WO1998003652A9 - Cofacteur de transcription p/caf associe a p300/cbp et utilisations - Google Patents

Cofacteur de transcription p/caf associe a p300/cbp et utilisations

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WO1998003652A9
WO1998003652A9 PCT/US1997/012877 US9712877W WO9803652A9 WO 1998003652 A9 WO1998003652 A9 WO 1998003652A9 US 9712877 W US9712877 W US 9712877W WO 9803652 A9 WO9803652 A9 WO 9803652A9
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pro
caf
gin
cbp
ser
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PCT/US1997/012877
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WO1998003652A2 (fr
WO1998003652A3 (fr
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Publication of WO1998003652A9 publication Critical patent/WO1998003652A9/fr

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  • the present invention provides a transcriptional co-factor, p300/CBP-associated factor (P/CAF), which modulates transcription through binding to the cellular transcription co-factors p300 and CBP and through acetylation of histones. Also provided are methods for screening for the presence of P/CAF and for substances which alter the transcription modulating effect and growth regulatory activity of P/CAF.
  • P/CAF transcriptional co-factor, p300/CBP-associated factor
  • Cellular proteins p300 and CBP are global transcriptional coactivators that are involved in the regulation of various DNA-binding transcriptional factors (Janknecht and Hunter, 1996). Recently, p300 was found to be very closely related to CBP, a factor that binds selectively to the protein kinase A-phosphorylated form of CREB (3-5). Cellular factors p300 and CBP exhibit strong amino acid sequence similarity and share the capacity to bind both CREB and E1A (6-8). Although neither p300 nor CBP by itself binds to DNA, each can be recruited to promoter elements via interaction with sequence-specific activators and functions to be a transcriptional adaptor. For simplicity, p300 and CBP will be termed p300/CBP in the context of discussing their shared functional properties.
  • p300/CBP is a large protein consisting of over 2,400 amino acids, known to interact with a variety of DNA-binding transcriptional factors including nuclear hormone receptors (13,57), CREB (3,4, 7), c-Jun/v-Jun (9,11), YY1 (10), c-Myb/v-Myb (12,58), Sap-la (59), c-Fos (11) and MyoD (60).
  • DNA-binding factors recruit p300/CBP not only by direct but also indirect interactions through cofactors; for example, nuclear hormone receptors recruit p300/CBP directly as well as through indirect interactions, via SRC-1, which stimulates transcription by binding to various nuclear hormone receptors (13,61).
  • El A transforming activity resides in two distinct domains, the targets of which include p300/CBP and products of the retinoblastoma (RB) susceptibility gene family (1,2). Interactions of El A with p300/CBP and RB are thought to influence functionally distinct growth regulatory pathways, allowing the two domains to contribute additively to transformation (1).
  • El A and functionally related viral proteins perturb cell growth regulation derives in large part from studies on their interactions with RB (1,2).
  • the molecular function of El A is based on its capacity to interfere with cellular protein- protein interactions. Since both E1A and various cellular targets bind to a site in RB termed the pocket domain (2), El A can competitively disrupt the complex formation between RB and its cellular targets.
  • E 1 A inhibits the p300/CBP-mediated transcriptional activation of many promoters (14).
  • the complex of p300 and YY1, El A inhibits transcription without disrupting the complex (10).
  • the present invention provides a cellular protein designated P/CAF which binds to p300/CBP and plays an important role in both transcription and cell cycle regulation associated with a histone acetyltransferase activity.
  • the present invention also provides a histone acetyltransferase activity in the p300/CBP cellular protein, thus providing targets for modulating transcription and cell cycle regulation in cells.
  • the present invention provides a purified protein designated P/CAF having a molecular weight of about 93,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and which acetylates histones and which also binds to the p300/CBP cellular protein.
  • the present invention further provides a nucleic acid encoding the P/CAF protein as well as a vector containing the nucleic acid and a host for the vector.
  • a purified antibody which specifically binds the P/CAF protein is also provided.
  • bioassay for screening substances for the ability to inhibit the transcription modulating activity of P/CAF and/or histone acetyltransferase activity comprising contacting the substance with a system in which histone acetylation by P/CAF can be determined; determining the amount of histone acetylation by P/CAF in the presence of the substance; and comparing the amount of histone acetylation by P/CAF in the presence of the substance with the amount of histone acetylation by P/CAF in the absence of the substance, a decreased amount of histone acetylation by P/CAF in the presence of the substance indicating a substance that can inhibit the transcription modulating activity and/or histone acetyltransferase activity of P/CAF.
  • the present invention provides a bioassay for screening substances for the ability to inhibit the transcription modulating activity and/or histone acetyltransferase activity of P/CAF comprising contacting the substance with a system in which the p300 binding of P/CAF can be determined; determining the amount of p300 binding of P/CAF in the presence of the substance; and comparing the amount of p300 binding of P/CAF in the presence of the substance with the amount of p300 binding of P/CAF in the absence of the substance, a decreased amount of p300 binding of P/CAF in the presence of the substance indicating a substance that can inhibit the transcription modulating activity and/or histone acetyltransferase activity of P/CAF.
  • Also provided is a method for determining the amount of P/CAF in a biological sample comprising contacting the biological sample with a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 under conditions whereby a P/CAF/p300 complex can be formed; and determining the amount of the P/CAF/p300 complex, the amount of the complex indicating the amount of P/CAF in the sample.
  • the present invention additionally provides a method for determining the amount of P/CAF in a biological sample comprising contacting the biological sample with an antibody which specifically binds P/CAF under conditions whereby a P/CAF/antibody complex can be formed; and determining the amount of the P/CAF/antibody complex, the amount of the complex indicating the amount of P/CAF in the sample.
  • an assay for screening substances for the ability to inhibit or stimulate the histone acetyltransferase activity of P/CAF comprising: contacting the substance with a system in which histone acetylation by P/CAF can be determined; determining the amount of histone acetylation by P/CAF in the presence of the substance; and comparing the amount of histone acetylation by P/CAF in the presence of the substance with the amount of histone acetylation by P/CAF in the absence of the substance, a decreased or increased amount of histone acetylation by P/CAF in the presence of the substance indicating a substance that can inhibit or stimulate, respectively, the histone acetyltransferase activity of P/CAF.
  • the present invention further provides an assay for screening substances for the ability to inhibit binding of P/CAF to p300/CBP comprising: contacting the substance with a system in which the P/CAF binding of P300/CBP can be determined; determining the amount of P/CAF binding of p300/CBP in the presence of the substance; and comparing the amount of binding of P/CAF to p300/CBP in the presence of the substance with the amount of binding of P/CAF to p300/CBP in the absence of the substance, a decreased amount of binding of P/CAF to p300/CBP in the presence of the substance indicating a substance that can inhibit the ability to inhibit binding of P/CAF to p300/CBP.
  • an assay for screening substances for the ability to inhibit or stimulate the histone acetyltransferase activity of p300/CBP, comprising: contacting the substance with a system in which histone acetylation by p300/CBP can be determined; determining the amount of histone acetylation by p300/CBP in the presence of the substance; and comparing the amount of histone acetylation by p300/CBP in the presence of the substance with the amount of histone acetylation by p300/CBP in the absence of the substance, a decreased or increased amount of histone acetylation by p300/CBP in the presence of the substance indicating a substance that can inhibit or stimulate, respectively, the histone acetyltransferase activity of p300/CBP.
  • the present invention provides an assay for screening substances for the ability to inhibit binding of a DNA-binding transcription factor to p300/CBP comprising: contacting the substance with a system in which the DNA-binding transcription factor binding of P300/CBP can be determined; determining the amount of DNA-binding transcription factor binding of p300/CBP in the presence of the substance; and comparing the amount of binding of DNA-binding transcription factor to p300/CBP in the presence of the substance with the amount of binding of DNA-binding transcription factor to p300/CBP in the absence of the substance, a decreased amount of binding of DNA-binding transcription factor to p300/CBP in the presence of the substance indicating a substance that can inhibit the ability to inhibit binding of DNA- binding transcription factor to p300/CBP.
  • a method for inhibiting the transcription modulating activity of P/CAF in a subject comprising administering to the subject a transcription modulating activity inhibiting amount of a substance in a pharmaceutically acceptable carrier.
  • Also provided in the present invention is a method for stimulating the transcription modulating activity of P/CAF in a subject, comprising administering to the subject a transcription modulating activity stimulating amount of a substance in a pharmaceutically acceptable carrier. Furthermore, the present invention provides a method for inhibiting the histone acetyltransferase activity of p300/CBP in a subject, comprising administering to the subject a histone acetyltransferase activity inhibiting amount of a substance in a pharmaceutically acceptable carrier.
  • the present invention additionally provides a method for stimulating the histone acetyltransferase activity of p300/CBP in a subject, comprising administering to the subject a histone acetyltransferase activity stimulating amount of a substance in a pharmaceutically acceptable carrier.
  • Figs. 1 A-B P/CAF-p300/CBP interaction in vivo.
  • Cell extract was immunoprecipitated with rabbit anti-P/CAF (lanes 1 , 4, and 7), rabbit anti-CBP (lanes 2 and 5), and mouse anti-p300 (lane 9) antibodies.
  • cell extract was precipitated with rabbit control IgG (lanes 3, 6, and 8) or mouse anti-HA monoclonal antibody (lane 10).
  • the precipitates were analyzed by immunoblotting with anti-P/CAF (lanes 1-3), anti-CBP (lanes 4-6), and anti-p300 (lanes 7-10) antibodies. The positions of non-specific bands are indicated by asterisks.
  • IB El A inhibits the P/CAF-p300 interaction in vivo. Osteosarcoma cells were transfected with either control vector (lanes 1 and 4) or El A- (lanes 2 and 5) or El A ⁇ N- (lanes 3 and 6) expression vectors. Extract from the transfected subpopulation was immunoprecipitated with anti-P/CAF (lanes 1-3) or control (lanes 4-6) IgG. The precipitates were analyzed by immunoblotting with anti-p300 and anti-P/CAF.
  • Figs. 2 A-F P/CAF and El A mediate antagonistic effects on cell cycle progression.
  • HeLa cells ATCC accession number CCL 2
  • These plasmids were constructed by subcloning FLAG-P/CAF and El A cDNAs into pCX (34) and pcDNAI (Invitrogen), respectively.
  • Fig. 3 Histone acetyltransferase activity of P/CAF.
  • Activity of hGCN5 (lanes 1 and 4) and P/CAF (lanes 2 and 5) that acetylates free histones (lanes 1-3) or histones in the nucleosome core particle (35) (lanes 4-6) was measured as described (36).
  • Each reaction contains 0.3 pmol of affinity purified FLAG-hGCN5 or FLAG-P/CAF, 4 pmol of the histone octamer or the nucleosome core particle and 10 pmol of [l- 14 C]acetyl- Co A.
  • the histone octamer dissociates into dimers or tetramers under assay conditions.
  • Acetylated histones were detected by autoradiography after separation by SDS-PAGE. The bands corresponding to acetylated histones H3 and H4 are indicated by arrows.
  • the present invention provides a purified protein designated P/CAF having a molecular weight of about 93,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and which acetylates histones.
  • the P/CAF protein can also bind to the amino acid region of SEQ LD NO:3 (amino acid (aa) residues 1753 - 1966) of the cellular transcriptional factor, p300 (which has the complete amino acid sequence of SEQ ID NO: 6 and the nucleotide sequence of SEQ ID NO: 12), and the amino acid region of SEQ ID NO: 6 (amino acid residues 1805 - 1854) of the cellular transcriptional factor, CBP (which has the complete amino acid sequence of SEQ LD NO: 7 and the nucleotide sequence of SEQ LD NO: 13).
