WO1998056806A1 - Proteine de coactivation de facteur de transcription, p/cip - Google Patents

Proteine de coactivation de facteur de transcription, p/cip Download PDF

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Publication number
WO1998056806A1
WO1998056806A1 PCT/US1998/012263 US9812263W WO9856806A1 WO 1998056806 A1 WO1998056806 A1 WO 1998056806A1 US 9812263 W US9812263 W US 9812263W WO 9856806 A1 WO9856806 A1 WO 9856806A1
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Prior art keywords
cip
ncoa
polypeptide
amino acid
substantially purified
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PCT/US1998/012263
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English (en)
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Michael G. Rosenfield
Christopher K. Glass
David W. Rose
Joseph Torchia
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The Regents Of The University Of California
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Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Priority to US09/445,353 priority Critical patent/US6812336B1/en
Priority to AU81421/98A priority patent/AU8142198A/en
Publication of WO1998056806A1 publication Critical patent/WO1998056806A1/fr
Priority to US10/971,982 priority patent/US7211402B2/en

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to molecular biology and biochemistry and more specifically to a coactivator protein, p/CIP, which is involved in regulating gene expression by CBP/p300-dependent transcription factors, and to methods of using the coactivator protein to selectively regulate gene expression.
  • p/CIP coactivator protein
  • Regulation of gene expression is mediated by sequence-specific transcription factors that bind to target genes and activate or repress transcription. Many of these factors are controlled by extracellular signals that switch the factors between inactive and active states. Such signals can result in post-translational modification as observed, for example, with the members of the STAT family of transcription factors, or can result in ligand-induced conformational changes as observed, for example, with members of the nuclear receptor family of transcription factors.
  • Coactivator proteins have been identified that are recruited to the active forms of such transcription factors and are required for their transcriptional effects.
  • the coactivators, CBP and p300 serve essential roles in transcriptional activation by several classes of regulated transcription factors, including nuclear receptors, STAT factors, AP-1 proteins, NF-KB and CREB.
  • CBP and p300 serve essential roles in transcriptional activation by several classes of regulated transcription factors, including nuclear receptors, STAT factors, AP-1 proteins, NF-KB and CREB.
  • NoA nuclear receptor coactivator
  • NCoA-1 and NCoA-2 appear to have relatively selective roles in mediating the transcriptional effects of nuclear receptors.
  • Evidence indicates, however, that additional factors are required for the transcriptional activities of many CBP-dependent transcription factors, including
  • the present invention provides a substantially purified nucleic acid molecule having a nucleotide sequence encoding a transcriptional coactivator protein, designated p/CIP, which binds to CBP/p300-dependent transcription factors and regulates their activity.
  • p/CIP transcriptional coactivator protein
  • the invention provides a substantially purified nucleic acid molecule having the nucleotide sequence shown in Figure 1, which encodes murine p/CIP, and a nucleotide sequence complementary to that shown in Figure 1.
  • the invention also provides a substantially purified nucleic acid molecule encoding an active fragment of a p/CIP polypeptide, which has a nucleotide sequence encoding substantially the same amino acid sequence as a portion of a p/CIP polypeptide.
  • a nucleic acid molecule can encode, for example, an active fragment including a CBP interaction domain, such as a fragment having about amino acids 758 to 1115 of p/CIP shown in Figure 1, or a nuclear receptor interaction domain, such as a fragment having about amino acids 591 to 803 or about amino acids 680 to 740 of p/CIP shown in Figure 1.
  • a substantially purified nucleic acid molecule having a nucleotide sequence encoding a full length mouse NCoA-2 protein, which is related to p/CIP.
  • the invention also provides a substantially purified NCoA-2 active fragment, having a nucleotide sequence encoding substantially the same amino acid sequence as a portion of a NCoA-2 polypeptide.
  • Such a NCoA-2 active fragment can include, for example, a nuclear receptor interaction domain.
  • the invention also provides vectors comprising a nucleic acid molecule of the invention and host cells containing such vectors.
  • the invention provides a substantially purified p/CIP nucleotide sequence having at least about 14 consecutive nucleotides of the nucleotide sequence shown in Figure 1, or a nucleotide sequence complementary thereto.
  • the present invention also provides a substantially purified p/CIP polypeptide, which can bind to a CBP/p300-dependent transcription factor and regulate its activity.
  • the invention provides a substantially purified p/CIP polypeptide having substantially the same amino acid sequence as p/CIP shown Figure 1.
  • the invention additionally provides a substantially purified p/CIP active fragment having substantially the same amino acid sequence as a portion of a p/CIP polypeptide.
  • a particularly useful p/CIP active fragment can include, for example, a CBP interaction domain or a nuclear receptor interaction domain, or can be an portion of a p/CIP polypeptide useful for eliciting production of an antibody that specifically binds to p/CIP.
  • the invention further provides a substantially purified NCoA-2 polypeptide having substantially the same amino acid sequence as amino acid sequence shown in
  • Figure 2a Active fragments of a NCoA-2 polypeptide of the invention also are provided herein.
  • the invention also provides anti-p/CIP antibodies that specifically bind to p/CIP, as well as p/CIP-binding fragments of such antibodies.
  • the invention further provides anti-NCoA-2 antibodies and antigen binding fragments thereof.
  • the invention provides cell lines producing anti-p/CIP antibodies or anti-NCoA-2 antibodies.
  • the present invention further provides methods of identifying an effective agent that alters the association of p/CIP or NCoA-2 polypeptide with a second protein, such as a nuclear receptor or a CBP, which associates with the p/CIP or NCoA-2 polypeptide in vi tro or in vivo .
  • the method includes the steps of contacting a p/CIP or NCoA-2 polypeptide with an agent under conditions that allow the p/CIP or NCoA-2 polypeptide to associate with the second protein, and detecting an altered association of the p/CIP or NCoA-2 polypeptide with the second protein.
  • An agent that alters the association of p/CIP, for example, with a second protein can be a peptide, a polypeptide, a peptidomimetic or an organic molecule, such an effective agent being useful, for example, for modulating the level of transcription in a cell.
  • a peptide portion of p/CIP comprising a helical leucine-rich, charged domain (LCD)
  • LCD helical leucine-rich, charged domain
  • a second LCD of p/CIP can inhibit signal transduction induced by interferon ⁇ , but not signal transduction induced by retinoic acid.
  • selected peptide portions of p/CIP or of NCoA-2 can be valuable for regulating gene expression in a cell, and these and other effective agents can have therapeutic efficacy.
  • Figure 1 shows the nucleotide sequence and deduced amino acid sequence of p/CIP.
  • Figures 2a to 2d provide a characterization of p/CIP and a related member of the NCoA family, NCoA-2.
  • Figure 2a compares the amino acid sequences of the full length murine p/CIP and murine NCoA-2 polypeptides .
  • the conserved bHLH, PAS "A" domain, the nuclear receptor interaction domains and the minimal nuclear receptor and CBP interaction domains are boxed, and repeat motifs involved in critical interactions are bracketed.
  • Figures 2b and 2c provide western blot analyses of total cell extracts for p/CIP, NCoA-1 and NCoA-2 in various tissues and cell lines, showing widespread expression of all three proteins, although relative levels differ.
  • Figure 2d provides schematic diagrams showing regions of homology of p/CIP with NCoA-1 and NCoA-2.
  • the asterisks refer to the repeated peptide motifs that appear to be of functional importance (see Figures 6 and 7) .
  • Figure 3 shows the results of biochemical analysis of p/CIP and NCoA Factors.
  • Figure 3a demonstrates interactions between recombinant GST proteins and NCoA proteins from HeLa whole cell extracts detected using an anti-p/CIP antibody (left) or an anti-NCoA-1 antibody (right) .
  • Figure 3b shows co-immunoprecipitation of CBP/p300 and p/CIP.
  • Anti-p/CIP, anti-NCoA-1 or anti-NCoA-2 IgG was incubated with HeLa whole cell extracts and immunocomplexes were separated by SDS-PAGE and probed using anti-CBP/p300 IgG.
  • Figure 3b shows the detection of CBP/p300 in supernatant following immunodepletion of whole cell extracts with specific anti-NCoA antibodies.
  • Figure 3c shows the results of yeast two-hybrid assays mapping regions of interaction between p/CIP and the CBP C-terminus (aa 2058-2170) or liganded estrogen receptor (LBD) .
  • Figure 3d demonstrates that a common nuclear receptor interaction domain is found in p/CIP, NCoA-1 and NCoA-2 by yeast two-hybrid assay.
  • Ligands (+) were estradiol (IO “6 M) , Triac (IO “6 M) or retinoic acid (IO “6 M) .
  • Figure 3e shows p/CIP, NCoA-1 or NCoA-2 interactions with nuclear receptors in vi tro.
  • Recombinant GST-nuclear receptor proteins were incubated with whole cell extract in the presence (+) or absence (-) of ligand, then western blot analysis was performed using p/CIP-, NCoA-1- or NCoA-2-specific IgG.
  • Figure 3f shows the results of transcription activation studies, in which reporter genes containing the minimal prolactin promoter (P-36 luciferase) , alone, or two copies of the indicated response elements, and plasmids expressing p/CIP, NCoA-1 or NCoA-2 were transfected into HeLa cells in the presence of the corresponding ligand.
  • the effect of varying amounts of plasmid expressing GAL4 (1-147) , GAL4-NCoA-l or GAL4-p/CIP fusion proteins on a minimal (UAS) 6 -dependent reporter are shown in the right panel.
  • Figure 4 demonstrates a role of P/CIP in the function of CBP-dependent transcription factors.
