the control of RetinOR nuclear receptors.
  • the systems typically include a robotic armature which transfers fluid from a source to a destination, a controller which controls the robotic armature, a label detector, a data storage unit which records label detection, and an assay component such as a microtiter dish comprising a well having a reaction mixture or a substrate.
  • This Example describes the cloning and characterization of two novel human nuclear hormone receptors.
  • EST clone W27871 (SEQ ID NO: 8) was identified as a putative novel nuclear receptor clone from human retina. This sequence exhibited some similarity to the DNA binding domains of a number of nuclear receptors. EST clone W27871 was used in a BLAST search of the database. The closest potential homologs were identified as being a putative C. elegans nuclear receptor and the human tailless nuclear receptor.
  • Oligonucleotides FCT27871.fl TzoI-sense of nucleotides 29-55 of W27871) and FCT27871.rl ( ⁇ l-antisense of nucleotides 308-332) spanning the putative P-box of the putative nuclear receptor were used to PCR amplify the intervening sequence of W27871 from human retina cDNA (Clontech, Palo Alto CA) using standard PCR procedures. PCR amplification yielded a single ⁇ 300 bp DNA fragment that was then cloned into pBluescript KS + II and sequenced. The sequence of the -300 bp sequence (SEQ ID NO: 9) confirmed the similarity of the clone to the above-mentioned nuclear receptors.
  • RetinOR RetinOR
  • RetinORl is identical to RetinOR2 except for the presence in RetinOR2 of a 40 bp insertion at base pair 1101 relative to the translation start site ( Figure 1).
  • the 40 bp insertion introduces a stop codon into the coding sequence, thus resulting in a truncated version of RetinOR that is 38 amino acids shorter than RetinORl ( Figure 2).
  • the truncated RetinOR2 polypeptide lacks part of helix 10 and helices 11 and 12 of the canonical ligand binding domain structure (Wurtz et al. (1996) Nat. Struct. Biol. 3: 87-94).
  • RetinOR gene expression in various human tissues was analyzed by PCR. Expression of RetinOR mRNA was detected only in the retina. No expression was observed in brain, heart, liver, kidney, lung, pancreas, placenta, and skeletal muscle. However, the possibility remains that RetinOR is found in additional tissues. Because human RetinOR gene expression is found in the retina, it is likely that this novel receptor plays a critical role in the development of the visual system. Furthermore, the similarity between RetinOR and other nuclear receptors suggests that RetinOR is or can be regulated by a ligand.
  • a vector includes a reporter gene construct which contains multiple copies of a 17-mer GAL4 upstream activating sequence (UAS G ) located upstream of a promoter.
  • the promoter is operably linked to a luciferase reporter gene.
  • a suitable reporter gene construct is that present in the pG5luc vector (Promega Corporation, Madison WI).
  • a polynucleotide that encodes an hRetinORl ligand binding domain is cloned into an expression vector which also includes a coding region for a yeast GAL4 DNA binding domain (amino acids 1-147) (e.g., pBIND (Promega Corporation)), such that the GAL4 DNA binding domain and the RetinORl ligand binding domain are expressed as a fusion protein.
  • a vector which encodes a GAL4 DNA binding domain alone is provided as a positive control.
  • Kits from which the vectors used in this and subsequent Examples can be constructed are commercially available (e.g., MATCHMAKERTM Two-Hybrid Assay Kit(User Manual PT3002-1; Catalog # K1602-1, CLONTECH Laboratories, Inc., Palo Alto CA).
  • Mammalian cells e.g., HeLa, CHO, NIH 3T3 are maintained in DMEM supplemented with 10% fetal bovine serum. Twenty-four hours before transfection, cells re seeded in 24 well tissue culture plates at a density of approximately 5 x 10 4 cells per well. Cells are transfected with vectors using an appropriate protocol (e.g., LipofectinTM, Life Technologies, Grand Island NY) according to the manufacturer's guidelines. Typically, about 1.8 ⁇ g of total DNA is used, which includes 0.3 ⁇ g of reporter and 0.05 to 1.0 ⁇ g of expression vector per 35 mm diameter dish.
  • an appropriate protocol e.g., LipofectinTM, Life Technologies, Grand Island NY
  • transfection reagent The recommended amount of transfection reagent is added to a mixture of DNA and medium, vortexed briefly and allowed to incubate at room temperature for 10 minutes.
  • the growth medium is removed from the cells; the transfection mixture is added; and the cells are incubated at 37°C. After a one-hour incubation, 1 ml of complete medium is added to the cells and incubation continued for 48 hours, at which time reporter gene expression is assessed.
  • Luciferase expression is assessed using a luminometer.
  • the vector that contains the reporter gene construct and either 1) the vector that encodes the RetinOR 1-GAL4 fusion protein or 2) a control vector are transfected into mammalian host cells as described above. Cells are grown in complete medium. Luciferase expression is then assessed.
  • Luciferase is expressed at its high basal level in control cells that contain the reporter gene vector and the control vector that expresses the GAL4 DNA binding domain. If, as predicted, RetinORl is a repressor of transcription, luciferase expression is suppressed in cells that contain the reporter gene vector and the vector that encodes the RetinORl - GAL4 fusion protein. To determine whether the repression is mediated by corepressors, cells are transfected with the reporter gene vector, the RetinOR 1-GAL4 fusion protein expression vector, and a third vector that encodes a RetinORl ligand binding domain in the absence of a DNA binding domain.
  • RetinOR2 which lacks an AD-2 domain
  • the expression vector that encodes the RetinOR2 ligand binding domain is introduced into the mammalian host cells along with the reporter gene and RetinORl -GAL4 fusion protein vectors. Luciferase expression is not derepressed in cells that contain the RetinOR2 ligand binding domain, due to the failure of RetinOR2 to bind to the corepressors and thus prevent their interaction with the DNA-bound RetinORl -GAL4 fusion protein.
  • This Example describes an assay for identifying modulators of RetinOR- mediated regulation of transcriptional activity.
  • the vector that contains the reporter gene construct and either 1) the vector that encodes the RetinORl -GAL4 fusion protein or 2) a control vector are transfected into mammalian host cells as described above. After transfection, the cells are grown in complete medium in the presence or absence of a candidate modulator of RetinOR-mediated transcriptional regulation.
  • a cell that expresses the positive control GAL4 DNA binding domain unlinked to a RetinORl ligand binding domain will produce luciferase at a high level.
  • a cell that contains a vector that expresses the RetinOR 1-GAL4 fusion protein is not expected to express high levels of luciferase in the absence of a modulator of RetinORl activity, due to the predicted repressive activity of the RetinORl ligand binding domain in the absence of a ligand for RetinORl .
  • a candidate compound that modulates the interaction between the RetinORl ligand binding domain and the corepressors and/or coactivators that mediate the regulatory effect of RetinORl will relieve the repression of luciferase expression.
  • the candidate compound in wells that exhibit higher levels of luciferase compared to wells in which no candidate modulator is present is identified as a lead compound for a modulator of RetinORl activity.
  • a compound that acts as a ligand for RetinORl can cause a change in the conformation of the RetinORl ligand binding domain which eliminates the interaction with corepressors and results in the ligand binding domain interacting with coactivators.
  • a reporter gene construct and a vector that encodes a GAL4 DNA binding domain-RetinORl ligand binding domain fusion protein as described in Example 3 are provided. Also provided is a third vector that contains a coding region for an activation domain from, for example, a herpes simplex virus type 1 VP16 (amino acids 411-456) (e.g., pACT, Promega Corporation).
  • a polynucleotide sequence that encodes all or part of a putative corepressor and/or coactivator of RetinORl is cloned in-frame with the activation domain coding region. The polynucleotide sequence can be derived from a known corepressor and/or coactivator, or can be a member of a cDNA library.
  • the vector that contains the reporter gene construct is transfected into mammalian cells. Into control cells is also transfected the vector that encodes the GAL4- RetinORl fusion protein. Experimental cells are transfected with these two vectors as well as a vector that encodes the activation domain fusion polypeptide.
  • Cells that contain the reporter gene vector and a vector that expresses a GAL4 DNA binding domain alone will exhibit a high level of luciferase expression.
  • Expression of the GAL4 DNA binding domain fused to the RetinORl ligand binding domain are expected to exhibit repression of luciferase expression.
  • a cell that contains, in addition to the reporter gene vector and the GAL4- RetinORl fusion protein expression vector, a vector that encodes the VP16 AD fused to a polypeptide that interacts with RetinORl will exhibit activation of luciferase expression.
  • the interaction between the AD fusion polypeptide and the DNA-bound RetinORl ligand binding domain results in the VP 16 AD being placed in close proximity to the promoter, resulting in transcription activation.