  • the P/CAF protein can be defined by any one or more of the typically used parameters.
  • these parameters include, but are not limited to molecular weight (calculated or empirically determined), isoelectric focusing point, specific epitope(s), complete amino acid sequence, sequence of a specific region (e.g., N-terrninus) of the amino acid sequence and the like.
  • the P/CAF protein can consist of the amino acid sequence of SEQ ID NO:l or the P/CAF protein can comprise the amino acid sequence of SEQ D NO: 2 which represents the carboxy terminal end of the P/CAF protein and contains the histone acetyltransferase activity, or the amino acid sequence of SEQ D NO: 4, which represents the amino terminal end of the P/CAF protein, containing the binding site for p300/CBP. Because the amino-terminal region is specific for P/CAF it can be used to define and identify P/CAF.
  • purified refers to a protein (polypeptide, peptide, etc.) that is sufficiently free of contaminants or cell components with which it normally occurs to distinguish it from the contaminants or other components of its natural environment.
  • the purified protein need not be homogeneous, but must be sufficiently free of contaminants to be useful in a clinical or research setting, for example, in an assay for detecting antibodies to the protein. Greater levels of purity can be obtained using methods derived from well known protocols. Specific methods for purifying P/CAF proteins are known in the art.
  • the invention also includes those P/CAF polypeptides having slight variations in amino acid sequence which yield polypeptides equivalent to the P/CAF protein defined herein.
  • variations may arise naturally as allelic variations (e.g., due to genetic polymorphism) or may be produced by human intervention (e.g., by mutagenesis of cloned DNA sequences), such as induced point, deletion, insertion and substitution mutants.
  • Minor changes in amino acid sequence are generally preferred, such as conservative amino acid replacements, small internal deletions or insertions, and additions or deletions at the ends of the molecules.
  • Substitutions may be designed based on, for example, the model of Dayhoff, et al. (37). These modifications can result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide other specific mutations.
  • Modifications to any of the P/CAF proteins or fragments can be made, while preserving the specificity and activity (function) of the native protein or fragment thereof.
  • "native" describes a protein that occurs in nature.
  • the modifications contemplated herein can be conservative amino acid substitutions, for example, the substitution of a basic amino acid for a different basic amino acid.
  • Modifications can also include creation of fusion proteins with epitope tags or known recombinant proteins or genes encoding them created by subcloning into commercial or non-commercial vectors (e.g., polyhistidine tags, flag tags, myc tag, glutathione-S- transferase [GST] fusion protein, xylE fusion reporter construct).
  • modifications can be such as do not affect the function of the protein or the way the protein accomplishes that function (e.g., its secondary structure or the ultimate result of the protein's activity). These products are equivalent to the P/CAF protein.
  • the means for determining the function, way and result parameters are well known.
  • the invention also enables the purification of P/CAF homologs from other species and allelic variants from individuals within a species.
  • an antibody raised against the exemplary human P/CAF protein can be used routinely to screen preparations from different humans for allelic variants of the P/CAF protein that react with the P/CAF protein- specific antibody.
  • an antibody raised against an epitope for example, from a conserved amino acid region of the human P/CAF protein can be used to routinely screen for homologs of the P/CAF protein in other species.
  • a P/CAF protein can be routinely identified in and obtained from other species and from individuals within a species using the methods taught herein and others known in the art.
  • the DNA encoding a conserved amino acid sequence can be used to probe genomic DNA or DNA libraries of an organism to predictably obtain the P/CAF gene for that organism.
  • the gene can then be cloned and expressed as the P/CAF protein and purified according to any of a number of routine, predictable methods.
  • routine protein purification methods available in the art can be found in Pei et al. (38).
  • a purified polypeptide fragment of the P/CAF protein is also provided.
  • fragment as used herein regarding a P/CAF protein, means a molecule of at least five contiguous amino acids of P/CAF protein that has at least one function shared by P/CAF protein or a region thereof. These functions can include antigenicity, binding capacity, acetyltransferase activity and structural roles, among others.
  • the P/CAF fragment can be specific for a recited source. As used herein to describe an amino acid sequence (protein, polypeptide, peptide, etc.), "specific" means that the amino acid sequence is not found identically in any other source.
  • a P/CAF fragment can be species-specific (e.g., found in the P/CAF protein of humans, but not of other species).
  • a fragment of the P/CAF protein having histone acetyltransferase activity can consist of the amino acid sequence of SEQ LD NO:2.
  • a fragment of the P/CAF protein which binds to the amino acid sequence of SEQ LD NO: 3 on p300 and the amino acid sequence of SEQ LD NO: 9 on CBP can consist of the amino acid sequence of SEQ ID NO:4.
  • SEQ ID NO:4 To the extent that these fragments are specific for P/CAF, they can be used to identify and define P/CAF.
  • An antigenic fragment of P/CAF protein is provided.
  • An antigenic fragment has an amino acid sequence of at least about five consecutive amino acids of a P/CAF protein amino acid sequence and binds an antibody or elicits an immune response in an animal.
  • An antigenic fragment can be selected by applying the routine technique of epitope mapping to P/CAF protein to determine the regions of the proteins that contain epitopes reactive with antibodies or are capable of eliciting an immune response in an animal. Once the epitope is selected, an antigenic polypeptide containing the epitope can be synthesized directly, or produced recombinantly by cloning nucleic acids encoding the antigenic polypeptide in an expression system, according to standard methods.
  • an antigenic fragment of the antigen can be isolated from the whole P/CAF protein or a larger fragment of the P/CAF protein by chemical or mechanical disruption. Fragments can also be randomly chosen from a known P/CAF protein sequence and synthesized. The purified fragments thus obtained can be tested to determine their antigenicity and specificity by routine methods.
  • isolated nucleic acid that encodes a P/CAF protein is also provided.
  • isolated means a nucleic acid separated or substantially free from at least some of the other components of the naturally occurring organism, for example, the cell structural components commonly found associated with nucleic acids in a cellular environment and/or other nucleic acids.
  • the isolation of nucleic acids can therefore be accomplished by techniques such as cell lysis followed by phenol plus chloroform extraction, followed by ethanol precipitation of the nucleic acids (39).
  • the isolated nucleic acids are necessarily totally free of all non- nucleic acid components or all other nucleic acids, but that the isolated nucleic acids are isolated to a degree of purification to be useful in clinical, diagnostic, experimental, or other procedures such as, for example, gel electrophoresis, Southern, Northern or dot blot hybridization, or polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • lysis of the cell followed by gel filtration or anion exchange chromatography, binding DNA to silica in the form of glass beads, filters or diatoms in the presence of high concentrations of chaotropic salts, or ethanol precipitation of the nucleic acids.
  • the nucleic acids of the present invention can include positive and negative strand RNA as well as DNA and can include genomic and subgenomic nucleic acids found in the naturally occurring organism.
  • the nucleic acids contemplated by the present invention include double stranded and single stranded DNA of the genome, complementary positive stranded cRNA and mRNA, and complementary cDNA produced therefrom and any nucleic acid which can selectively or specifically hybridize to the isolated nucleic acids provided herein.
  • Stringent conditions are used to distinguish selectively or specifically hybridizing nucleic acids from non-selectively and non-specifically hybridizing nucleic acids.
  • An isolated nucleic acid that encodes a P/CAF protein can be species-specific (i.e., does not encode the P/CAF protein of other species and does not occur in other species).
  • Examples of the nucleic acids contemplated herein include the nucleic acid of SEQ LD NO: 10 as well as the nucleic acids that encode each of the P/CAF proteins or fragments thereof described herein.
  • P/CAF proteins and protein fragments can be routinely obtained as'described herein and their structure (sequence) determined by routine means including the methods as used herein.
  • P/CAF protein-encoding nucleic acids can be isolated from an organism in which they are normally found (e.g., humans), using any of the routine techniques. For example, a genomic DNA or cDNA library can be constructed and screened for the presence of the nucleic acid of interest using one of the present P/CAF protein-encoding nucleic acids as a probe. Methods of constructing and screening such libraries are well known in the art and kits for performing the construction and screening steps are commercially available (for example, Stratagene Cloning Systems, La Jolla, CA). Once isolated, the nucleic acid can be directly cloned into an appropriate vector, or if necessary, be modified to facilitate the subsequent cloning steps. Such modification steps are routine, an example of which is the addition of oligonucleotide linkers, which contain restriction sites, to the termini of the nucleic acid (See, for example, ref. 39).
  • P/CAF protein-encoding nucleic acids can also be synthesized.
  • a method of obtaining a DNA molecule encoding a specific P/CAF protein is to synthesize a recombinant DNA molecule which encodes the P/CAF protein.
  • nucleic acid synthesis procedures are routine in the art and oligonucleotides coding for a particular protein region are readily obtainable through automated DNA synthesis.
  • a nucleic acid for one strand of a double-stranded molecule can be synthesized and hybridized to its complementary strand.
  • One can design these oligonucleotides such that the resulting double-stranded molecule has either internal restriction sites or appropriate 5' or 3' overhangs at the termini for cloning into an appropriate vector.
  • Oligonucleotides complementary to or identical with the P/CAF protein- encoding nucleic acid sequence can be synthesized as primers for amplification reactions, such as PCR, or as probes to detect P/CAF protein encoding nucleic acids by various hybridization protocols (e.g., Northern blot; Southern blot; dot blot, colony screening, etc.).
  • various hybridization protocols e.g., Northern blot; Southern blot; dot blot, colony screening, etc.
  • Double-stranded molecules coding for relatively large proteins can readily be synthesized by first constructing several different double-stranded molecules that code for particular regions of the protein, followed by ligating these DNA molecules together.
  • Cunningham, et al. (40) have constructed a synthetic gene encoding the human growth hormone by first constructing overlapping and complementary synthetic oligonucleotides and ligating these fragments together.
  • Ferretti, et al. (41) wherein synthesis of a 1057 base pair synthetic bovine rhodopsin gene from synthetic oligonucleotides is disclosed.
  • P/CAF protein-encoding nucleic acid By constructing a P/CAF protein-encoding nucleic acid in this manner, one skilled in the art can readily obtain any particular P/CAF protein with modifications at any particular position or positions. See also, U.S. Patent No. 5,503,995 which describes an enzyme template reaction method of making synthetic genes. Techniques such as this are routine in the art and are well documented. DNA encoding the P/CAF protein or P/CAF protein fragments can then be expressed in vivo or in vitro.
  • the nucleic acid encoding the P/CAF protein can be any nucleic acid that functionally encodes the P/CAF protein.
  • the nucleic acid can include, but is not limited to, expression control sequences, such as an origin of replication, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers which can serve to select for cells containing the vector or the vector containing the insert, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcription termination sequences as well as any other sequence which may facilitate the expression of the inserted nucleic acid.
  • expression control sequences such as an origin of replication, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic
  • Preferred expression control sequences are promoters derived from metallothionine genes, actin genes, immunoglobulin genes, CMV, SV40, adenovirus, bovine papilloma virus, etc.
  • a nucleic acid encoding a P/CAF protein can readily be determined based upon the genetic code for the amino acid sequence of the P/CAF protein and many nucleic acid sequences will encode a P/CAF protein. Modifications in the nucleic acid sequence encoding the P/CAF protein are also contemplated. Modifications that can be useful are modifications to the sequences controlling expression of the P/CAF protein to make production of P/CAF protein inducible or repressible as controlled by the appropriate inducer or repressor.