  • Figure 4a shows the effect of microinjection of affinity-purified anti-p/CIP IgG on ligand-dependent gene activation by RAR in Rat-1 cells.
  • Figure 4b shows experiments as in Figure 4a, but performed using minimal promoters with four copies of the estrogen (ERE) , thyroid hormone (TRE) or progesterone (PRE) receptor response elements.
  • ERP estrogen
  • TRE thyroid hormone
  • PRE progesterone
  • Figure 4c demonstrates that both CBP and p/CIP expression vectors are required to rescue anti-p/CIP IgG inhibition of RAR-dependent gene activation.
  • Figure 4d shows the effect of expression of the p/CIP core CBP interaction domain (947-1084) on RAR dependent transcription (left) or on SP-1 or CMV dependent transcription (right) .
  • Figure 4e shows the effect of anti-p/CIP IgG ( ⁇ p/CIP) on an interferon ⁇ dependent promoter (GAS/LacZ) 12 (left) and the effect of p/CIP (aa 947-1084) on interferon ⁇ stimulated transcriptions and failure of CBP expression vector to rescue this inhibition (right) .
  • Figure 4f shows the effect of anti-NCoA-1 IgG ( ⁇ 1) on GAS and cAMP-dependent (2 x CRE) promoters. All were performed at least three separate times, with >200 cells injected; error bars are ⁇ 2 x SEM.
  • Figure 5 demonstrates a role for NCoA-1 and NCoA-2 in nuclear receptor function.
  • Figure 5a demonstrates that microinjection of affinity-purified anti-NCoA-1, but not of anti-NCoA-2, IgG blocked ligand-dependent gene activation by RAR
  • Figure 5b shows experiments as in Figure 5a, except using minimal promoters with two copies of the estrogen (ERE) or T3R (TRE) response elements with less profound effects upon progesterone (PRE) mediated transcription .
  • ERP estrogen
  • TRE T3R
  • Figure 5c demonstrates that anti-NCoA-1 IgG blocked retinoic acid-dependent activation of the RARE/LacZ reporter was not rescued by CMV expression vectors expressing p/CIP or CBP; however, expression was fully rescued by CMV-NCoA-1 and b CMV-NCoA-2.
  • Figure 5d shows photomicrographs of rhodamine- stained injected cells and the corresponding protein of XGal staining.
  • FIG. 6 shows the leucine-rich charged domains (LCD's) in p/CIP/NCoA/CBP.
  • Figure 6a shows that a repeated leucine-rich domain is required for protein-protein interactions between p/CIP, CBP, NCoA proteins and nuclear receptors. The sequence of some of these domains are noted, with the core hexapeptide motifs indicated by brackets. Helical wheels of NCoA-1 LCD2 and CBP LCD6 are shown.
  • Figure 6b shows that mutation of amino acids 70-73 in CBP (QLSELL-QLAAAA) resulted in a complete loss of ligand-dependent interaction with T3R.
  • Figure 6c shows results of the yeast two-hybrid assay of interactions between the NCoA-1 nuclear receptor interaction domains (aa 635-760) with nuclear receptors (left) .
  • Mutations of the LCD2 motif (RLHRLL-RLAAAA) abolished ligand-dependent interaction, while peptides encompassing LCD2 (37 amino acids "aa”) alone or LCD6 (59 amino acids) were sufficient for ligand-dependent interaction (center) .
  • 24-mer peptides encompassing LCD1, LCD2 or a control peptide were tested for ability to inhibit binding of 35 S-labeled NCoA interaction domain fragment (aa 635-760) to liganded RAR with TTNPB (1 ⁇ M) (right)
  • Figures 6d and 6e demonstrate the functional effect of plasmids expressing mutations in LCD2 (HRLL ⁇ AAAA) and LCD3 (RYLL ⁇ AAAA) of NCoA-1 on rescue of inhibition by microinjected anti-NCoA-1 IgG ( -1) on retinoic dependent transcription ( Figure 6d) and on estrogen dependent transcription ( Figure 6e) .
  • Figure 7 demonstrates that distinct helical motifs block transcriptional effects of specific signal transduction pathways.
  • Figure 7a shows that a 19-mer peptide, corresponding to NCoA-1 LCD4, but not a control peptide (CBP-622, control P) , inhibits retinoic acid induced, but not interferon ⁇ induced gene expression.
  • Figure 7b shows the effect of microinjection of the N-terminal 22 amino acids of CBP (CBP N'-Pl), a synthetic N-terminal CBP peptide, on retinoic acid and interferon gene activation events.
  • CBP N'-Pl CBP N'-P2
  • Figure 7c shows, similar to Figure 7b, that interferon ⁇ inhibition of retinoic acid-dependent activation of the RARE/LacZ reporter (right panel) was fully abolished by co-injection of the CBP N'-Pl peptide, which had no effect on retinoic acid dependent inhibition of the GAS/LacZ reporter by activated retinoic acid reporter.
  • Figure 7d provides a model of p/CIP/CBP (p300) function, indicating that several signal transduction pathways mediated by specific transcription factors require a functional p/CIP, CBP/p300 complex, and potentially p/CAF, with each partner required, but not sufficient, to mediate transcriptional effects. Nuclear receptor-specific requirements for distinct protein-protein associations via specific LCD's is suggested.
  • the present invention provides a substantially purified nucleic acid molecule encoding a transcriptional coactivator protein, designated p/CIP (j ⁇ 300/£BP/ co-integrator-associated £rotein) , which binds to CBP/p300-dependent transcription factors and regulates their activity.
  • p/CIP a transcriptional coactivator protein
  • the invention provides a substantially purified nucleic acid molecule having the nucleotide sequence shown in Figure 1, which encodes p/CIP, and a nucleotide sequence complementary to that shown in Figure 1.
  • p/CIP is a member of the NCoA (Nuclear receptor Co-Activator) gene family and is involved in regulating the transcriptional activities of various CBP-dependent transcription factors, including STAT 1, AP-1 and CREB.
  • the invention provides a substantially purified nucleic acid molecule encoding a full length murine NCoA-2 polypeptide having the amino acid sequence shown in Figure 2a.
  • substantially purified when used in reference to a nucleic acid molecule of the invention, means that the nucleic acid molecule is relatively free from contaminating lipids, proteins, nucleic acids or other cellular material normally associated with a nucleic acid molecule in a cell.
  • a substantially purified nucleic acid molecule of the invention can be obtained, for example, by chemical synthesis of the nucleotide sequence shown in Figure 1 or by cloning the molecule using methods such as those disclosed in Example I .
  • CBP is present in a complex with p/CIP, which is required for transcriptional activity of nuclear receptors and other
  • CBP/p300-dependent transcription factors including STAT and AP-1.
  • the related nuclear receptor coactivator protein, NCoA-1 also is specifically required for ligand-dependent gene activation by nuclear receptors.
  • p/CIP, NCoA-1, and CBP contain related leucine-rich charged helical interaction motifs that are required for receptor-specific mechanisms of gene activation. The disclosure of these leucine-rich motifs permits selective inhibition of distinct signal transduction pathways.
  • CBP and p300 are functionally conserved proteins that have intrinsic acetylase activity and serve essential roles in activation by a large number of regulated transcription factors, including nuclear receptors, CREB, AP-1, bHLH and STAT proteins (see, for example, Chakravarti et al . , Nature 383:99-103 (1996); Kwok et al., Nature 370:223-226 (1994); Arias et al . , Nature 370: 226-229 (1994); Eckner et al . , Genes and Devel . 10(19): 2478-2490 (1996), each of which is incorporated herein by reference) .
  • NCoA-1 nuclear receptor coactivator-1
  • p/CIP is a NCoA/SRC family member that forms a complex with CBP in a cell.
  • both p/CIP and NCoA-1 are required for the function of nuclear receptors, while p/CIP, but not NCoA-1, is required for function of other CBP-dependent transcription factors.
  • a series of helical leucine-rich, charged residue-rich domains (LCD's) within these factors serve as interaction motifs that are involved in assembly of a coactivator complex and that contribute to the specificity of nuclear receptor activation events.
  • the invention provides methods of identifying agents that modulate the activity of specific classes of transcription factors.
  • the cloned p/CIP cDNA is a novel member of the NCoA gene family.
  • NCoA-1 SRC-1
  • NCoA-2 TRF2, GRIP2
  • p/CIP interacts with several nuclear receptors in a ligand-dependent manner.
  • Analysis of p/CIP, NCoA-1 and NCoA-2 has led to identification of a series of helical interaction motifs that mediate interactions between NCoA proteins and nuclear receptors, and a separate series of helical motifs that mediate interactions between NCoA proteins and CBP (see, for example, Figure 2a) .
  • Mutations within these motifs reduce the ability of the coactivator to mediate transcriptional activation.
  • microinjection of peptides cornerbanding to specific helical motifs exert selective inhibitory effects on transcription by different classes of transcription factors.
  • effective agents such as peptides have been identified that selectively block STAT 1 activity but not nuclear receptor-dependent transcription.
  • effective agents have been identified that selectively block retinoic acid receptor activity but not STAT 1 activity.
  • the invention also provides vectors comprising a nucleic acid molecule of the invention and host cells containing such vectors.
  • the invention provides nucleotide sequences that bind to a nucleic acid molecule of the invention, such nucleotide sequences being useful, for example, as probes, which can identify the presence of a nucleic acid molecule encoding p/CIP in a sample or as antisense molecules, which can inhibit the expression of a nucleic acid molecule encoding a p/CIP.