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Abstract

La présente invention concerne de nouveaux récepteurs hormonaux nucléaires, RetinOr, qui sont présents dans le tissu rétinien. L'invention concerne également des acides nucléiques codant pour les récepteurs RetinOr ainsi que des techniques de dosage d'éléments qui réagissent aux récepteurs RetinOr en vue de l'identification de ligands desdits récepteurs et de composés capables de moduler l'activité régulatrice de l'expression génique de récepteurs RetinOR.
PCT/US1999/017885 1998-08-07 1999-08-06 Nouveau recepteur hormonal nucleaire retinien Ceased WO2000008052A1 (fr)

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AU55500/99A AU5550099A (en) 1998-08-07 1999-08-06 A novel retinal nuclear hormone receptor
IL13979800A IL139798A0 (en) 1998-08-07 2000-05-11 Gas jet removal of particulated soil from fabric

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001098319A1 (fr) * 2000-05-19 2001-12-27 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, site de liaison de la retine 12, et polynucleotide codant ce polypeptide
WO2002070561A3 (fr) * 2001-03-05 2002-12-19 Inpharmatica Ltd Domaine de liaison du ligand du recepteur hormonal nucleaire
WO2001067855A3 (fr) * 2000-03-16 2003-02-13 Deltagen Inc Souris transgeniques contenant des interruptions de genes ciblees

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998028418A1 (fr) * 1996-12-23 1998-07-02 Smithkline Beecham Plc Recepteur d'hormone nucleaire sans queue (recepteur de tlx)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998028418A1 (fr) * 1996-12-23 1998-07-02 Smithkline Beecham Plc Recepteur d'hormone nucleaire sans queue (recepteur de tlx)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001067855A3 (fr) * 2000-03-16 2003-02-13 Deltagen Inc Souris transgeniques contenant des interruptions de genes ciblees
WO2001098319A1 (fr) * 2000-05-19 2001-12-27 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, site de liaison de la retine 12, et polynucleotide codant ce polypeptide
WO2002070561A3 (fr) * 2001-03-05 2002-12-19 Inpharmatica Ltd Domaine de liaison du ligand du recepteur hormonal nucleaire
WO2002070560A3 (fr) * 2001-03-05 2002-12-19 Inpharmatica Ltd Domaine de liaison du ligand du recepteur hormonal nucleaire

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