  • the nucleic acids can be generated by means standard in the art, such as by recombinant nucleic acid techniques, as exemplified in the examples herein, and by synthetic nucleic acid synthesis or in vitro enzymatic synthesis. After a nucleic acid encoding a particular P/CAF protein of interest, or a region of that nucleic acid, is constructed, modified, or isolated, that nucleic acid can then be cloned into an appropriate vector, which can direct the in vivo or in vitro synthesis of that wild-type and/or modified P/CAF protein.
  • the vector is contemplated to have the necessary functional elements that direct and regulate transcription of the inserted nucleic acid, as described above.
  • the vector containing the P/CAF nucleic acid or nucleic acid fragment can be in a host (e.g., cell or transgenic animal) for expressing the nucleic acid.
  • the P/CAF protein or fragment thereof can thus be produced in a host system containing the expression vector and its functional activity as described herein can be demonstrated according to methods well known in the art.
  • E. coli Esscherichia coli
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteria, such as Salmonella, Serratia, as well as various Pseudomonas species.
  • These prokaryotic hosts can support expression vectors which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence and have ribosome binding site sequences, for example, for initiating and completing transcription and translation. If necessary, an amino terminal methionine can be provided by insertion of a Met codon 5' and in-frame with the gene sequence. Also, the carboxy-terminal extension of the protein can be removed using standard oligonucleotide mutagenesis procedures.
  • yeast expression can be used. There are several advantages to yeast expression systems. First, evidence exists that proteins produced in yeast secretion systems exhibit correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast secretory systems.
  • the Saccharomyces cerevisiae pre- pro-alpha-factor leader region (encoded by the MFa-1 gene) is routinely used to direct protein secretion from yeast (42).
  • the leader region of pre-pro-alpha-factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene.
  • This enzyme cleaves the precursor protein on the carboxyl side of a Lys- Arg dipeptide cleavage-signal sequence.
  • the polypeptide coding sequence can be fused in-frame to the pre-pro-alpha-factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter.
  • the protein coding sequence is followed by a translation termination codon which is followed by transcription termination signals.
  • the polypeptide encoding sequence of interest can be fused to a second protein coding sequence, such as Sj26 or ⁇ -galactosidase, used to facilitate purification of the resultant fusion protein by affinity chromatography.
  • the insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast.
  • Efficient post-translational glycosylation and expression of recombinant proteins can also be achieved in Baculovirus expression systems in insect cells.
  • Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures and secretion of active protein.
  • Vectors useful for the expression of proteins in mammalian cells are characterized by insertion of the protein encoding sequence between a strong viral promoter and a polyadenylation signal.
  • the vectors can contain genes conferring either gentamicin or methotrexate resistance for use as selectable markers.
  • the antigen and immunoreactive fragment coding sequence can be introduced into a Chinese hamster ovary (CHO) cell line using a methotrexate resistance-encoding vector.
  • Presence of the vector RNA in transformed cells can be confirmed by Northern blot analysis and production of a cDNA or opposite strand RNA corresponding to the protein encoding sequence can be confirmed by Southern and Northern blot analysis, respectively.
  • suitable host cell lines capable of secreting intact proteins include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, and the like.
  • Expression vectors for these cells can include expression control sequences, as described above.
  • the vectors containing the nucleic acid sequences of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cell host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cell hosts.
  • vectors for the expression of protein in mammalian cells similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, hepatitis B virus surface antigen, protease Nexin 1, and eosinophil major basic protein, can be employed.
  • the vector can include CMV promoter sequences and a polyadenylation signal available for expression of inserted nucleic acid in mammalian cells (such as COS7).
  • the nucleic acid sequences can be expressed in hosts after the sequences have been positioned to ensure the functioning of an expression control sequence.
  • These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • expression vectors can contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired nucleic acid sequences (see, e.g., U.S. Patent 4,704,362).
  • the nucleic acids produced as described above can also be expressed in a host which is a non-human animal to create a transgenic animal, containing, in a germ or somatic cell, a nucleic acid comprising the coding sequence for all or a portion of the P/CAF protein, as well as all of the other regulatory elements required for expression of the P/CAF protein-encoding sequence.
  • the animal will express the P/CAF gene or portion thereof to produce the P/CAF protein or protein fragment and such expression can be detected by determination of a particular phenotype unique to the transgenic animal expressing the transferred nucleic acid.
  • the nucleic acid can be the nucleic acid of SEQ LD NO: 10, a nucleic acid having a nucleotide sequence which encodes the P/CAF protein, a nucleic acid having a nucleotide sequence which encodes the protein of SEQ LD NO:l, as well as the nucleic acids that encode the proteins comprising the fragments of SEQ ID NOS:2 and 4.
  • the nucleic acids of the invention can contain substitutions or deletions which provide a particular phenotype of interest.
  • various deletions or base substitutions can be introduced into the nucleic acid encoding the P/CAF protein for the purpose of studying the effects of these particular deletions or substitutions on the transcription modulation activity of the P/CAF protein. These effects can be monitored by observation of such characteristics as growth and development of the animal, the ability to develop tumors, survival rates and the like.
  • the gene construct introduced into the animal cells to produce the transgenic animal can contain any of the regulatory elements described above to modulate expression of the foreign genes.
  • the term "phenotype" includes morphology, biochemical profiles, changes in tumor formation and other parameters that are affected by the presence of the P/CAF protein.
  • the transgenic animals of the invention can also be used in a method for determining the effectiveness of administering a nucleic acid encoding a functional P/CAF protein to a subject in need of a functional P/CAF protein.
  • a nucleic acid encoding a nonfunctional P/CAF protein can be introduced into the animal's cells and expressed to yield a characteristic phenotype.
  • a nucleic acid encoding a functional P/CAF protein can be introduced into the animal's cells and the effects on the animal's phenotypic characteristics can be determined.
  • nucleic acid that encodes a P/CAF protein an isolated nucleic acid that encodes a fragment of P/CAF protein is also provided.
  • the nucleic acid encoding the fragment can be obtained using any of the methods applicable to the nucleic acid encoding the entire P/CAF protein.
  • the nucleic acid fragment can encode a species-specific P/CAF protein fragment (e.g., found in the P/CAF protein of humans, but not in the P/CAF proteins of other species). Nucleic acids encoding species-specific fragments of P/CAF protein are themselves species- specific or allele-specific fragments of the P/CAF gene.
  • fragments of a nucleic acid encoding a fragment of the P/CAF protein can include the nucleic acid sequences which encode the amino acid sequences of the fragments of SEQ ID NOS:2 or 4. The same routine computer analyses used to select these examples of fragments can be routinely used to obtain others. Fragments of P/CAF-encoding nucleic acids can be primers for PCR or probes, which can be species- specific, gene-specific or allele-specific. P/CAF-encoding nucleic acid fragments can encode antigenic or immunogenic fragments of P/CAF protein that can be used in therapeutic assays or screening protocols. P/CAF gene fragments can encode fragments of P/CAF protein having histone acetylase activity and/or p300/CBP binding activity as described above, as well as other uses that may become apparent.
  • an isolated nucleic acid of at least ten nucleotides that selectively hybridizes with the nucleic acid of SEQ LD NO: 10 under selected conditions is provided.
  • the conditions can be PCR amplification conditions and the hybridizing nucleic acid can be a primer consisting of a specific fragment of the reference sequence or a nearly identical nucleic acid that hybridizes only to the exemplified P/CAF-encoding nucleic acid or allelic variants thereof.
  • the invention provides an isolated nucleic acid that selectively hybridizes with the P/CAF-encoding nucleic acid sequence of SEQ LD NO: 10 under stringent conditions.
  • the hybridizing nucleic acid can be a probe that hybridizes only to the exemplified P/CAF-encoding nucleic acid sequence.
  • the hybridizing nucleic acid can be a naturally occurring species-specific allelic variant of the exemplified P/CAF gene.
  • the hybridizing nucleic acid can also include insubstantial base substitutions that do not prevent hybridization under the stated stringent conditions or affect either the function of the encoded protein, the way the protein accomplishes that function (e.g., its secondary structure) or the ultimate result of the protein's activity. The means for determining these parameters are well known.
  • the term "selectively hybridizes" excludes the occasional randomly hybridizing nucleic acids as well as nucleic acids that encode other known homologs of the P/CAF protein.
  • the selectively hybridizing nucleic acids of the invention can have at least 70%, 73%, 78%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity with the segment and strand of the sequence to which it hybridizes. This list is not intended to exclude percent complementarity values between these values.
  • the nucleic acids can be at least 10, 15, 16, 17, 18, 20, 21, 23, 24, 25, 30, 35, 40, 50, 100, 150, 200, 300, 500, 550, 750, 900, 950, or 1000 nucleotides in length or any intervening length, depending on whether the nucleic acid is to be used as a primer, probe or for protein expression.
  • the hybridizing nucleic acid can comprise a region of at least ten nucleotides (up to full length) that is completely complementary to a unique region of the nucleic acid to which it hybridizes.
  • the nucleic acid can be an alternative coding sequence for the P/CAF protein, or can be used as a probe or primer for detecting the presence of or obtaining the P/CAF protein. If used as primers, the invention provides compositions including at least two nucleic acids which selectively hybridize with different regions of the nucleic acid so as to amplify a desired region. Depending on the length of the probe or primer, it can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions.
  • the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes (P/CAF DNA in a sample) should be at least enough to exclude hybridization with a nucleic acid from another species.
  • the invention provides examples of these nucleic acids of P/CAF, so that the degree of complementarity required to distinguish selectively hybridizing from nonselectively hybridizing nucleic acids under stringent conditions can be clearly determined for each nucleic acid. It should also be clear that the hybridizing nucleic acids of the invention will not hybridize with nucleic acids encoding unrelated proteins (hybridization is selective) under stringent conditions.
  • washing conditions refers to the washing conditions used in a hybridization protocol.
  • the washing conditions should be a combination of temperature and salt concentration chosen so that the denaturation temperature is approximately 5- 20 °C below the calculated T m of the nucleic acid hybrid under study.
  • the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to the probe or protein encoding nucleic acid of interest and then washed under conditions of different stringencies.
  • the nucleic acid sequence of SEQ LD NO: 10 was used as a specific radiolabeled probe for the detection of messenger RNA transcribed from the P/CAF gene by performing hybridizations under stringent conditions.
  • T m of such an oligonucleotide can be estimated by allowing 2°C for each A or T nucleotide, and 4°C for each G or C. For example, an 18 nucleotide probe of 50% G+C would, therefore, have an approximate T m of 54 °C.
  • the invention provides an isolated nucleic acid that selectively hybridizes with the P/CAF gene shown in the sequence set forth as SEQ LD NO: 10 under stringent conditions.
  • the invention further provides an isolated nucleic acid complementary to the nucleotide sequence set forth in SEQ LD NO: 10.
  • a purified antibody and an antiserum containing polyclonal antibodies that specifically bind the P/CAF protein or antigenic fragment are also provided.
  • the term "bind” means the well understood antigen/antibody binding as well as other nonrandom association with an antigen.
  • "Specifically bind” as used herein describes an antibody or other ligand that does not cross react substantially with any antigen other than the one specified, in this case, an antigen of the P/CAF protein.
  • Antibodies can be made as described in Harlow and Lane (33). Briefly, purified P/CAF protein or an antigenic fragment thereof can be injected into an animal in an amount and in intervals sufficient to elicit a humoral immune response.