  • a substantially purified nucleic acid molecule of the invention is exemplified by the nucleotide sequence shown in Figure 1, which encodes p/CIP protein, also shown in Figure 1. Due to the degeneracy of the genetic code and in view of the disclosed amino acid sequence of a p/CIP protein, additional nucleic acid molecules of the invention would be well known to those skilled in the art. Such nucleic acid molecules have a nucleotide sequence that is different from that shown in Figure 1 but, nevertheless, encode the amino acid sequence shown in Figure 1. Thus, the invention provides a substantially purified nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of murine p/CIP as shown in Figure 1. Similarly, the invention provides a substantially purified nucleic acid molecule encoding a full length NCoA-2 polypeptide having the amino acid sequence shown in Figure 2a.
  • a nucleic acid molecule encoding p/CIP indicates 1) the polynucleotide sequence of one strand of a double stranded DNA molecule comprising the nucleotide sequence that codes, for example, for p/CIP and can be transcribed into an RNA that encodes the coactivator, or 2) an RNA molecule, which can be translated, for example, into p/CIP. It is recognized that a double stranded DNA molecule also comprises a second polynucleotide strand that is complementary to the coding strand and that the disclosure of a polynucleotide sequence comprising a coding sequence necessarily discloses the complementary polynucleotide sequence.
  • polynucleotide sequences including, for example, polydeoxyribonucleotide or polyribonucleotide sequences that are complementary to the nucleotide sequence shown in Figure 1 or to a nucleic acid molecule encoding p/CIP having the amino acid sequence shown in Figure 1.
  • polynucleotide is used in its broadest sense to mean two or more nucleotides or nucleotide analogs linked by a covalent bond.
  • oligonucleotide also is used herein to mean two or more nucleotides or nucleotide analogs linked by a covalent bond, although those in the art will recognize that oligonucleotides generally are less than about fifty nucleotides in length and, therefore, are a subset within the broader meaning of the term "polynucleotide.”
  • nucleotides comprising a polynucleotide are naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to 2 ' -deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose.
  • a polynucleotide also can comprise nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides.
  • nucleotide analogs are well known in the art and commercially available, as are polynucleotides containing such nucleotide analogs (Lin et al., Nucl. Acids Res. 22:5220-5234 (1994); Jellinek et al., Biochemistry 34:11363-11372 (1995); Pagratis et al . , Nature Biotechnol . 15:68-73 (1997)).
  • the covalent bond linking the nucleotides of a polynucleotide generally is a phosphodiester bond.
  • the covalent bond also can be any of numerous other bonds, including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides (see, for example, Tarn et al . , Nucl. Acids Res. 22:977-986 (1994); Ecker and Crooke, BioTechnolo ⁇ y 13:351360 (1995)).
  • bonds including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides (see, for example, Tarn et al . , Nucl. Acids Res. 22:977-986 (1994); Ecker and Crooke, BioTechnolo ⁇ y 13:351360 (1995)).
  • nucleotide of the invention where it is desired to synthesize a polynucleotide of the invention, the artisan will know that the selection of particular nucleotides or nucleotide analogs and the covalent bond used to link the nucleotides will depend, in part, on the purpose for which the polynucleotide is prepared. For example, where a polynucleotide will be exposed to an environment containing substantial nuclease activity, the artisan will select nucleotide analogs or covalent bonds that are relatively resistant to the nucleases.
  • a polynucleotide comprising naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized or can be produced with recombinant DNA methods using an appropriate polynucleotide as a template.
  • a polynucleotide comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally will be chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template (Jellinek et al . , supra, 1995).
  • the invention also provides nucleotide sequences that can bind to a nucleic acid molecule encoding p/CIP. Such nucleotide sequences are useful, for example, as probes, which can hybridize to a nucleic acid molecule encoding a p/CIP and allow the identification of the nucleic acid molecule in a sample.
  • a nucleotide sequence of the invention is characterized, in part, in that it is at least nine nucleotides in length, such sequences being particularly useful as primers for the polymerase chain reaction (PCR) , and can be, for example, at least fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or twenty-one nucleotides in length.
  • a nucleotide sequence of the invention can have at least twenty-five, thirty, thirty-five, forty or fifty nucleotides of the nucleotide sequence shown in Figure 1.
  • Such a nucleotide sequence of the invention is useful as a hybridization probe or as a primer for PCR and can be used, for example, to identify homologous nucleic acid molecules encoding p/CIP proteins in other eukaryotes, particularly other mammals, including humans .
  • p/CIP is a member of the
  • nucleic acid molecule encoding p/CIP shares regions of substantial homology with a nucleic acid molecule encoding an NCoA protein such as NCoA-2.
  • a comparison of the nucleic acid molecules encoding p/CIP and NCoA-2 also will reveal nucleotide sequences that are unique to p/CIP, such nucleotide sequences being encompassed within the invention.
  • a substantially purified nucleotide sequence of the invention can comprise a portion of a coding sequence of a nucleic acid molecule encoding p/CIP or of a sequence complementary thereto, depending on the purpose for which the nucleotide sequence is to be used.
  • a mixture of a coding sequence and its complementary sequence can be prepared and, if desired, can be allowed to anneal to produce double stranded oligonucleotides.
  • the invention also provides antisense nucleic acid molecules, which are complementary to a nucleic acid molecule encoding p/CIP and can bind to and inhibit the expression of the nucleic acid molecule.
  • a nucleic acid molecule of the invention can be introduced into a cell by methods of transfection, or can be contained " in a plasmid or viral vector, which can be introduced into the cell, such that the nucleic acid molecule is stably or transiently expressed (see, for example, Sambrook et al . , Molecular Cloning : A laboratory manual (Cold Spring Harbor Laboratory Press 1989); Ausubel et al . , Current Protocols in Molecular Biolo ⁇ y (Green Publ . , NY 1994), each of which is incorporated herein by reference) . Accordingly, the invention provides vectors comprising a nucleic acid molecule of the invention and host cells, which are appropriate for maintaining such vectors .
  • Vectors which can be cloning vectors or expression vectors, are well known in the art and commercially available.
  • An expression vector comprising a nucleic acid molecule of the invention, which can encode a p/CIP or can be an antisense molecule, can be used to express the nucleic acid molecule in a cell.
  • an expression vector contains the expression elements necessary to achieve, for example, transcription of the nucleic acid molecule, although such elements also can be inherent to the nucleic acid molecule cloned into the vector.
  • an expression vector contains or encodes a promoter sequence, which can provide constitutive or, if desired, inducible expression of a cloned nucleic acid sequence, a poly-A recognition sequence, and a ribosome recognition site, and can contain other regulatory elements such as an enhancer, which can be tissue specific.
  • the vector also contains elements required for replication in a procaryotic or eukaryotic host system or both, as desired.
  • Such vectors which include plasmid vectors and viral vectors such as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, semliki forest virus and adeno-associated virus vectors, are well known and can be purchased from a commercial source (Promega, Madison WI; Stratagene, La Jolla CA; GIBCO/BRL, Gaithersburg MD) or can be constructed by one skilled in the art (see, for example, Meth. Enzymol .. Vol. 185, D.V. Goeddel, ed. (Academic Press, Inc., 1990); Jolly, Cane. Gene Ther. 1:51-64 (1994); Flotte, iL .
  • a nucleic acid molecule including a vector, can be introduced into a cell by any of a variety of methods known in the art (Sambrook et al . , supra, 1989, and in Ausubel et al . , Current Protocols in Molecular Biology. John Wiley and Sons, Baltimore, MD (1994) , which is incorporated herein by reference) . Such methods include, for example, transfection, lipofection, microinjection, electroporation and infection with recombinant vectors or the use of liposomes.
  • Introduction of a nucleic acid molecule by infection with a viral vector is particularly advantageous in that it can efficiently introduce the nucleic acid molecule into a cell ex vivo or in vivo .
  • viruses are very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the nucleic acid molecule contained in the vector to specific cell types. Viral or non-viral vectors also can be modified with specific receptors or ligands to alter target specificity through receptor mediated events.
  • a nucleic acid molecule also can be introduced into a cell using methods that do not require the initial introduction of the nucleic acid molecule into a vector.
  • a nucleic acid molecule encoding a p/CIP can be introduced into a cell using a cationic liposome, which also can be modified with specific receptors or ligands as described above (Morishita et al . , J. Clin. Inves .. 91:2580-2585 (1993), which is incorporated herein by reference; see, also, Nabel et al . , supra,
  • a nucleic acid molecule can be introduced into a cell using, for example, adenovirus-polylysine DNA complexes (see, for example, Michael et al . , J. Biol. Chem.. 268:6866-6869 (1993), which is incorporated herein by reference) .
  • Other methods of introducing a nucleic acid molecule into a cell such that the encoded p/CIP or antisense nucleic acid molecule can be expressed are well known (see, for example, Goeddel, supra , 1990) .
  • Selectable marker genes encoding, for example, a polypeptide conferring neomycin resistance (Neo R ) also are readily available and, when linked to a nucleic acid molecule of the invention or incorporated into a vector containing the nucleic acid molecule, allow for the selection of cells that have incorporated the nucleic acid molecule.
  • Other selectable markers such as that conferring hygromycin, puromycin or ZEOCIN (Invitrogen, Carlsbad CA) resistance are known to those in the art of gene transfer as markers useful for identifying cells containing the nucleic acid molecule, including the selectable marker gene.
  • a "suicide” gene also can be incorporated into a vector so as to allow for selective inducible killing of a cell containing the gene.
  • a gene such as the herpes simplex virus thymidine kinase gene (TK) can be used as a suicide gene to provide for inducible destruction of such cells.
  • TK herpes simplex virus thymidine kinase gene
  • the cells can be exposed to a drug such as acyclovir or gancyclovir, which can be administered to an individual.