  • Serum polyclonal antibodies can be purified directly, or spleen cells from the animal can be fused with an immortal cell line and screened for monoclonal antibody secretion, according to procedures well known in the art. Purified monospecific polyclonal antibodies that specifically bind the P/CAF antigen are also within the scope of the present invention.
  • the antibodies of the present invention can bind the protein of claim 1, the protein of claim 2, the protein of claim 3 and/or the protein of claim 4, as well as any other proteins of the present invention.
  • a ligand that specifically binds the antigen is also contemplated.
  • the ligand can be a fragment of an antibody, such as , for example, an Fab fragment which retains P/CAF binding activity, or a smaller molecule designed to bind an epitope of the P/CAF antigen.
  • the antibody or ligand can be bound to a substrate or labeled with a detectable moiety or both bound and labeled.
  • the detectable moieties contemplated within the compositions of the present invention include those listed above in the description of the diagnostic methods, including fluorescent, enzymatic and radioactive markers.
  • the antibody can be bound to a solid support substrate or conjugated with a detectable moiety or therapeutic compound or both bound and conjugated.
  • conjugation techniques are well known in the art.
  • conjugation of fluorescent, radioactive or enzymatic moieties can be performed as described in the art (33,43).
  • the detectable moieties contemplated in the present invention can include fluorescent, radioactive and enzymatic markers and the like.
  • Therapeutic drugs contemplated with the present invention can include cytotoxic moieties such as ricin A chain, diphtheria toxin, pseudomonas exotoxin and other chemotherapeutic compounds.
  • the present invention also provides a method for determining the presence and thus the amount of P/CAF protein in a biological sample.
  • a biological sample includes any tissue or cell which would contain the P/CAF protein. Examples of cells include tissues taken from surgical biopsies, isolated from a body fluid or prepared in an in vitro tissue culture environment.
  • determining the amount of P/CAF in a biological sample can comprise contacting the biological sample with a polypeptide comprising the amino acid sequence of SEQ LD NO: 3 under conditions whereby a P/CAF/p300 complex can be formed; and determining the amount of the P/CAF/p300 complex, the amount of the complex indicating the amount of P/CAF in the sample. Determination of the amount of P/CAF/p300 complex can be accomplished through techniques standard in the art. For example, the complex may be precipitated out of a solution and detected by the addition of a detectable moiety conjugated to the p300 protein or by the detection of an antibody which binds p300 or the P/CAF protein, as taught in the Examples herein.
  • Antibodies which bind p300 or the P/CAF protein can be either monoclonal or polyclonal antibodies and can be obtained as described herein. Detection of P/CAF/p300 complexes by the detection of the binding of antibodies reactive with p300 or the P/CAF protein can be accomplished using various immunoassays as are available in the art, as described below.
  • determination of the amount of P/CAF in a biological sample can comprise contacting the biological sample with a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 under conditions whereby a P/CAF/CBP complex can be formed; and determining the amount of the P/CAF/CBP complex, the amount of the complex indicating the amount of P/CAF in the sample. Determination of the amount of P/CAF/CBP complex can be accomplished through techniques standard in the art. For example, the complex may be precipitated out of a solution and detected by the addition of a detectable moiety conjugated to the CBP protein or by the detection of an antibody which binds either CBP or the P/CAF protein, as taught in the Examples herein.
  • Antibodies which bind CBP or the P/CAF protein can be either monoclonal or polyclonal antibodies and can be obtained as described herein. Detection of P/CAF/CBP complexes by the detection of the binding of antibodies reactive with CBP or the P/CAF protein can be accomplished using various immunoassays as are available in the art, as described below.
  • Another example of determining the amount of P/CAF in a biological sample comprises contacting the biological sample with an antibody which specifically binds P/CAF under conditions whereby a P/CAF/ antibody complex can be formed and determining the amount of the P/CAF/antibody complex, the amount of the complex indicating the amount of P/CAF in the sample.
  • Antibodies which bind P/CAF can be either monoclonal or polyclonal antibodies and can be obtained as described herein. Determination of P/CAF/antibody complexes can be accomplished using various immunoassays as are available in the art, as described below.
  • Immunoassays such as immunofluorescence assays, radioimmunoassays (RIA), immunoblotting and enzyme linked immunosorbent assays (ELISA) can be readily adapted for detection and measurement of P/CAF in a biological sample. Both polyclonal and monoclonal antibodies can be used in the assays. Available immunoassays are well known in the art and are extensively described in the patent scientific literature. See, for example, U.S. Patent Nos.
  • the present invention also provides a bioassay for screening substances for the ability to inhibit the histone acetyltransferase activity of P/CAF comprising contacting a system, in which histone acetylation by P/CAF can be determined, with the substance under conditions whereby histone acetylation by P/CAF can occur; determining the amount of histone acetylation by P/CAF in the presence of the substance; and comparing the amount of histone acetylation by P/CAF in the presence of the substance with the amount of histone acetylation by P/CAF in the absence of the substance, a decreased amount of histone acetylation by P/CAF in the presence of the substance indicating a substance that can inhibit the histone acetyltransferase activity of P/CAF.
  • the acetylation of histones by P/CAF can be determined in a system including, for example, either core histones (histones H2A, H2B, H3 and H4) or the nucleosome core particles (146 base pairs of DNA wrapped around the octamer of core histones) as substrates, the P/CAF protein and radiolabeled acetyl-CoA (e.g., [l- 14 C]acetyl CoA).
  • the presence of acetylated histones can be detected by autoradiography after separation by SDS-PAGE as described herein in the Examples.
  • the compound to be tested for the ability to inhibit the histone acetyltransferase activity of P/CAF can be added to this system and assayed for inhibiting ability.
  • the present invention also provides a bioassay for screening substances for the ability to inhibit the transcription modulating activity of P/CAF, comprising contacting a system, in which histone acetylation by P/CAF can be determined, with the substance under conditions whereby histone acetylation by P/CAF can occur; determining the amount of histone acetylation by P/CAF in the presence of the substance; and comparing the amount of histone acetylation by P/CAF in the presence of the substance with the amount of histone acetylation by P/CAF in the absence of the substance, a decreased amount of histone acetylation by P/CAF in the presence of the substance indicating a substance that can inhibit the transcription modulating activity and cell cycle progression suppressing activity of P/CAF.
  • the acetylation of histones by P/CAF can be determined in a system including, for example, either core histones (histones H2A, H2B, H3 and H4) or the nucleosome core particles (146 base pairs of DNA wrapped around the octamer of core histones) as substrates, the P/CAF protein and radiolabeled acetyl-CoA (e.g., [l- 14 C]acetyl CoA).
  • core histones histones H2A, H2B, H3 and H4
  • nucleosome core particles 146 base pairs of DNA wrapped around the octamer of core histones
  • radiolabeled acetyl-CoA e.g., [l- 14 C]acetyl CoA
  • the presence of acetylated histones can be detected by autoradiography after separation by SDS-PAGE as described herein in the Examples.
  • the compound to be tested for the ability to inhibit the transcription modulating activity of P/CAF by interfering with the histone acetyltransferase activity of P/CAF can be added to this system and assayed for inhibiting ability.
  • a bioassay for screening substances for the ability to inhibit the binding of p300 to P/CAF comprising contacting a system in which the binding of p300 to P/CAF can be determined, with the substance under conditions whereby the binding of p300 and P/CAF can occur; determining the amount of p300 binding to P/CAF in the presence of the substance; and comparing the amount of p300 binding to P/CAF in the presence of the substance with the amount of p300 binding to P/CAF in the absence of the substance, a decreased amount of p300 binding to P/CAF in the presence of the substance indicating a substance that can inhibit the binding of p300 to P/CAF.
  • the binding of p300 to P/CAF can be determined in a system, for example, which can include a cell free reaction mixture comprising a fragment of the p300 protein comprising the amino acid sequence of SEQ ID NO: 3 and P/CAF.
  • the system can comprise a cell extract produced from cells producing both p300 and P/CAF. Determination of the binding of p300 to P/CAF can be carried out as taught herein.
  • a bioassay for screening substances for the ability to inhibit the binding of CBP to P/CAF comprising contacting a system in which the binding of CBP to P/CAF can be determined, with the substance under conditions whereby the binding of CBP to P/CAF can occur; determining the amount of CBP binding to P/CAF in the presence of the substance; and comparing the amount of CBP binding to P/CAF in the presence of the substance with the amount of CBP binding to P/CAF in the absence of the substance, a decreased amount of CBP binding to P/CAF in the presence of the substance indicating a substance that can inhibit the binding of CBP to P/CAF.
  • the binding of CBP to P/CAF can be determined in a system, for example, which can include a cell free reaction mixture comprising a fragment of the CBP protein comprising the amino acid sequence of SEQ LD NO: 9 and P/CAF.
  • the system can comprise a cell extract produced from cells producing both CBP and P/CAF. Determination of the binding of CBP to P/CAF can be carried out as taught herein.
  • the present invention further contemplates a bioassay for screening substances for the ability to stimulate the histone acetyltransferase activity of P/CAF comprising contacting a system, in which histone acetylation by P/CAF can be determined, with the substance; determining the amount of histone acetylation by P/CAF in the presence of the substance; and comparing the amount of histone acetylation by P/CAF in the presence of the substance with the amount of histone acetylation by P/CAF in the absence of the substance, an increased amount of histone acetylation by P/CAF in the presence of the substance indicating a substance that can stimulate the histone acetyltransferase activity of P/CAF.
  • the acetylation of histones by P/CAF can be determined in a system including, for example, either core histones (histones H2A, H2B, H3 and H4) or the nucleosome core particles (146 base pairs of DNA wrapped around the octamer of core histones) as substrates, the P/CAF protein and radiolabeled acetyl- CoA (e.g., [l- 14 C]acetyl CoA).
  • the presence of acetylated histones can be detected by autoradiography after separation by SDS-PAGE as described herein in the Examples.
  • the compound to be tested for the ability to stimulate the histone acetyltransferase activity of P/CAF can be added to this system and assayed for stimulating ability.
  • the present invention further contemplates a bioassay for screening substances for the ability to stimulate the transcription modulating activity of P/CAF comprising contacting a system, in which histone acetylation by P/CAF can be determined, with the substance; determining the amount of histone acetylation by P/CAF in the presence of the substance; and comparing the amount of histone acetylation by P/CAF in the presence of the substance with the amount of histone acetylation by P/CAF in the absence of the substance, an increased amount of histone acetylation by P/CAF in the presence of the substance indicating a substance that can stimulate the transcription modulating activity of P/CAF.
  • the acetylation of histones by P/CAF can be determined in a system including, for example, either core histones (histones H2A, H2B, H3 and H4) or the nucleosome core particles (146 base pairs of DNA wrapped around the octamer of core histones) as substrates, the P/CAF protein and radiolabeled acetyl-CoA (e.g., [l- 14 C]acetyl CoA).
  • the presence of acetylated histones can be detected by autoradiography after separation by SDS-PAGE as described herein in the Examples.
  • the compound to be tested for the ability to stimulate the transcription modulating activity of P/CAF by increasing the histone acetyltransferase activity of P/CAF can be added to this system and assayed for stimulating ability.
  • the present invention further provides a bioassay for screening substances for the ability to stimulate binding of p300 to P/CAF, comprising contacting a system in which the binding of p300 to P/CAF can be determined, with the substance under conditions whereby the binding of p300 to P/CAF can occur; determining the amount of p300 binding to P/CAF in the presence of the substance; and comparing the amount of p300 binding to P/CAF in the presence of the substance with the amount of p300 binding to P/CAF in the absence of the substance, an increased amount of p300 binding to P/CAF in the presence of the substance indicating a substance that can stimulate the binding of p300 to P/CAF.