  • TILs tumor infiltrating lymphocytes
  • the present invention also provides a substantially purified p/CIP polypeptide, which forms a complex with CBP/p300 in a cell and regulates CBP/p300-dependent transcriptional activity.
  • a p/CIP polypeptide of the invention is exemplified herein by murine p/CIP, which is a protein of about 152 kDa that has a conserved amino-terminal basic helix-loop-helix domain, PAS "A" domain, a serine/ threonine-rich region and a carboxy-terminal glutamine-rich region.
  • Murine p/CIP is related to SRC-l/NCoA-1 and NCoA-2/TIF-2 , showing overall amino acid identity of 31% and 36%, respectively.
  • CBP-dependent transcription factors including nuclear receptors such as the retinoic acid receptor, estrogen receptor, thyroid receptor and progesterone receptor, and other CBP-dependent transcription factors such as STAT 1 (see Example I) .
  • the present invention provides a substantially purified p/CIP polypeptide.
  • a polypeptide can have, for example, substantially the same amino acid sequence as murine p/CIP shown in Figure 1 (see, also, Figure 2a) .
  • a substantially purified p/CIP active fragment having substantially the same amino acid sequence as a portion of a p/CIP polypeptide.
  • Such an active fragment can include, for example, a CBP interaction domain or a nuclear receptor interaction domain.
  • LCD peptide portions of p/CIP also are provided (see Example I) .
  • substantially purified means that the polypeptide or polypeptide fragment is relatively free from contaminating lipids, proteins, nucleic acids or other cellular material normally associated with a polypeptide in a cell .
  • p/CIP or "p/CIP polypeptide” means the polypeptide referred to herein as "p300/CBP/CoIntegrator-associated Protein” and includes the murine p/CIP polypeptide shown in Figure 1.
  • murine p/CIP displays homology to SRC-l/NCoA-1 and NCoA-2/TIF-2 , sharing basic helix-loop-helix domains, a PAS "A" domain, serine/threonine-rich region and glutamine-rich region with NCoA-1 and NCoA-2.
  • p/CIP encompasses murine p/CIP and is intended to include related polypeptides having substantial amino acid sequence similarity to this polypeptide. Such related polypeptides will exhibit greater sequence similarity to p/CIP than to SRC-l/NCoA-1 or to NCoA-2/TIF-2 and include alternatively spliced forms of p/CIP and isotype variants of the amino acid sequence shown in Figure 1.
  • the term p/CIP also encompasses homologous polypeptides obtained from different mammalian species, such as a human homolog of the murine p/CIP polypeptide disclosed in Figure 1.
  • a p/CIP polypeptide generally has an amino acid identity of greater than about 40%, preferably greater than about 50%, more preferably greater than about 60%, and can have an amino acid identity of greater than about 70%, 75%, 80%, 85%, 90% or 95% with the murine p/CIP amino acid sequence disclosed in Figure 1.
  • an active fragment of a p/CIP polypeptide which includes substantially the same amino acid sequence as a portion of a p/CIP polypeptide.
  • active p/CIP fragment means a peptide or polypeptide which has substantially the same amino acid sequence as a portion of a p/CIP polypeptide, provided that the fragment retains at least one biological activity of a p/CIP polypeptide.
  • an active fragment generally has an amino acid sequence of about 15 to about 400 contiguous residues and can have, for example, an amino acid sequence of at least about 18, 20, 25, 30, 35, 40, 50, 100, 150, 200, 250, 300, 350 or 400 contiguous residues.
  • a particularly useful active fragment has from about 80 to about 150 amino acids.
  • a biological activity of a p/CIP polypeptide that is retained by an active p/CIP fragment can be, for example, measurable activity in binding to a CBP protein, binding to a nuclear receptor, activity in retinoic acid, estrogen, thyroid or progesterone dependent transcription, activity in other CBP-dependent transcription, or other inherent biological activity.
  • An isolated p/CIP active fragment of the invention can include, for example, a CBP interaction domain.
  • a CBP-binding active fragment can have, for example, an amino acid sequence that is identical or substantially the same as a portion of p/CIP shown in Figure 1, for example, substantially the same as about amino acids 758 to 1115 of p/CIP, about amino acids 947 to 1084 of p/CIP, or about amino acids 163 to 610 of p/CIP shown in Figure 1.
  • Additional p/CIP active fragments having a CBP interaction domain can readily be identified, for example, using yeast two-hybrid assays or microinjection assays, as set forth in Example I. As disclosed herein, such an active fragment can block CBP-dependent gene activation, for example, interferon- ⁇ or TPA stimulated gene activation or retinoic acid dependent gene activation.
  • a substantially purified p/CIP active fragment of p/CIP can include a nuclear receptor interaction domain.
  • a nuclear receptor-binding active fragment of p/CIP can have, for example, an amino acid sequence that is identical or substantially the same as a portion of p/CIP shown in Figure 1, and can bind a nuclear receptor such as the estrogen receptor, for example, in a ligand-dependent manner.
  • An example of a p/CIP active fragment having a nuclear receptor interaction domain is a fragment having substantially the same amino acid sequence as about amino acids 591 to 803 of p/CIP or about amino acids 680 to 740 of p/CIP shown in Figure 1.
  • NCoA-2 is a novel member of the nuclear receptor co-activator family designated NCoA-2.
  • murine NCoA-2 is a polypeptide of about 160 kDa that interacts with a 100 amino acid region in the carboxy termini of CBP (amino acids 2058-2170) , as well as with the liganded estrogen receptor.
  • the invention provides a substantially purified NCoA-2 polypeptide having substantially the same amino acid sequence as the amino acid sequence shown in Figure 2a.
  • the invention provides a substantially purified NCoA-2 active fragment having substantially the same amino acid sequence as a portion of a NCoA-2 polypeptide.
  • NCoA-2 polypeptide can include, for example, a nuclear receptor interaction domain .
  • NCoA-2 or "NCoA-2 polypeptide” is intended to mean a polypeptide having substantial similarity to the murine NCoA-2 polypeptide shown in Figure 2a.
  • a NCoA-2 polypeptide has a basic helix-loop-helix domain, a PAS "A" domain, a serine/threonine-rich region and a glutamine-rich region.
  • NCoA-2 encompasses murine NCoA-2 and is intended to include related polypeptides having substantial amino acid sequence similarity to this polypeptide. Such related polypeptides will exhibit greater sequence similarity to NCoA-2 than to SRC-l/NCoA-1 or to p/CIP and include alternatively spliced forms of NCoA-2 and isotype variants of the amino acid sequences shown in Figure 2a.
  • NCoA-2 also encompasses homologous polypeptides obtained from different mammalian species, although the human TIF-2 and GRIP-1 polypeptides described in Voegel et al . , EMBO J. 15:3667-3675 (1996) and Hong et al . , Proc. Natl.
  • NCoA-2 polypeptide generally has an amino acid sequence having an amino acid identity of greater than about 70%, preferably greater than about 75%, more preferably greater than about 80%, and can have an amino acid identity of greater than about 85%, 90% or 95% with the murine NCoA-2 amino acid sequence disclosed in Figure 2a.
  • an active fragment of a p/CIP or NCoA-2 polypeptide can be produced by any of several methods well known in the art.
  • an active fragment of the invention can be produced by enzymatic cleavage of a p/CIP polypeptide using a proteolytic enzyme such as trypsin, chymotrypsin or the like, or a combination of such enzymes. The resulting enzymatic digestion subsequently can be purified using well known methods.
  • An active fragment also can be produced using methods of solution or solid phase peptide synthesis or can be expressed from a nucleic acid molecule such as a portion of the coding region of the nucleic acid sequence shown in Figure 1, or can be purchased from a commercial source .
  • the invention also provides an LCD peptide portion of p/CIP, which includes a helical leucine-rich, charged domain (LCD) and which can inhibit the transcriptional activity of one type of nuclear receptor, such as the retinoic acid receptor, but not of a second, related nuclear receptor such as the estrogen receptor.
  • An LCD peptide portion of p/CIP also can selectively inhibit signal transduction induced by interferon ⁇ without inhibiting signal transduction induced by retinoic acid.
  • an LCD peptide portion of p/CIP or of another NCoA can be useful for regulating gene expression in a cell.
  • An LCD peptide portion of p/CIP or NCoA-2 is characterized, in part, as containing one or more copies of the consensus core sequence, LXXLL, where L is leucine and X is independently selected to be any amino acid.
  • an LCD peptide portion contains at least three copies of the consensus core sequence LXXLL.
  • An LCD peptide portion of p/CIP can include, for example, one or more of the following amino acid sequences : KGHKKLLQLLTCS , LLQEKHRILHKLLQN, KKNNALLRYLLDRDD, LRNSLDDLLGPPS or RALLDQLHTFL.
  • An LCD peptide portion of NCoA-2 can include, for example, one or more of the following amino acid sequences : KGQTKLLQLLTTK, SLKEKHKILHRLLQD, KKENALLRYLLDKDD, FGSSPDDLLCPHP or GALLDQLYLAL.
  • An LCD peptide portion of p/CIP or of NCoA-2 can be a helical domain with amphipathic characteristics and can have a length of eight, nine, ten, twelve, fourteen, sixteen, twenty, forty, sixty, eighty or more residues.
  • amino acid includes both amino acids and amino acid equivalents.
  • An amino acid equivalent is a compound which departs from the structure of a naturally occurring amino acid, but which has substantially the structure of an amino acid, such that it can be substituted within a peptide or protein which retains its biological activity.
  • amino acid equivalents can include amino acids having side chain modifications or substitutions, and also can include related organic acids, amides or the like.
  • Amino acid equivalents include amino acid mimetics, which are those structures which exhibit substantially the same spatial arrangement of functional groups as amino acids but do not necessarily have both the -amino and -carboxyl groups characteristic of amino acids .
  • the invention also provides anti-p/CIP antibodies and anti-murine NCoA-2 antibodies, as well as antigen binding fragments of such antibodies.
  • the invention provides cells lines such as isolated cell lines that produce antibodies of the invention, particularly monoclonal antibodies.
  • antibody is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies.
  • antigen means a p/CIP protein, polypeptide .or peptide portion thereof.
  • An anti-p/CIP antibody, or antigen binding fragment of such an antibody is characterized by having specific binding activity for p/CIP or a peptide portion thereof of at least about 1 x 10 s M "1 .
  • An anti-p/CIP antibody can have specific binding activity for p/CIP without binding other NCoA polypeptides such as NCoA-1 or NCoA-2.
  • Fab, F(ab') 2 , Fd and Fv fragments of an anti-p/CIP antibody, which retain specific binding activity for p/CIP, are included within the definition of an antibody. Similar antibodies can be identified with respect to the full length murine NCoA-2 polypeptide disclosed herein.
  • antibody as used herein includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof.
  • non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al . , Science 246:1275-1281 (1989), which is incorporated herein by reference.
  • Anti-p/CIP antibodies can be raised using as an immunogen a substantially purified full length p/CIP " protein, which can be prepared from natural sources or produced recombinantly, or a peptide portion of a p/CIP polypeptide as defined herein, including synthetic peptides as described above.
  • a non-immunogenic peptide portion of p/CIP can be made immunogenic by coupling the hapten to a carrier molecule such bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) , or by expressing the peptide portion as a fusion protein.
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • Various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art and described, for example, by Harlow and Lane, supra,
  • Particularly useful antibodies of the invention are those that bind to uncomplexed p/CIP, but not to a p/CIP in a complex, for example, with CBP, and, conversely, those that bind to the complexed form of p/CIP, but not to the uncomplexed form.
  • An anti-p/CIP antibody is useful, for example, for determining the presence or level of a p/CIP in a tissue sample, which can be a lysate or a histological section. The identification of the presence or level of a p/CIP in the sample can be made using well known immunoassay and immunohistochemical methods (Harlow and Lane, supra, 1988) .
  • An anti-p/CIP antibody also can be used to substantially purify p/CIP from a sample and, in addition, can be used to copurify a protein such as a transcription factor that is complexed with the p/CIP polypeptide.
  • An anti-p/CIP antibody can be used to detect a p/CIP polypeptide in a sample of cells or in an organism.
  • kits incorporating an anti-p/CIP antibody which can be specific for the complexed or uncomplexed form of p/CIP, can be particularly useful.
  • a kit can contain, in addition to an anti-p/CIP antibody, a reaction cocktail that provides the proper conditions for performing the assay, control samples that contain known amounts of p/CIP and, if desired, a second antibody specific for the anti-p/CIP antibody.
  • a protein such as an anti-p/CIP antibody, as well as p/CIP or a peptide portion thereof, can be labeled so as to be detectable using methods well known in the art (Hermanson, "Bioco jugate Techniques” (Academic Press 1996) , which is incorporated herein by reference; Harlow and Lane, supra, 1988; chap. 9).
  • a protein can be labeled with various detectable moieties including a radiolabel, an enzyme, biotin, or a fluorochrome or fluorescent moiety, such as a green fluorescent protein (see U.S. Patent No.
  • Reagents for labeling a protein such as an anti-p/CIP antibody can be included in a kit containing the protein or can be purchased separately from a commercial source.
  • a labeled antibody Following contact, for example, of a labeled antibody with a sample such as a tissue homogenate or a histological section of a tissue, specifically bound labeled antibody can be identified by detecting the particular moiety.
  • a labeled second antibody can be used to identify specific binding of an unlabeled anti-p/CIP antibody.
  • a second antibody generally will be specific for the particular class of the first antibody. Such second antibodies are readily available from commercial sources.
  • the second antibody can be labeled using a detectable moiety as described above.
  • a sample is labeled using a second antibody
  • the sample is first contacted with a first antibody, which is an anti-p/CIP antibody, then the sample is contacted with the labeled second antibody, which specifically binds to the anti-p/CIP antibody and results in a labeled sample.
  • polyclonal antibodies for example, in a rabbit, goat, mouse or other mammal
  • monoclonal antibodies can be obtained using methods that are well known and routine to the skilled person (Harlow and Lane, supra, 1988) .
  • spleen cells from a p/CIP-immunized mouse can be fused to an appropriate myeloma cell line such as SP/02 myeloma cells to produce hybridoma cells.
  • Cloned hybridoma cell lines can be screened using labeled p/CIP protein to identify clones that secrete anti-p/CIP monoclonal antibodies.
  • Hybridomas expressing anti-p/CIP monoclonal antibodies having a desirable specificity and affinity can be isolated and utilized as a continuous source of the antibodies, which are useful, for example, for preparing standardized kits as described above.
  • a recombinant phage that expresses, for example, a single chain anti-p/CIP antibody also provides a monoclonal antibody that can used for preparing standardized kits.
  • a monoclonal anti-p/CIP antibody can be used to prepare anti-idiotypic antibodies, which presents an epitope that mimics the epitope recognized by the monoclonal antibody used to prepare the anti-idiotypic antibodies.
  • the epitope recognized by the monoclonal antibody includes, for example, an LCD
  • the anti-idiotypic antibody can act as an inhibitor of p/CIP binding to CBP or p/CIP binding to a transcription factor, thus providing a means to regulate a signal transduction pathway.
  • the present invention also provides a method of identifying an effective agent that alters the association of a p/CIP polypeptide with a second protein such as CBP or a transcription factor, or that alters the formation of a complex containing two or three of these proteins.
  • the method includes the steps of contacting a p/CIP polypeptide and a second protein with an agent under conditions that allow the p/CIP polypeptide to associate with the second protein and detecting an altered association of the p/CIP polypeptide and said second protein.
  • the altered association indicates that the agent is an effective agent.
  • the p/CIP polypeptide can have, for example, the amino acid sequence shown in Figure 1, and the second protein can be, for example, a CBP protein, a nuclear receptor or a CBP/p300 -dependent transcription factor.
  • An altered association can be detected, for example, by measuring the transcriptional activity of a reporter gene.
  • a p/CIP polypeptide can be contacted with an agent in vi tro or in a cell, including a prokaryotic cell such as a yeast cell and a eukaryotic cell, such as a mammalian cell, for example, a human cell.
  • the present invention further provides a method of identifying an effective agent that alters the association of a NCoA-2 polypeptide with a second protein.
  • the method includes the steps of contacting a NCoA-2 polypeptide and a second protein with an agent under conditions that allow the NCoA-2 polypeptide to associate with the second protein and detecting an altered association of the NCoA-2 polypeptide and said second protein.
  • second protein refers to a protein that specifically associates with a p/CIP or NCoA-2 polypeptide. It is recognized, however, that p/CIP and NCoA-2 can associate with more than one additional protein at the same time to form a complex.
  • a second protein is exemplified herein by CBP proteins, by nuclear receptors and by other CBP/p300-dependent transcription factors, which form a complex with p/CIP or NCoA-2.
  • Effective agents that alter the association, for example, of p/CIP, CBP and a transcription factor can be extremely valuable in that the agent can modulate transcriptional activity of the transcription factor.
  • agent means a biological or chemical compound such as a simple or complex organic molecule, a peptide, a peptidomimetic, a polypeptide, a nucleic acid, a chemical or a small molecule .
  • the screening assays described herein are particularly useful in that they can be automated, facilitating high through-put screening of randomly or rationally designed agents or libraries of agents, such as chemicals, small molecules, drugs, peptides, peptidomimetics or polypeptides, in order to identify those agents that alter the association of p/CIP or NCoA-2 with a second protein. If desired, an agent can be screened individually, or can be screened in combination with other agents, for example, in a library.
  • the term “associate” or “association, " when used in reference to a p/CIP or NCoA-2 polypeptide and a second (or second and third) protein means that the p/CIP or NCoA-2 polypeptide and the second protein have a binding affinity for each other such that they form a bound complex in vivo or in vi tro, including in a cell in culture or in a reaction comprising substantially purified reagents.
  • bind or “interact” is used interchangeably with the term "associate.
  • the screening assays disclosed herein provide a method of identifying an "effective agent," which is an agent that can increase or decreased the affinity of an association between a p/CIP or NCoA-2 polypeptide and a second protein and that has presumptive therapeutic activity.
  • an "effective agent” is an agent that can increase or decreased the affinity of an association between a p/CIP or NCoA-2 polypeptide and a second protein and that has presumptive therapeutic activity.
  • modulate or “alter, " as used herein in reference to the association of a p/CIP or NCoA-2 polypeptide and one or two other proteins, means that the affinity of the association is increased or decreased.
  • Effective agents that can alter such association and, therefore, complex formation of p/CIP, CBP and a transcription factor can be useful for modulating a signal transduction pathway and, therefore, expression of genes in the pathway.
  • an effective agent that alters the association of p/CIP with a second protein such as a CBP protein may, additionally alter the association of other proteins with p/CIP.
  • an effective agent can selectively alter the association of, for example, p/CIP with a CBP protein without altering the association of p/CIP with other proteins .
  • an effective agent can function directly or indirectly and by a variety of mechanisms to alter the association of a p/CIP polypeptide, or NCoA-2 polypeptide, with a second protein.