  • the binding of p300 to P/CAF can be determined in a system, for example, which can include a cell free reaction mixture comprising a fragment of the p300 protein comprising the amino acid sequence of SEQ LD NO: 3 and P/CAF.
  • the system can comprise a cell extract produced from cells producing both p300 and P/CAF. Determination of the binding of p300 to P/CAF can be carried out as taught herein.
  • a bioassay for screening substances for the ability to stimulate the binding of CBP to P/CAF comprising contacting a system in which the binding of CBP to P/CAF can be determined, with the substance under conditions whereby the binding of CBP to P/CAF can occur; determining the amount of CBP binding to P/CAF in the presence of the substance; and comparing the amount of CBP binding to P/CAF in the presence of the substance with the amount of CBP binding to P/CAF in the absence of the substance, an increased amount of CBP binding to P/CAF in the presence of the substance indicating a substance that can stimulate the binding of CBP to P/CAF.
  • the binding of CBP to P/CAF can be determined in a system, for example, which can include a cell free reaction mixture comprising a fragment of the CBP protein comprising the amino acid sequence of SEQ ID NO: 9 and P/CAF.
  • the system can comprise a cell extract produced from cells producing both CBP and P/CAF. Determination of the binding of CBP to P/CAF can be carried out as taught herein.
  • the present invention contemplates a method for inhibiting the transcription modulating activity of P/CAF in a subject, comprising administering to the subject a transcription modulating activity inhibiting amount of a substance in a pharmaceutically acceptable carrier.
  • the substance can be identified according to the protocols provided herein as one that can inhibit the transcription modulating activity of P/CAF by preventing the binding of P/CAF to p300/CBP or by inhibiting the histone acetyltransferase activity of P/CAF as well as by any other inhibitory mechanism as identified by the protocols provided herein.
  • Inhibition of the transcription modulating activity of P/CAF in a subject is desirable, for example, to inhibit HIV TAT-mediated transcription and therefore, the method of the present invention can be used to treat HIV-infected subjects.
  • the substance can be in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the substance, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
  • P/CAF can be inhibited in a subject by administering to the subject a substance which binds p300/CBP at the P/CAF binding site or a substance which binds the P/CAF protein at the p300/CBP binding site, the ultimate result being that P/CAF and p300/CBP do not bind with one another and P/CAF cannot exert its transcription modulating and/or histone acetyltransferase effect.
  • the substance can be a protein, such as an antibody which binds the P/CAF protein binding site at or near the p300/CBP binding site, thereby preventing its binding or an antibody which binds the p300/CBP protein at or near the P/CAF binding site, thereby preventing its binding.
  • the substance can also bind the histone acetyltransferase site on P/CAF or at the acetylation site on the histone, thereby preventing acetylation by P/CAF.
  • the substance which binds p300/CBP, the P/CAF protein or the histone and has the net effect of inhibiting the transcription modulating effect and or histone acetyltransferase activity of P/CAF in the cell can be delivered to a cell in the subject by mechanisms well known in the art.
  • nucleic acid encoding a protein which binds either to p300/CBP or the P/CAF protein and has the net effect of inhibiting the transcription modulating effect and/or histone acetyltransferase activity of P/CAF in the cell can be delivered to a cell in the subject by gene transduction mechanisms well known in the art.
  • nucleic acid can be introduced by liposomes as well as via retroviral or adeno-associated viral vectors, as described below.
  • the substance which inhibits the transcription modulating effect and/or histone acetyltransferase activity of P/CAF can be an antisense RNA or an antisense DNA which binds the RNA or DNA of P/CAF, thereby preventing translation or transcription of the RNA or DNA encoding P/CAF and having the net effect of inhibiting the transcription modulating effect and/or histone acetyltransferase activity of P/CAF by inhibiting P/CAF production.
  • the antisense RNA of the present invention can be generated from the nucleic acid of SEQ ID NO: 14 (human) or SEQ ID NO: 15 (mouse).
  • the antisense DNA can be a phosphorothioate oligodeoxyribonucleotide having the nucleotide sequence of SEQ LD NO: 16 (human) or of SEQ ID NO: 17 (mouse).
  • the mouse antisense RNA can be used to inhibit the activity of mouse P/CAF, having the nucleotide sequence of SEQ LD NO: 18 and the amino acid sequence of SEQ ID NO: 8.
  • the present invention also contemplates an antisense nucleic acid sequence which can bind the DNA or RNA of any of the transcription factors or other proteins now known or later identified to bind P/CAF, thereby inhibiting expression of the gene products of these proteins and having the net effect of inhibiting the transcription modulating effect and/or histone acetyltransferase activity of P/CAF.
  • the antisense nucleic acid can comprise a typical nucleic acid, but the antisense nucleic acid can also be a modified nucleic acid or a derivative of a nucleic acid such as a phosphorothioate analogue of a nucleic acid.
  • the composition can comprise, for example, an antisense RNA that specifically binds an RNA encoded by the gene encoding the serum protein. Antisense RNAs can be synthesized and used by standard methods (62).
  • Antisense RNA can inhibit gene expression by forming an RNA/RNA duplex between the antisense RNA and the RNA transcribed from the target gene.
  • the precise mechanism by which this duplex formation decreases the production of the protein encoded by the endogenous gene probably involves binding of complementary regions of the normal sense mRNA and the antisense RNA strand with duplex formation in a manner that blocks RNA processing and translation.
  • Alternative mechanisms include the formation of a triplex between the antisense RNA and duplex DNA or the formation of an DNA-RNA duplex with subsequent degradation of DNA-RNA hybrids by RNAse H.
  • an antigene effect can result from certain DNA-based oligonucleotides via triple-helix formation between the oligomer and double-stranded DNA which results in the repression of gene transcription.
  • the present invention results in inhibition of expression of the P/CAF gene by the introduced and replicated DNA resulting in inhibition of the transcription modulating and/or histone acetyltransferase activity of P/CAF, by a reduction in the expression of the nucleic acid to which the antisense nucleic acid is hybridized, and therefore a reduction of the gene product from the targeted gene.
  • the antisense nucleic acid may be obtained by any number of techniques known to one skilled in the art.
  • One method of constructing an antisense nucleic acid is to synthesize a recombinant antisense DNA molecule.
  • oligonucleotide synthesis procedures are routine in the art and oligonucleotides coding for a particular protein or regulatory region are readily obtainable through automated DNA synthesis.
  • a nucleic acid for one strand of a double-stranded molecule can be synthesized and hybridized to its complementary strand.
  • One can design these oligonucleotides such that the resulting double-stranded molecule has either internal restriction sites or appropriate 5' or 3' overhangs at the termini for cloning into an appropriate vector.
  • Double-stranded molecules coding for relatively large proteins or regulatory regions can be synthesized by first constructing several different double-stranded molecules that code for particular regions of the protein or regulatory region, followed by ligating these DNA molecules together. Once the appropriate DNA molecule is synthesized, this DNA can be cloned downstream of a promoter in an antisense orientation. Techniques such as this are routine in the art and are well documented.
  • An example of another method of obtaining an antisense nucleic acid is to isolate that nucleic acid from the organism in which it is found and clone it in an antisense orientation.
  • a DNA or cDNA library can be constructed and screened for the presence of the nucleic acid of interest. Methods of constructing and screening such libraries are well known in the art and kits for performing the construction and screening steps are commercially available (for example, Stratagene Cloning Systems, La Jolla, CA).
  • the nucleic acid can be directly cloned into an appropriate vector in an antisense orientation, or if necessary, be modified to facilitate the subsequent cloning steps. Such modification steps are routine, an example of which is the addition of oligonucleotide linkers which contain restriction sites to the termini of the nucleic acid.
  • General methods are set forth in Sambrook et al. (39).
  • the DNA that is introduced into the cell is in an expression orientation that is antisense to a corresponding endogenous DNA or RNA of the cells.
  • an endogenous DNA comprises a gene which encodes for a particular protein
  • the introduced DNA is in an expression orientation opposite the expression of the endogenous DNA; that is the DNA operatively linked to a promoter is in an antisense expression orientation relative to the corresponding endogenous gene.
  • the introduced DNA may be homologous to the entire transcribed gene or homologous to only part of the transcribed gene.
  • the sequence of the introduced DNA may be divergent to that of the endogenous DNA but only divergent to the extent that hybridization of the nucleic acids occurs, thereby preventing transcription.
  • One skilled in the art can determine the maximum extent of this divergence by routine screening of antisense DNAs corresponding to an endogenous DNA of the cell. In this manner, one skilled in the art can readily determine which fragments, or alternatively the extent of homology of the fragments or the entire gene that is necessary to inhibit gene expression.
  • the antisense nucleic acids of the present invention can be made according to protocols standard in the art, as well as described in the Examples provided herein.
  • the antisense nucleic acids can be administered to a subject according to the gene transduction protocols standard in the art, as described below.
  • the present invention also contemplates a method for stimulating the transcription modulating activity and/or histone acetyltransferase activity of P/CAF in a subject comprising administering to the subject a substance, in a pharmaceutically acceptable carrier, determined according to the methods taught herein, to have a stimulatory affect on the transcription modulating and/or histone acetyltransferase activity of P/CAF.
  • the substance can be one which has been identified, according to the protocols provided herein, to stimulate histone acetyltransferase activity in P/CAF or promote binding of P/CAF to p300/CBP.
  • the stimulation of the transcription modulation activity and/or histone acetyltransferase activity of P/CAF in a subject is desirable, for example, to activate tumor suppressor p53 (which promotes apoptosis) or to activate the muscle differentiation factor, MyoD.
  • the method of the present invention can be employed to treat cancer and to promote muscle differentiation in conditions where muscle differentiation is desired.
  • the substance can be delivered to a cell in the subject by mechanisms well known in the art.
  • a method for promoting binding of P/CAF to p300/CBP in a subject comprising administering to the subject a substance identified by the methods provided herein to promote binding of P/CAF to either p300 or CBP.
  • nucleic acid encoding a protein which stimulates the transcription modulating activity and/or histone acetyltransferase activity of P/CAF can be delivered to a cell in the subject by gene transduction mechanisms, as described below.
  • Also provided in the present invention is a method of inhibiting the cell cycle progression inducing effect of an oncoprotein which binds p300/CBP in a subject comprising transducing the cells of the subject with a vector comprising a nucleic acid encoding the P/CAF protein; inducing expression of the nucleic acid in the cell to produce the P/CAF in an amount which will allow the P/CAF gene product to replace the oncoprotein bound to p300/CBP, whereby the replacement of the oncoprotein bound to p300/CBP by the P/CAF gene product inhibits the cell cycle progression inducing effect of the oncoprotein.
  • the oncoprotein which binds p300/CBP in the cell can be the adenovirus El A oncoprotein.
  • a method for providing a functional P/CAF protein to a subject in need of the functional P/CAF protein comprising transducing the cells of the subject with a vector comprising a nucleic acid encoding the P/CAF protein and inducing expression of the nucleic acid to produce the functional P/CAF protein in the cell, thereby providing the functional P/CAF protein to the subject.
  • the transduction of the vector nucleic acid into the subject's cells can be carried out according to standard gene therapy protocols well known in the art (see, for example, U.S. Patent No. 5,339,346).
  • the present invention also provides a bioassay for screening substances for the ability to inhibit the histone acetyltransferase activity of p300/CBP comprising contacting a system, in which histone acetylation by p300/CBP can be determined, with the substance under conditions whereby histone acetylation by p300/CBP can occur; determining the amount of histone acetylation by p300/CBP in the presence of the substance; and comparing the amount of histone acetylation by p300/CBP in the presence of the substance with the amount of histone acetylation by p300/CBP in the absence of the substance, a decreased amount of histone acetylation by p300/CBP in the presence of the substance indicating a substance that can inhibit the histone acetyltransferase activity of p300/CBP.
  • the acetylation of histones by p300/CBP can be determined in a system including, for example, either core histones (histones H2A, H2B, H3 and H4) or the nucleosome core particles (146 base pairs of DNA wrapped around the octamer of core histones) as substrates, the P300/CBP protein and radiolabeled acetyl-CoA (e.g., [l- 14 C]acetyl CoA).
  • the presence of acetylated histones can be detected by autoradiography after separation by SDS-PAGE as described herein in the Examples.
  • the compound to be tested for the ability to inhibit the histone acetyltransferase activity of p300/CBP can be added to this system and assayed for acetyltransferase inhibiting ability.
  • a bioassay for screening substances for the ability to inhibit the binding of a transcriptional factor to p300/CBP comprising contacting a system in which the binding of a transcriptional factor to p300/CBP can be determined, with the substance under conditions whereby the binding of the transcriptional factor and p300/CBP can occur; determining the amount of transcriptional factor binding to p300/CBP in the presence of the substance; and comparing the amount of transcriptional factor binding to p300/CBP in the presence of the substance with the amount of transcriptional factor binding to p300/CBP in the absence of the substance, a decreased amount of transcriptional factor binding to p300/CBP in the presence of the substance indicating a substance that can inhibit the binding of a transcriptional factor to p300/CBP.
  • the binding of a transcriptional factor to p300/CBP can be determined in a system, for example, which can include a cell free reaction mixture comprising a transcriptional factor which binds p300/CBP and p300/CBP.
  • the system can comprise a cell extract produced from cells producing both a transcriptional factor which binds ⁇ 300/CBP and ⁇ 300/CBP.
  • the transcriptional factor which binds p300/CBP can be selected from, but is not limited to the group consisting of nuclear hormone receptors, CREB, c-Jun/v-Jun, c-Myb/v-Myb, YYI, Sap- la, c-Fos, MyoD and SRC-1, as well as any other transcriptional factor now known or later identified to bind p300/CBP.
  • the screening assay of the present invention can also be used to identify substances which inhibit the binding of p300/CBP to other components to which it is known to bind, for example, P/CAF, PP9U RSK , TFIIB, El A, SV40 large T antigen, as well as any other substances now known or later identified to bind p300/CBP. Determination of the binding of a transcriptional factor or other substance to p300/CBP can be carried out as taught in the Examples herein as well as by protocols described in the literature.
  • the present invention further contemplates a bioassay for screening substances for the ability to stimulate the histone acetyltransferase activity of p300/CBP comprising contacting a system, in which histone acetylation by p300/CBP can be determined, with the substance; determining the amount of histone acetylation by p300/CBP in the presence of the substance; and comparing the amount of histone acetylation by p300/CBP in the presence of the substance with the amount of histone acetylation by p300/CBP in the absence of the substance, an increased amount of histone acetylation by p300/CBP in the presence of the substance indicating a substance that can stimulate the histone acetyltransferase activity of p300/CBP.
  • the acetylation of histones by p300/CBP can be determined in a system including, for example, either core histones (histones H2A, H2B, H3 and H4) or the nucleosome core particles (146 base pairs of DNA wrapped around the octamer of core histones) as substrates, the p300/CBP protein and radiolabeled acetyl-CoA (e.g., [l- 14 C]acetyl CoA).
  • the presence of acetylated histones can be detected by autoradiography after separation by SDS-PAGE as described herein in the Examples.
  • the compound to be tested for the ability to stimulate the histone acetyltransferase activity of p300/CBP can be added to this system and assayed for stimulating ability.
  • the present invention further provides a bioassay for screening substances for the ability to stimulate binding of a component, which binds p300/CBP, to p300/CBP, comprising contacting a system in which the binding of the component to p300/CBP can be determined, with the substance under conditions whereby the binding of the component to p300/CBP can occur; determining the amount of component binding to p300/CBP in the presence of the substance; and comparing the amount of component binding to p300/CBP in the presence of the substance with the amount of component binding to p300/CBP in the absence of the substance, an increased amount of component binding to p300/CBP in the presence of the substance indicating a substance that can stimulate the binding of the component to p300/CBP.
  • the binding of the component to p300/CBP can be determined in a system, for example, which can include a cell free reaction mixture comprising the component and p300/CBP.
  • the system can comprise a cell extract produced from cells producing both the component and p300/CBP.
  • the component which binds p300/CBP can be any of the transcriptional factors or other proteins which are known or are identified in the future to bind p300/CBP, as set forth above. Determination of the binding of the component to p300/CBP can be carried out as taught in the Examples provided herein and according to protocols available in the literature.
  • a method for inhibiting the histone acetyltransferase activity of p300/CBP in a subject comprising administering to the subject a histone acetyltransferase activity inhibiting amount of a substance in a pharmaceutically acceptable carrier.
  • the mechanism of the inhibitory action of the substance can be the inhibition of the binding of a DNA-binding transcription factor, such as, for example, a nuclear hormone receptor, CREB, c-Jun/v-Jun, c-Myb/v-Myb, YY1, Sap- la, c-Fos, MyoD or SRC-1, to p300/CBP.
  • the histone acetyltransferase activity of p300/CBP can be inhibited in a subject by administering to the subject a substance which binds p300/CBP at the transcription factor binding site or a substance which binds the transcription factor protein at the p300/CBP binding site, the ultimate result being that the transcription factor and p300/CBP do not bind with one another and p300/CBP cannot acetylate histones.
  • the substance which binds either to the transcription factor or the p300/CBP protein and has the net effect of inhibiting the histone acetyltransferase activity of p300/CBP in the cell can be identified according to the screening methods provided herein and delivered to a cell in the subject by mechanisms well known in the art.
  • the substance can be a protein, such as an antibody which binds the p300/CBP protein binding site at or near the DNA-binding transcription factor binding site, thereby preventing its binding or an antibody which binds the DNA-binding transcription factor at or near the p300/CBP binding site, thereby preventing its binding.
  • the substance can also bind the histone acetyltransferase site on p300/CBP (aa 1195-1673 on p300 or aa 1174-1850 on CBP) or at the acetylation site on the histone, thereby preventing acetylation by p300/CBP.
  • the substance can be a nucleic acid which can be expressed in the cell to produce a protein which inhibits the histone acetyltransferase activity of p300/CBP.
  • a nucleic acid encoding a protein which binds either to a transcription factor or the p300/CBP protein and has the net effect of inhibiting the histone acetyltransferase activity of p300/CBP in the cell can be delivered to a cell in the subject by gene transduction mechanisms well known in the art.
  • nucleic acid can be introduced by liposomes as well as via retroviral or adeno-associated viral vectors, as described below.
  • the substance which inhibits the histone acetyltransferase activity of p300/CBP can be an antisense RNA or an antisense DNA which binds the RNA or DNA of p300/CBP thereby preventing translation or transcription of the RNA or DNA encoding p300/CBP and having the net effect of inhibiting the histone acetyltransferase activity of P/CAF by inhibiting p300/CBP production.
  • the antisense RNA or DNA of the present invention can be produced and introduced into cells according to the same methods as set forth above for P/CAF antisense nucleic acids.
  • the present invention also contemplates a method for stimulating the histone acetyltransferase activity of p300/CBP in a subject comprising administering to the subject a histone acetyltransferase activity stimulating amount of a substance, in a pharmaceutically acceptable carrier, determined according to the methods taught herein, to have a stimulatory affect on the histone acetyltransferase activity of p300/CBP.
  • the substance can exert a stimulatory effect by promoting the binding of a DNA-binding transcription factor of the present invention to p300/CBP.
  • the substance can be delivered to a cell in the subject by mechanisms well known in the art.
  • a nucleic acid encoding a protein which stimulates the transcription modulating activity of p300/CBP can be delivered to a cell in the subject by gene transduction mechanisms, as described below.
  • the nucleic acids of the present invention can be in a vector for delivering the nucleic acids to the site for expression of the P/CAF protein.
  • the vector can be one of the commercially available preparations, such as the pGM plasmid (Promega).
  • Vector delivery can be by liposome, using commercially available liposome preparations or newly developed liposomes having the features of the present liposomes.
  • vector delivery can be via a viral system, including, but not limited to, retroviral, adenoviral and adeno-associated viral systems. Other delivery methods can be adopted and routinely tested according to the methods taught herein.
  • the modes of administration of the liposome will vary predictably according to the disease being treated and the tissue being targeted. For example, for treating cancer in either the lung or the liver, which are both sinks for liposomes, intravenous delivery is reasonable. For other localized cancers, as well as precancerous conditions, catheterization of an artery upstream from the target organ is a preferred mode of delivery, because it avoids significant clearance of the liposome by the lung and liver. For cancerous lesions at a number of other sites (e.g., skin cancer, localized dysplasias), topical delivery is expected to be effective and may be preferred, because of its convenience. Leukemias and other disorders involving dysregulated proliferation of certain isolatable cell populations may be more readily treated by ex vivo administration of the nucleic acid
  • the liposomes may be administered topically, parenterally (e g , intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally or the like, although intravenous or topical administration is typically preferred
  • parenterally e g , intravenously
  • intramuscular injection by intraperitoneal injection, transdermally, extracorporeally or the like
  • intravenous or topical administration is typically preferred
  • the exact amount of the liposomes required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disease being treated, the particular compound used, its mode of administration and the like Thus, it is not possible to specify an exact amount However, an appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein
  • Parenteral administration if used, is generally characterized by injection
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant level of dosage is maintained See, e.g , U.S Patent No 3,610,795, which is incorporated by reference herein
  • Topical administration can be by creams, gels, suppositories and the like
  • Ex vivo (extracorporeal) delivery can be as typically used in other contexts
  • CBP binds to c-Jun in a phosphorylation-dependent manner in association with stimulation of transcription (9).
  • GCN4 is believed to be a c- Jun counterpart on the basis of similarities in DNA recognition (15) as well as the participation of both proteins in UV signaling pathways (16).
  • Yeast genetic screening has led to the isolation of various cofactors for GCN4, including GCN5 (yGCN5), ADA2 (yADA2) and AD A3 (yADA3) (17-19). These factors are considered to function as a complex (or in a common pathway) based on genetic and protein-protein interaction studies (18-22).
  • p300/CBP and yADA2 exhibit significant sequence similarity within a 50 amino acid region including a Zn 2+ finger motif (3). Human counterparts to yGCN5, yADA2, or yADA3 that interact with p300/CBP to mediate transcriptional activation by c-Jun were searched for in various nucleotide sequence databases.
  • human fetal liver and fetal brain cDNA libraries (Clontech) were screened with ETS05039 and NLB2000-5R, respectively and complete clones were isolated from the human fetal liver cDNA library.
  • the former encodes an N-terminal region with no sequence similarity to any proteins in the databases besides the yGCN5- related C-terminal region, whereas the latter encodes only the yGCN5-related region.
  • P/CAF p300/CBP-associated factor
  • human GCN5 hGCN5
  • RNA blotting indicated that transcripts detected by the P/CAF and hGCN5 cDNAs are ubiquitously expressed, but the former is most abundant in heart and skeletal muscle, whereas the latter is most abundant in pancreas and skeletal muscle.