  • An effective agent can function, for example, as a competitor of the binding interaction between a p/CIP polypeptide and a second protein, or between a
  • NCoA-2 polypeptide and a second protein can be an effective agent that decreases the affinity of the association of a p/CIP polypeptide with a second protein, as can be a fragment of a CBP protein or nuclear receptor that alters the association of p/CIP with a second protein.
  • a peptide portion of p/CIP comprising an LCD, for example, amino acids 947 to 1084 of p/CIP (see Figure 2A) is an example of such an agent, since the peptide inhibits, for example, retinoic acid-dependent gene activation (see Example I) .
  • Additional peptide effective agents which can be peptides as small as about five amino acids, can be identified, for example, by screening a peptide library (see, for example, Ladner et al., U.S. Patent No: 5,223,409, which is incorporated herein by reference) using one of the assays described herein.
  • An effective agent also can bind to a p/CIP or NCoA-2 polypeptide at a site distant from the site of interaction, thereby altering the three-dimensional conformation of the polypeptide such that the affinity of the association with a second protein is increased or decreased.
  • An effective agent also can produce an altered association by promoting a modification such as phosphorylation of a p/CIP or NCoA-2 polypeptide.
  • an effective agent can sequester or alter the subcellular localization of a p/CIP or NCoA-2 polypeptide, thereby modulating the effective concentration of the polypeptide and the extent to which the polypeptide can associate with a second protein.
  • a variety of in vivo and in vi tro screening assays for detecting an altered association are well known in the art including, for example, the two hybrid assay, coimmunoprecipitation assays, reporter assays and other well known methods such as equilibrium dialysis.
  • methods for distinguishing the specific association of a p/CIP, for example, and a second protein from a non-specific interaction are routine and, generally, include performing the appropriate control experiments to demonstrate the absence of non-specific protein binding.
  • An effective agent can be identified by an altered level of reporter gene transcription as compared to a control level of transcription in the absence of the agent.
  • a particularly useful reporter gene is cytosolic ⁇ -lactamase, which can be detected by the CCF2/AM substrate, as described in Tsien et al . (U.S. patent No. 5,741,657, which is incorporated herein by reference).
  • a reporter gene can encode a protein expressed, for example, on the cell surface, and an altered level of reporter gene transcription detected by FACS analysis.
  • a two-hybrid system such as the yeast two hybrid system, can be particularly useful for screening a panel of agents in order to detect an altered association of a p/CIP or NCoA-2 polypeptide with a second protein (see Example I) .
  • an effective agent is identified by an altered level of transcription of a reporter gene (see Fields and Song, Nature 340:245-246 (1989), which is incorporated herein by reference) .
  • the level of transcription of a reporter gene due to the bridging of a DNA-binding domain p/CIP or NCoA-2 polypeptide hybrid and a transactivation domain-second protein hybrid can be determined in the absence and presence of an agent .
  • an agent may not be able to cross the yeast cell wall and, therefore, cannot enter a yeast cell to alter the association of a p/CIP or NCoA-2 polypeptide with the second protein.
  • yeast spheroplasts which are yeast cells that lack a cell wall, can circumvent this problem (Ausubel et al . , supra, 1994) , which is incorporated herein by reference) .
  • an agent upon entering a cell, may require "activation" by a cellular mechanism, which may not be present in yeast. Activation of an agent can include, for example, metabolic processing of the agent or a modification such as phosphorylation of the agent, which can be necessary to convert the agent into an effective agent.
  • a mammalian cell line can be used to screen a panel of agents.
  • a transcription assay such as the yeast two hybrid system described in Example I can be adapted for use in mammalian cells using well known methods (see, for example, Fearon et al . , Proc. Natl. Acad. Sci., USA 89:7958-7962 (1992), which is incorporated herein by reference; see, also, Sambrook et al., supra, 1989; Ausubel et al . , supra, 1994).
  • vi tro screening assays can utilize, for example, p/CIP or a p/CIP fusion polypeptide such as a histidine-p/CIP fusion protein.
  • the p/CIP or p/CIP fusion polypeptide should have an affinity for a solid substrate as well as the ability to associate with a second protein.
  • Convenient solid substrates include columns, beads, filters and other materials well known in the art. If desired, the solid substrate can contain a covalently attached anti-p/CIP antibody.
  • a fusion polypeptide such as a His-p/CIP fusion polypeptide
  • a nickel chelate substrate which is bound by the histidine component of the fusion protein, can be used (Invitrogen, Carlsbad, CA) .
  • GST glutathione-S-transferase
  • GST glutathione-S-transferase
  • a glutathione affinity resin Stratagene, La Jolla, CA
  • DAKO Anti-GST antibody
  • FLAG fusion polypeptides
  • AU fusion polypeptides
  • Myc tag fusion polypeptides which can be immobilized on a substrate using anti-Myc antibody, commercially available from Invitrogen.
  • the second protein can be immobilized on a solid substrate using a fusion protein strategy or antibody, as described above.
  • An in vi tro screening assay can be performed by allowing a p/CIP or NCoA-2 polypeptide or p/CIP or NCoA-2 fusion polypeptide, for example, to bind to a solid substrate, then adding the second protein, and an agent to be tested. Control reactions, which do not contain an agent, can be performed in parallel. Incubation is performed under suitable conditions, which include, for example, an appropriate buffer concentration, pH, incubation time and temperature.
  • association of the p/CIP or NCoA-2 polypeptide and the second protein in the absence and presence of an agent can be detected, for example, by attaching a detectable moiety such as a radionuclide, fluorescent or antigenic label to the p/CIP or NCoA-2 polypeptide, and measuring the amount of label that is associated with the solid support .
  • a detectable moiety such as a radionuclide, fluorescent or antigenic label
  • agents to be screened according to a method of the invention can include a variety of biological or chemical compounds such as organic molecules, peptides and peptidomimetics, polypeptides or nucleic acids.
  • agents to be screened include fragments of p/CIP or NCoA-2 polypeptides and fragments of CBP proteins or nuclear receptors .
  • Such fragments can be produced by chemical or proteolytic cleavage of the isolated polypeptide. Methods for chemical and proteolytic cleavage and for purification of the resultant polypeptide fragments are well known in the art as described above. (See, for example, Lieber, Methods in Enzymology. Vol. 182, "Guide to Protein Purification," San Diego: Academic Press, Inc.
  • Polypeptide libraries include, for example, tagged chemical libraries comprising peptides and peptidomimetic molecules.
  • Polypeptide libraries also comprise those generated by phage display technology. Phage display technology includes the expression of polypeptide molecules on the surface of phage as well as other methodologies by which a protein ligand is or can be associated with the nucleic acid which encodes it.
  • phage display libraries including vectors and methods of diversifying the population of peptides which are expressed, are well known in the art (see, for example, Smith and Scott, Methods Enzymol . 217:228-257 (1993); Scott and Smith, Science 249:386-390 (1990); and Huse, WO 91/07141 and WO 91/07149, each of which is incorporated herein by reference) .
  • These or other well known methods can be used to produce a phage display library which can be screened, for example, with one of the disclosed assays to identify an effective agent that alters the association of a p/CIP or NCoA-2 polypeptide with a second protein.
  • a peptide portion of p/CIP comprising a helical leucine-rich, charged domain (LCD) , can inhibit the transcriptional activity of one type of nuclear receptor, such as the retinoic acid receptor, but not of a second, related nuclear receptor such as the estrogen receptor, whereas a second LCD of p/CIP can inhibit signal transduction induced by interferon y, but not signal transduction induced by retinoic acid.
  • selected peptide portion of p/CIP or of an NCoA can be useful for selecting regulating gene expression in a cell . Because ⁇ ) t to H H H
  • polypeptide to associate with the nuclear receptor and detecting an altered association between the NCoA polypeptide, or nuclear receptor binding fragment thereof, and the nuclear receptor, where an increased association indicates that the agent is an agonistic ligand of the nuclear receptor and a decreased association indicates that the agent is an antagonistic ligand of the nuclear receptor.
  • an increased association indicates that the agent is an agonistic ligand of the nuclear receptor and a decreased association indicates that the agent is an antagonistic ligand of the nuclear receptor.
  • NCoA polypeptide means a nuclear receptor coactivator protein that is characterized, in part, as containing one or more "LXXLL” motifs and by its ability to mediate ligand-dependent nuclear receptor activation.
  • a NCoA polypeptide can be, for example, a SRC-l/NCoA-1, p/CIP or NCoA-2 polypeptide, or a polypeptide having substantial similarity to one of these polypeptides.
  • a NCoA polypeptide can have, for example, at least about 30% amino acid identity with a SRC-l/NCoA-1, p/CIP or NCoA-2 polypeptide and, further, can have at least about 40%, 50%, 60%, 70%, 80% or 90% amino acid identity with a SRC-l/NCoA-1, p/CIP or NCoA-2 polypeptide.
  • the agent to be tested for agonist or antagonist activity can be provided in purified form, or in impure form as a pool of different agents.
  • an agent can be a biological or chemical compound such as a simple or complex organic molecule, a peptide, a peptidomimetic, a polypeptide or a nucleic acid.
  • the nuclear receptor can be, for example, a steroid hormone receptor, retinoid receptor or fatty acid metabolite receptor. Retinoic acid receptors, estrogen receptors, progesterone receptor and thyroid receptors are examples of nuclear receptors useful in the claimed methods.
  • the NCoA polypeptide can be, for example, a p/CIP, NCoA-1 or NCoA-2 polypeptide, or an active fragment of a NCoA polypeptide with nuclear receptor binding activity. Active p/CIP and NCoA-2 polypeptides including a nuclear receptor interaction domain have been described hereinabove. Such a fragment can be, for example, a LCD peptide that contains one or more LXXLL motifs. In the presence of an agent that is an agonist, the nuclear receptor can undergo a conformational change, whereby there is an increased association of the nuclear receptor with the NCoA polypeptide or nuclear receptor interaction domain thereof, or with another coactivator containing one or more LXXLL motifs.
  • the nuclear receptor can be immobilized on a solid substrate, for example, by expressing the nuclear receptor as a GST fusion protein and capturing the fusion protein on a glutathione affinity matrix.
  • the nuclear receptor fusion protein can be incubated, for example, with labeled NCoA polypeptide such as labeled p/CIP polypeptide in the presence of an agent to be tested. Following incubation and subsequent washing of the glutathione affinity matrix, specifically bound NCoA polypeptide can be detected quantitatively, semi-quantitatively, or qualitatively.
  • a ligand that is an agonist the association of the p/CIP or other NCoA polypeptide with the GST matrix is increased.
  • the association of the NCoA polypeptide with the GST matrix is decreased.
  • a NCoA polypeptide such as p/CIP can be labeled with a variety of labels including fluorescent labels or radiolabels such as 35 S-labeled amino acids, which can be incorporated by in vi tro translation using a rabbit reticulocyte translation system.
  • a LCD peptide containing at least one LXXLL motif also can be used, if desired, in place of or in addition to, the NCoA polypeptide.
  • Such a LCD peptide can be modified to contain N- or C-terminal tyrosine residues that do not substantially influence interaction with the nuclear receptor but which can be conveniently labeled, for example, using radioiodination.
  • a short sequence tag suitable for phosphorylation of an LCD peptide with 32 P-ATP also can be used as a label.
  • Fluorescent detection for example using green fluorescent protein, can be particularly useful in the methods of the invention.
  • Useful fluorescent detection methods include florescence polarization as well as fluorescence resonance energy transfer (FRET) -based assays.
  • FRET-based assays are particularly advantageous for high throughput screening approaches since such assays are homogeneous and do not require a washing step and, in addition, can be useful for detecting nuclear receptor interactions within a cell.
  • the ligand to be identified can be an antagonist.
  • the present invention provides, for example, a method of identifying an antagonist of a nuclear receptor. The method includes the steps of contacting a NCoA polypeptide or nuclear receptor interaction domain thereof and a nuclear receptor with an agonist of the nuclear receptor and an agent under conditions that allow the NCoA polypeptide or nuclear receptor interaction domain thereof to associate with said nuclear receptor; and detecting an altered association of the NCoA polypeptide or nuclear receptor interaction domain thereof and the nuclear receptor, where a decreased association indicates that the agent is an antagonist of the nuclear receptor.
  • An agent that is an antagonist can compete with the agonist for binding to the nuclear receptor without inducing the conformation change required for interaction of the NCoA polypeptide, or nuclear receptor interaction domain thereof, and the nuclear receptor.
  • a LCD peptide containing one or more LXXLL motifs can be substituted for a NCoA polypeptide in the methods of the invention.
  • the invention provides a method of identifying a ligand with mixed agonist and antagonist properties with respect to a particular nuclear receptor.
  • a method includes the steps of: contacting a first NCoA polypeptide, or nuclear receptor interaction domain thereof, and a nuclear receptor with an agent to form a first complex; detecting an altered association of the first complex in the presence and absence of the agent; contacting a second NCoA polypeptide, or nuclear receptor interaction domain thereof, and a nuclear receptor with the agent to form a second complex; and detecting an altered association of the second complex in the presence and absence of the agent, wherein an increased association of the first complex combined with a decreased association of the second complex or a decreased association of the first complex combined with an increased association of the second complex indicates that the agent is a ligand of the nuclear receptor having mixed agonist and antagonist activity.
  • a ligand identified by this method can be particularly useful since it can exhibit different effects on nuclear receptor function in different cell types and can be useful for differentially modulating different classes of transcription factors.
  • This example provides methods for isolating and characterizing the nucleic acid molecule encoding p/CIP, which regulates the activity of CBP/p300-dependent transcription factors. Additional details related to these methods are provided in Torchia et al . , June 1997, at http://www.Nature.com, which is incorporated herein by reference.
  • Expression cloning was performed, using a 32 P-labeled GST-CBP (2058-2170) or 3 P-labeled GST-ER ligand binding domain probe in the presence of IO "6 M estradiol (Kamei et al . , Cell 85:1-12 (1996), which is incorporated herein by reference).
  • cDNA's corresponding to p/CIP, NCoA-1 and NCoA-2 were assembled into PCMX and tested by in vi tro translation, generating products which all migrated at approximately 160 kDa.
  • Databank accession numbers for p/CIP and NCoA-2 sequences are AF000581 (p/CIP) and AF000582 (NCoA-2) , each of which is incorporated herein by reference.
  • the yeast strain EGY 48, the LexA- ⁇ -galactosidase reporter construct (PSH 18-34) and the B42 parental vectors (pEG 202 and pJG 4-5) were all previously described (Gyuris et al . , Cell 75:791-803 (1993) , which is incorporated herein by reference; Kamei et al., supra, 1996). Nuclear receptor ligand binding domains and various CBP fragments were subcloned into PEG 202 bait vector. DNA fragments encompassing the entire p/CIP-NCoA-1 or NCoA-2 proteins were generated either by using an appropriate restriction digest or by PCR and subcloned into pJG 4-5 prey vectors.
  • EGY 48 cells were transformed with the lac Z reporter plasmid pSH 18-34 with the appropriate bait and prey vectors and plated out on -Ura-His-Trp medium containing 2% galactose. Isolated yeast colonies were allowed to grow in the same liquid medium, followed by assaying for ⁇ galactosidase, as previously described (Ausubel et al . , supra, 1994).
  • Transfection experiments were conducted in either HELA or CV-1 cells using the standard calcium phosphate procedure. Typically, 1 ug of a RARE- or ERE-driven luciferase reporter were cotransfected with 1 ug of the indicated vectors. The final DNA concentration was adjusted to 10 ug/60 mm dish, incubated for 24 hr, then the appropriate ligands were administered for 24 hr at a concentration of IO "6 M. Alternatively, cotransfection experiments were conducted using a PCMX p/CIP, NCoA-1 or PCR-generated NCoA-1 fragments fused to the GAL 4 DNA binding domain (aa 1-147) . Cells were transfected with 1 ug of a (UAS) 6 -luciferase reporter and the indicated concentrations of GAL4 fusion proteins, then harvested 48 hrs later.
  • UAS UAS 6 -luciferase reporter
  • cDNA fragments corresponding to p/CIP (544-851), NCoA-1 (424-789) or NCoA-2 (787-1129) were subcloned into the pM vector containing an in-frame His tag and recombinant His-tagged proteins were generated and purified by nickel chelate chromatography. The purified recombinant proteins were injected into rabbits and antibodies were generated and affinity purified using standard procedures (Harlow and Lane, supra, 1988.
  • NCoA-1 LCD1 (aa 631-647) ; NCoA-1 LCD2 (687-706) ; NCoA-1 LCD4 (aa 907-926); CBP N'-Pl (aa 1-19); and CBP N--P2 (aa 8-19) .
  • GST-RAR (143-462), GST-ER (251-595) and GST-CBP (2058-2170) were generated as described (Kamei et al., supra, 1996). 25 ul of GST SEPHAROSE beads containing 10 ug of the GST recombinant proteins were incubated in the presence or absence of the appropriate ligand for 30 min at room temperature, followed by the addition of 1 mg of cell extract and incubated for an additional 1 hr at 4°C. The complexes were then centrifuged, washed three times in NET-N buffer, separated by SDS-PAGE and western blotted with the appropriate antibodies (1 ug/ml) .
  • NCoA-1 and CBP were introduced by site-directed mutagenesis using the quick change mutagenesis kit according to the manufacturers instructions (Stratagene; La Jolla CA) .
  • Double stranded oligonucleotides were designed such that the wild type sequence corresponding to amino acids 695 to 698 and amino acids 756 to 759 in pCMX NCoA-1 and pJG4-5-4
  • NCoA-l(aa 635-760) were substituted with alanines .
  • a similar protocol was used to replace amino acids 70 to 73 in PJG4-5 CBP(aa 1-101).
  • Rat-1 fibroblasts were seeded on acid washed glass coverslips at subconfluent density and grown in MNE/F12 medium supplemented with 10% fetal bovine serum, gentamicin and methotrexate . Prior to the injection, the cells were rendered quiescent by incubation in serum-free medium for 24-36 hr.
  • Plasmids were injected into the nuclei of cells at a final concentration of 100 mg/ml. Peptides were injected at a concentration of 200 mM. Either preimmune IgG of the appropriate species or antibodies directed against p/CIP, NCoA-1 or NCoA-2 were co-injected and allowed the unambiguous identification of the injected cells.
  • Microinjections were carried out using an Eppendorf semiautomated microinjection system mounted on an inverted Zeiss microscope. Approximately 1 hr after injection, the cells were stimulated, where indicated, with the appropriate ligand. In the case of rescue experiments, the cells were stimulated with ligand 6 hr after injection, to allow protein expression. After overnight incubation, the cells were fixed, then stained to detect injected IgG and ⁇ -galactosidase expression (Rose et al . , J. Cell. Biol. 119:1405-1411 (1992), which is incorporated herein by reference; Kamei et al . , supra,
  • Injected cells were identified by staining with tetramethylrhodamine-conjugated donkey anti-rabbit IgG.
  • the initial expression screening strategy for identifying members of the pl60 gene family was based on the observation that the biochemically-identified pl60 proteins interacted with a 100 amino acid region in the C-termini of CBP (aa 2058-2170) , as well as the liganded estrogen receptor (ER; Ogryzko et al . , Cell 87:953-960 (1996) , which is incorporated herein by reference) .