  • the P/CAF binding site was presumed to reside in the C terminal one third of CBP (residues 1,678-2,442) because it was observed that this region, when fused to a DNA binding domain, activates transcription (4) in a manner repressed by coexpression of l2S ElA.
  • This region was divided into 6 overlapping fragments and each was expressed in E. coli as a glutathione-S-transferase (GST) fusion protein.
  • GST-CBP fusions were incubated with recombinant P/CAF protein and, subsequently, purified using glutathione-Sepharose. Co-purified P/CAF was detected by immunoblotting analysis.
  • CBP and p300 were amplified by PCR.
  • a series of deletions of the CBP segment B was created by site-directed in vitro mutagenesis (30). These fragments were subcloned into pGEX-2T (Pharmacia). GST- fusions were expressed in E.
  • buffer B [20 mM Tris-HCl (pH 8.0), 5 mM MgCl 2 , 10% glycerol, 1 mM AEBSF, 0.1% NP40, 10 ⁇ g/ml of aprotinin, 10 ⁇ g/ml of leupeptin, 1 ⁇ g/ml of pepstatin A, 1 mM DTT] containing 0.1 M KCl for these experiments.
  • GST-CBP-segment B was purified by glutathione-Sepharose and phenyl- Sepharose chromatographic steps, P/CAF, hGCN5, and El A were expressed as FLAG- fusions in Sf9 cells via baculovirus vectors and affinity-purified with M2-agarose (ref. 30; Kodak-LBI).
  • P/CAF, hGCN5, and El A were expressed as FLAG- fusions in Sf9 cells via baculovirus vectors and affinity-purified with M2-agarose (ref. 30; Kodak-LBI).
  • M2-agarose ref. 30; Kodak-LBI
  • a crude E. coli extract containing 20 pmol of GST- fusion was incubated with 40-60 pmol of P/CAF or El A in a total volume of 50 ⁇ l of buffer B with 0.1 M KCl on ice for 10 min.
  • segment B' the segment spanning residues 1763-1966 (segment B') of p300, which is analogous to the CBP segment-B, was used. Twenty percent of the P/CAF and hGCN5 inputs and 100% of the El A input were also analyzed. In the GST precipitation assays, almost identical amounts of the GST fusions were recovered in all samples. Interaction between P/CAF and CBP (segment B) was determined in the absence and in the presence of El A. Control reactions with GST-CBP alone and without GST-CBP were also performed. Input proteins were analyzed.
  • hADA2 a human structural homolog of yADA2, termed hADA2 (25) has revealed that, unlike the sequence similarity between p300/CBP and yADA2, which is restricted to a 50 amino acid region, hADA2 shares extensive similarity (30% identity, 52% similarity) to yADA2 over the entire protein sequence.
  • a computer search of the complete genomic sequence of Saccharomyces cerevisiae revealed that yeast does not have counterparts of p300/CBP or P/CAF.
  • the p300/CBP-P/CAF pathway may have been acquired during metazoan evolution.
  • Osteosarcoma cells were transfected with either control vector or El A- or ElA ⁇ N-expression vectors. Extract from the transfected subpopulation was immunoprecipitated with anti-P/CAF or control IgG. The precipitates were analyzed by immunoblotting with anti-p300 and anti-P/CAF antibodies.
  • Rabbit anti-P/CAF antibody was raised to the P/CAF segment spanning residues 125-397 and purified by immunoaffinity chromatography (33).
  • a mixture of monoclonal antibodies raised to the human p300 segment spanning residues 1572-2371 (5) and rabbit polyclonal antibodies raised to the mouse CBP segment spanning residues 2-23 (for immunoprecipitation) and 1736-2179 (immunoblotting) were purchased from Upstate Biotechnology.
  • Anti-P/CAF antibody specifically detected a 95 kDa protein, which is very close to the calculated value for the full-length P/CAF, in the immunoprecipitates.
  • Anti- P/CAF antibody co-immunoprecipitated both CBP and p300.
  • anti-CBP antibody also co-immunoprecipitated P/CAF.
  • anti-p300 antibody did not co- immunoprecipitate P/CAF. This is most likely due to steric interference since the anti- p300 antibody was raised to the p300 segment spanning residues 1572-2371 which includes the P/CAF binding region.
  • HeLa cells were transfected by electroporation with 7 ⁇ g of P/CAF-expression plasmid and/or 3 ⁇ g of the full-length or the N-terminally deleted ( ⁇ 2-36) E1A 12S- expression plasmid as indicated. These plasmids were constructed by subcloning FLAG-P/CAF and El A cDNAs into pCX (34) and pcDNAI (Invitrogen), respectively. All samples, in addition, contained 1 ⁇ g of sorting plasmid (pCMV-IL2R) (31) and carrier plasmid (pCX) to normalize the total amount of DNA to 11 ⁇ g.
  • sorting plasmid pCMV-IL2R
  • pCX carrier plasmid
  • transfected subpopulation was purified by magnetic affinity cell sorting and nuclei were analyzed by dual parameter flow cytometry as described (32).
  • Histone acetyltransferase activity in P/CAF Acetylation of the N-terminal histone tails has been considered to play a crucial role in accessibility of transcription factors to nucleosomal templates (26-27).
  • yGCN5 has been identified as a histone acetyltransferase (28).
  • intrinsic histone acetyltransferase activity in P/CAF and hGCN5 was examined.
  • the core histones histones H2A, H2B, H3 and H4
  • nucleosome core particles 146 base pairs of DNA wrapped around the octamer of core histones
  • hGCN5 and P/CAF that acetylates free histones or histones in the nucleosome core particle (35) was measured as described (36).
  • Each reaction contained 0.3 pmol of affinity purified FLAG-hGCN5 or FLAG-P/CAF, 4 pmol of the histone octamer or the nucleosome core particle and 10 pmol of [l- 14 C]acetyl-CoA.
  • the histone octamer dissociated into dimers or tetramers under assay conditions. Acetylated histones were detected by autoradiography after separation by SDS-PAGE.
  • Direct function of P/CAF is likely to involve its intrinsic histone acetyltransferase activity. Although exact molecular mechanisms by which acetylation of core histones contribute to transcription remains undefined, acetylation of the histones is considered to play an important role in transcriptional regulation (26-27).
  • the positively charged N- terminal tails of core histones are believed to affect nucleosome structure by interacting with DNA at or near the nucleosome-spacer junction. Acetylation of the histone tails presumably destabilizes the nucleosome and facilitates access by regulatory factors.
  • there is a general correlation between the level of acetylation and transcriptional activity of nucleosomal domains. The findings of the present invention provide insights into the mechanisms of targeted histone acetylation.
  • Cellular factor p300/CBP binds to various sequence-specific factors that are involved in cell growth and/or differentiation, including CREB (3,4), c-Jun (9), Fos (11), c-Myb (12) and nuclear receptors (13). P/CAF could stimulate the activation function of these factors via promoter-specific histone acetylation.
  • the present invention demonstrates that El A appears to perturb normal cellular regulation by disrupting the connection between p300/CBP and its associated histone acetyltransferase.
  • FLAG-epitope tagged El A (or ⁇ E1A) was expressed in Sf9 cells (ATCC accession number CRL 1711) by infecting recombinant baculovirus (43). All purification steps were carried out at 4°C. Extract was prepared from infected cells by one cycle of freeze and thaw in buffer B (20 mM Tris-HCl, pH 8.0; 5 mM MgCl 2 ; 10% glycerol; 1 mM PMSF; 10 mM ⁇ -mercaptoethanol; 0.1% Tween 20) containing 0.1 M KCl and the complete protease inhibitor cocktail (Boehringer Mannheim).
  • the extract was incubated with M2 anti-FLAG antibody agarose (Kodak-LBI) for four hours with rotating and subsequently washed with the same buffer three times.
  • the resulting beads were incubated with HeLa (ATCC accession number CCL 2) nuclear extract for four to eight hours and thereafter washed with the same buffer six times.
  • FLAG-E1 A was eluted from the beads along with associated polypeptides by incubating with the same buffer containing 0.1 mg/ml FLAG peptide.
  • eluted polypeptides were dialyzed in 0.05 M KCl-buffer B and subsequently loaded onto a SMART Mono Q column (Pharmacia) equilibrated with the same 0.05 M KCl-buffer B. After washing, the column was developed with a linear gradient of 0.05-1.0 M KCl in buffer B. Mono Q fractions were concentrated with a MICROCON spin-filter (Amicon) and consequently loaded onto a SMART Superdex 200 column (Pharmacia) equilibrated with 0.1 M KCl-buffer B.
  • Filter binding assays were performed as described (80) with minor modifications. Samples were incubated at 30°C for 10-60 minutes in 30 ml of assay buffer containing 50 mM Tris-HCl, pH 8.0; 10% glycerol; 1 mM DTT; 1 mM PMSF; 10 mM sodium butyrate; 6 pmol of [ 3 H]acetyl CoA (4.3 mCi/mmole, Amersham Life Science Inc.); and 33 mg/ml of calf thymus histones (Sigma Chemical Co.). In experiments where synthetic peptides were substituted for core histones, 50 pmol of each peptide were used.
  • reaction mixture was spotted onto Whatman P-81 phosphocellulose filter paper and washed for 30 minutes with 0.2 M sodium carbonate buffer pH 9.2 at room temperature with 2-3 changes of the buffer.
  • the dried filters were counted in a liquid scintillation counter.
  • cDNAs corresponding to p300 portions of aa 1-670, aa 671-1194 and aa 1135-2414 were amplified by PCR (EXPAND High Fidelity PCR System; Boehringer Mannheim) as Kpnl-Notl fragments. The resulting fragments were subcloned into a baculovirus transfer vector having the FLAG-tag sequence (43).
  • the recombinant viruses were isolated using the BACULOGOLD system (Pharmingen), according to the manufacturer's protocol and were infected into Sf9 cells (ATCC accession number CRL 1711) to express FLAG-p300. Recombinant proteins were affinity purified with M2 anti-FLAG antibody-immobilized agarose (Kodak-LBI) according to the manufacturer's protocol.
  • cDNAs encoding the p300 portions and the CBP portion were first subcloned into the baculovirus transfer vector having the FLAG-tag as described above. Thereafter, the Xhol and Notl fragments encoding FLAG-p300 or FLAG-CBP fusions were resubcloned into the E. coli expression vector pET-28c (Novagene) digested with Sail and Notl. Recombinant proteins were expressed in E. coli BL21(DE3) and affinity purified with M2-antibody agarose.
  • E1A 12S protein inhibits transcription in a variety of genes via direct binding to p300/CBP (45)
  • El A also stimulates transcription in some contexts (46).
  • p300/CBP-bound El A was tested to determine whether it might recruit histone acetyltransferases or deacetylases to regulate transcription.
  • experiments were conducted as described below to determine if p300/CBP per se is a histone acetyltransferase.
  • affinity purified El A-binding polypeptides were separated by Mono Q ion-exchange column. Both p300/CBP and the acetyltransferase activity were coeluted at 140 mM KCl, while most of polypeptides were eluted at 260 mM KCl. The active fraction of Mono Q column (-140 mM KCl) was further separated by Superdex-200 gel filtration column. Both p300/CBP and the acetyltransferase activity coeluted after the void volume, indicating that p300/CBP is involved in the histone acetyltransferase activity.
  • p300 is a histone acetyltransferase
  • p300 per se or a polypeptide(s) associated with p300, possesses histone acetyltransferase activity.
  • the acetyltransferase activity of recombinant p300 was measured.