  • This strategy allowed isolation of the previously reported NCoA-l/SRC-1 protein and of a second related factor, NCoA-2 ( Figure 2a) , which has a molecular mass of
  • p/CIP is a 152 kDa protein that is highly related to SRC-l/NCoA-1 and NCoA-2/TIF-2 , showing an overall amino acid identity of 31% and 36%, respectively ( Figure 2a) .
  • p/CIP has a conserved N-terminal bHLH, PAS "A" domain (50-60% amino acid identity) , a serine/ threonine rich region, and a C-terminal glutamine-rich region, each of which also is present in NCoA-1 and NCoA-2.
  • Western blot analysis indicates that p/CIP, NCoA-1 and NCoA-2 are widely expressed in adult tissues and in all cell lines evaluated ( Figures 2b and 2c) .
  • GST-CBP (2058-2170) was used to affinity purify interacting proteins from HeLa cell extracts.
  • p/CIP was consistently observed by immunoblotting using affinity purified anti-p/CIP IgG, whereas much smaller amounts of NCoA-1 were detected following immunoblotting with anti NCoA-1 IgG ( Figure 3a) .
  • CBP interaction domain in p/CIP, deletion mutants were generated and tested against CBP (2058-2170) using a yeast two-hybrid assay.
  • the major CBP interaction domain was located between amino acids 758 to 1115 of p/CIP, with an internal 200 amino acid domain still capable of interacting.
  • a less pronounced interaction was observed with the N-terminal region containing the PAS "A" domain ( Figure 3c) .
  • a single nuclear receptor interaction domain (aa 591-803) was localized N-terminal of the CBP/p300 interaction domain ( Figure 3c) .
  • Further mapping delineated a minimal nuclear receptor interaction region encompassing amino acids 680-740 in p/CIP that was sufficient for binding to the liganded nuclear receptors.
  • NCoA-1 is selectively required as a coactivator for the ligand-activated nuclear receptor gene expression events; the requirement for the CBP/p300/p/CIP complex reflects a more general obligatory role in gene activation events.
  • NCoA-1 helical motif sequences in the nuclear receptor interaction domain of NCoA-1
  • mutations in helical domains 2 or 3 were generated in the context of the holoprotein and tested for the ability to rescue anti-NCoA-1 IgG inhibition of retinoic acid receptor function.
  • an NCoA-1 holoprotein harboring clustered point mutations in helical domain 3 (LCD3-mut) was completely ineffective at rescuing retinoic acid receptor function.
  • NCoA-1 containing a helical domain 2 (LCD2-mut) mutation retained some residual efficacy (Figure 6d) , consistent with the residual ability of the helical domains to mediate nuclear receptor interactions.
  • NCoA-1 harboring the helical domain 3 mutation retained full functional ability in estrogen receptor-dependent gene activation, whereas LCD2-mut was completely ineffective at rescuing estrogen receptor function (Figure 6e) .
  • NCoA-1 harbors two additional related helical interaction motifs, and a peptide encompassing one of these motifs (LCD4) can block nuclear receptor transcription factor function and does not impair STAT function ( Figure 7a) . Furthermore, a mutation within this motif markedly impairs the function of this region of p/CIP (data not shown) . Thus, specific signal transduction pathways can be selectively blocked by distinct helical interaction motifs .
  • CBP N'Pl a synthetic peptide against the N-terminal 22 amino acids of CBP
  • MAENLLY The identical peptide, from which the N-terminal seven amino acids (MAENLLY) were deleted, abolished this effect (CBP N'-P2; Figure 7b) , indicating that this sequence encompassed a motif required for STAT interaction and function.
  • CBP N'-Pl CBP N-terminal 22 amino acid peptide
  • results disclosed herein indicate that p/CIP, which is associated with CBP/p300 in cell, is involved in regulating transcription by nuclear receptors and by other CBP-dependent factors, including STAT and AP-1. Furthermore, both the CBP/p/CIP complex and NCoA-1 are required to permit full ligand-activated gene transcription in the cells examined, while NCoA-l/SRC-1 is not required for other CBP-dependent transcription. Because CBP is capable of associating with a large number of additional factors, including myb, YY1, SREBP, myoD and the HLHl factors, it is likely that p/CIP and CBP are components of a larger complex inportant for integration of many signal transduction pathways.
  • CBP holoprotein which may be contributed by p/CIP
  • p/CAF is capable of interacting with NCoA-1 as well as CBP41, although its role in mediating the transcriptional activation by nuclear receptors is unclear.
  • the nuclear receptor and CBP interaction domains within NCoA-1, NCoA-2, and p/CIP contain putative helical domains, referred to as LCD's, that are required and, in at least in some cases, sufficient, for receptor-specific interactions.
  • the third helical domain in the nuclear receptor interaction domain of NCoA-1 is differentially utilized, being important for retinoic acid function, but not for estrogen receptor- dependent gene activation events. Similar LCD's are present in CBP and in other factors, including TIF-1 and RIP 140, as well as in the N-terminal interaction domain of p/CAF. Thus, many factors can have the ability to associate with the complexes formed on receptor homodimers or heterodimers bound to their cognate DNA site and contribute to the specificity of nuclear receptor pathways. Such an assembly of specific complexes of proteins based on these interaction motifs can provide a basis for receptor-specific and regulated aspects of nuclear receptor function.
  • helical interaction domains in CBP/p/CIP/NCoA proteins and other nuclear receptor interacting factors permitted the use of such domains to selectively block gene activation events in response to specific signal transduction pathways.
  • peptides corresponding to CBP interaction domains selectively block nuclear receptor or STAT-1 function.
  • the actions of specific inhibitory peptides indicates that partitioning of CBP accounts, at least in part, for trans-repression of nuclear receptor, STAT and AP-1 pathways.

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Abstract

La présente invention concerne une molécule d'acides nucléiques sensiblement purifiée codant pour un polypeptide de p/CIP, qui régule l'activité de facteurs de transcription dépendant de CBP/p300. L'invention concerne également un polypeptide de p/CIP sensiblement purifié et des fragments actifs de celui-ci. De plus, l'invention concerne des procédés permettant d'identifier un agent efficace qui modifie l'association d'un polypeptide de p/CIP avec une deuxième protéine. L'invention concerne en outre des procédés permettant d'inhiber de façon sélective des voies de transduction de signal au moyen d'un fragment actif d'un polypeptide de p/CIP ou d'une molécule d'acides nucléiques codant pour un tel fragment actif.
PCT/US1998/012263 1997-06-12 1998-06-12 Proteine de coactivation de facteur de transcription, p/cip WO1998056806A1 (fr)

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US7297777B2 (en) 2000-02-22 2007-11-20 Laboratoires Serono Sa Process of purification of hCG and recombinant hCG purified by that method
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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1021462A1 (fr) * 1997-10-07 2000-07-26 Merck & Co., Inc. Essais pour ligands a recepteurs nucleaires reposant sur le transfert d'energie de resonance en fluorescence
EP1021462A4 (fr) * 1997-10-07 2005-04-13 Merck & Co Inc Essais pour ligands a recepteurs nucleaires reposant sur le transfert d'energie de resonance en fluorescence
US7132258B1 (en) 1998-02-04 2006-11-07 University Of Massachusetts Nucleic acid encoding vitamin D receptor related polypeptide
US6599741B1 (en) 1999-09-14 2003-07-29 Avontec Gmbh Modulating transcription of genes in vascular cells
DE29916160U1 (de) 1999-09-14 2000-03-09 Cardiogene Gentherapeutische Systeme AG, 40699 Erkrath Modulation der Transkription von Genen in vaskulären Zellen
US7186556B2 (en) 1999-09-14 2007-03-06 Avontec Gmbh Modulating transcription of genes in vascular cells
WO2001021793A1 (fr) * 1999-09-22 2001-03-29 Nobuyuki Miyasaka TRAITEMENTS CONTRE LES RHUMATISMES A BASE DE p21?Cip1¿
US8697050B2 (en) 1999-09-22 2014-04-15 Nobuyuki Miyasaka Methods of treatment using a vector encoding p21/Cip1
WO2001047981A1 (fr) * 1999-12-23 2001-07-05 Institut Gustave Roussy SEQUENCES NUCLEOTIDIQUES CODANT POUR UN POLYPEPTIDE CAPABLE D'INTERAGIR AVEC DES PROTEINES DES FAMILLES p300/CBP ET Rb
FR2802944A1 (fr) * 1999-12-23 2001-06-29 Roussy Inst Gustave Sequences nucleotidiques codant pour un polypeptide capable d'interagir avec des proteines des familles p300/cbp et rb
US7297777B2 (en) 2000-02-22 2007-11-20 Laboratoires Serono Sa Process of purification of hCG and recombinant hCG purified by that method
WO2002012270A1 (fr) * 2000-08-03 2002-02-14 Inverness Medical Switzerland Gmbh Peptides capables de fonctionner en tant que mimotopes pour des analytes d'estradiol
WO2002042423A3 (fr) * 2000-11-27 2003-01-30 Isis Pharmaceuticals Inc Modulation antisens de l'expression de src-2
WO2002057409A3 (fr) * 2000-11-27 2002-09-12 Isis Pharmaceuticals Inc Modulation antisens de l'expression de src-2
WO2002057409A2 (fr) * 2000-11-27 2002-07-25 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de src-2
WO2002042423A2 (fr) * 2000-11-27 2002-05-30 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de src-2
US7981694B2 (en) 2003-01-28 2011-07-19 The Regents Of The University Of California Solid phase isolation of proteins, nucleic acids and other macromolecules

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