  • p300 was divided into three fragments, each of which was expressed in and purified from Sf9 cells via a baculovirus expression vector.
  • Histone acetyltransferase activity was readily detected in the C-terminal fragment containing amino acids 1135-2414, whereas no activity was found in the other fragments, demonstrating conclusively that p300 per se is a histone acetyltransferase.
  • p300/CBP-histone acetyltransferase domain
  • acetyltransferases including histone acetyltransferases GCN5 and
  • Substrate specificity for acetylation by p300 was also examined.
  • histone octamers and mononucleosomes 146 base pairs of DNA wrapped around the octamer of core histones
  • the histone octamer dissociates into dimers or tetramers under physiological conditions
  • the histone octamer is referred to here as core histones.
  • core histones were used, p300 acetylated all four proteins, but preferentially H3 and H4. More importantly, in a nucleosomal context, p300 acetylated all four core histones nearly stoichiometrically. In contrast, p300 acetylated neither BSA nor lysozyme.
  • Hyperacetylated histones are believed to be linked with transcriptionally active chromatin (26,27,50,51). Hyperacetylated forms are found in histones H4, H3 and H2B, which have multiple acetylation sites in vivo. Thus, the level of acetylation by p300 was also tested.
  • Mononucleosomes treated with p300 were analyzed by two-dimensional gel electrophoresis. A Coomassie blue-stained gel and the corresponding autoradiogram showed that a significant amount of histones, especially H4, were hyperacetylated. Importantly, acetylation levels by p300 were very close to those of hyperacetylated histones prepared from HeLa nuclei treated with sodium butyrate, a histone deacetylase inhibitor. In contrast, no acetylated forms were detected in the reaction without p300. These results indicate that p300 acetylates histones in mononucleosomes to the hyperacetylated state by targeting multiple lysine residues.
  • p300 acetylates the four lysines in the histone H4 N-terminal tail in vitro which are acetylated in vivo
  • Lysines at positions 5, 8, 12 and 16 of histone H4 are acetylated in vivo (51).
  • Recent studies with yeast histone acetyltransferases demonstrate the position-specific acetylation by distinct acetyltransferases, i.e., while cytoplasmic acetyltransferases for histone deposition and chromatin assembly modify positions 5 and 12, GCN5 modifies positions 8 and 16 (52). Accordingly, the positions of acetylation by p300 were also determined.
  • a series of synthetic peptides containing acetylated lysines at various positions was used to determine the acetylation site-specificity of p300.
  • p300 preferentially acetylates the N-terminal histone tail
  • Histone acetyltransferases modify specific lysine residues in the N-terminal tail of core histones but not the C-terminal globular domain in vivo (26,27,50,51).
  • Structural models of nucleosomes suggest that most of the lysine residues in the C-terminal globular domain are buried. Therefore, experiments were conducted to examine whether restricted acetylation of the N-terminal tail resulted from the substrate specificity of the enzyme or inaccessibility of the enzyme to the core domain in nucleosomes.
  • the globular domains of all core histones contain a long helix flanked on either side by a loop segment and short helix, termed the "histone fold" (54,55,56).
  • the histone fold is involved in formation of the stable H2A-H2B and H3-H4 hetero-dimers, consisting of extensive hydrophobic contacts between the paired molecules. Therefore, it is likely that a histone monomer cannot fold properly, thereby increasing access of the histone acetyltransferase to the core domain. Based on these considerations, experiments were conducted to determine whether p300 acetylates free histone H4 in a N-terminal-specific manner.
  • Histone H4 was acetylated with p300 and subsequently the histone tail was removed by partial digestion with trypsin. The distributions of radioactivity between intact and core histones were compared. While the globular core histone domain was predominant at the higher trypsin concentrations, radioactivity was detected mostly in the intact histone. These data demonstrate that p300 preferentially acetylates the N-terminal tail of histone H4. HI. P/CAF interaction with MyoD
  • C 2 C 12 mouse cells (ATCC accession number CRL 1772) were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum (FBS) until they reached confluence. Differentiation was induced by switching medium to differentiation medium (DM), consisting of DMEM containing 2% horse serum.
  • DM differentiation medium
  • C 3 H/10Tl/2 fibroblasts (ATCC accession number CCL 226) were grown in DMEM supplemented with 10% FBS. Cells were transfected by the calcium phosphate precipitation method. Total amounts of transfected DNA were equalized by empty vector DNA.
  • CAT Chloramphenicol acetyltransferase
  • Expression vectors used for transfection experiments are as follows: pCX-P/CAF for P/CAF (43); pCMV-bp300 for p300 (65), pCMV-p300 (1869-2414) (64) and pCMV-p300 (1514-1922) (60) for p300 wild type and mutants; pElA12S, pEl A12S R2G, pEl A12S D2-36 and pEl A12S D121-130 for El A wild type and mutants (66,67,68); and pEMSV-MyoD for MyoD (64).
  • the antisense P/CAF RNA expression vector, pcDNA3 P/CAF-AS, was created as follows. The 2.5 Kb EcoRI-Kpnl fragment containing the entire P/CAF open reading frame was isolated from pCX-P/CAF (43). This fragment was subcloned into the EcoRI-Kpnl sites of plasmid pcDNA3 (Invitrogen) so that the antisense P/CAF RNA is driven under the CMV promoter. Reporter genes employed were 4RE-CAT and MCK-CAT (69). 4RE-CAT is driven by a synthetic promoter containing 4 copies of the E-box, whereas MCK-CAT is driven by the native MCK promoter (nucleotides -1256 to +7).
  • Cells were grown on small glass slides, subdivided into numbered squares of 2 mm x 2 mm and microinjected with purified and concentrated antibodies, as previously described (70).
  • cells were fixed in either 2% paraformaldehyde or 1 :2 methanol/acetone solution, preincubated with 5% BS A/PBS and incubated with the primary antibodies for 30 min at 37° C. Subsequently, antibody was visualized by incubating with either rhodamine- or fluorescein-conjugated secondary antibody for 30 min at 37° C. Injected antibodies were stained with a rhodamine-conjugated secondary antibody and nuclei were counter-stained by DAPI as previously described (69).
  • Antibodies employed are as follows; rabbit polyclonal affinity purified anti-P/CAF antibody (43), rabbit polyclonal anti-p300/CBP antiserum (71), mouse monoclonal anti-MyoD antibody (clone 5.8 A, kindly provided by P. Houghton), goat polyclonal anti-c-Jun affinity purified antibody (Santa Cruz) and rabbit pre-immune serum.
  • Cells were resuspended in lysis buffer (20 mM NaPO 4 , 150 mM NaCl, 5mM MgCl 2 , 0.1% NP40, 1 mM DTT, 10 mM sodium fluoride, 0.1 mM sodium vanadate, 1 mM phenylmethylsulfonyl-fluoride and 10 mg/ml each of leupeptin, aprotinin and pepstatin). After 30 min incubation on ice, samples were centrifuged at 12,000 x g for 30 min and supematants were used as cell extracts.
  • lysis buffer 20 mM NaPO 4 , 150 mM NaCl, 5mM MgCl 2 , 0.1% NP40, 1 mM DTT, 10 mM sodium fluoride, 0.1 mM sodium vanadate, 1 mM phenylmethylsulfonyl-fluoride and 10 mg/ml each of leupeptin, a
  • Extracts were pre-cleared by incubating with rabbit pre-immune serum and protein A/G Plus- Agarose (Santa Cruz) for 2 h at 4 C.
  • protein A/G Plus- Agarose serum and protein A/G Plus- Agarose (Santa Cruz) for 2 h at 4 C.
  • the supematants were incubated with the respective antibodies for 3 h at 4 C.
  • Protein A/G Plus-Agarose was added, and incubation continued for 3 h.
  • the matrix was washed with lysis buffer, then boiled in 2 X SDS sample buffer. Immunoblotting was performed by using the ECL chemiluminescent detection kit (Amersham) according to the manufacturer's protocol.
  • E-box-bound complexes were done as previously described (69). Briefly, 100 ng of the biotinylated double stranded DNA containing the E-box were immobilized on streptavidin-conjugated magnetic beads and incubated with 500 mg of cell extracts in the presence of poly dl-dC. After extensive washing, bound proteins were eluted with SDS sample buffer and analyzed by immunoblotting.
  • MyoD and El A (43) were expressed as FLAG-fusion proteins in Sf9 cells via a baculovirus expression system and affinity-purified on M2 anti-FLAG antibody-agarose (Kodak-LBI). Crude E. coli extracts containing GST-fusions were incubated with various amounts of MyoD and/or El A in 50 ml of buffer B (20 mM Tris-HCl, pH 8.0, 0.1 M KCl, 5 mM MgCl 2 , 10% glycerol, and 0.1% Nonidet P-40) on ice for 10 min. GST-precipitation was performed as described (43). MyoD and El A were detected by immunoblotting with anti-FLAG M2 antibody.
  • buffer B (20 mM Tris-HCl, pH 8.0, 0.1 M KCl, 5 mM MgCl 2 , 10% glycerol, and 0.1% Nonidet P-40
  • the proteins were resolved on a 4%-20% gradient SDS-PAGE and subjected to immunoblotting with the anti-FLAG M2 antibody.
  • the blot was developed with the SUPERSIGNAL chemiluminescent substrates (Pierce). P/CAF coactivates muscle-specific transcription
  • P/CAF and MyoD were co-transfected into mouse C3H10T1/2 fibroblasts, and MyoD-mediated transcription was determined from reporter activity driven by the artificial (4RE) and the naturally-occurring muscle creatine kinase (MCK) promoters.
  • MCK creatine kinase
  • Overexpression of P/CAF stimulated MyoD-dependent transcription several folds in both promoters. Similar results were obtained for the myoD activated myogenin promoter Transcriptional activation was further stimulated by co-transfecting with MyoD, P/CAF and p300 expression vectors, suggesting that P/CAF may function by forming a complex with p300/CBP.
  • P/CAF As a myogenic coactivator in a more relevant environment, P/CAF was overexpressed in proliferating C2C12 myoblasts which express endogenous myogenic bHLH factors. As observed in fibroblasts, overexpression of P/CAF stimulated muscle specific transcription. Concomitant expression of exogenous p300 increased P/CAF-mediated coactivation. The repression exerted by wild type El A, but not mutant El A D2-36, on P/CAF coactivation of MyoD was also observed in muscle cells.
  • C3H10T1/2 fibroblasts were transiently transfected with P/CAF and MyoD expression vectors.
  • An expression vector for the green fluorescent protein (GFP) was co-transfected to identify transfected cells. After incubation in differentiation medium, the myogenic conversion of transfected cells was determined by simultaneous expression of the GFP and the differentiation-specific marker myosin heavy chain (MHC). Forced expression of MyoD in fibroblasts caused muscle differentiation in 12% of the transfected fibroblasts. This myogenic conversion was 20% by co-expressing MyoD and P/CAF.
  • GFP green fluorescent protein
  • MHC differentiation-specific marker myosin heavy chain
  • P/CAF is essential for myogenic transcription and differentiation
  • Microinjection of anti-P/CAF or anti-p300/CBP antibody did not interfere with induction of p53 by DNA damaging agents, showing specificity of the inhibition by the antibodies.
  • the injection of anti-MyoD antibody only partially inhibited differentiation, supporting the view of functional redundancy between MyoD and Myf-5 (75,76).
  • Injection of anti-c-Jun antibody or control antibody did not interfere with muscle differentiation.
  • RGFGIGS is an amino acid sequence required for acetyl coenzyme A binding and activity of human spermidine/spermine Nl acetyltransferase. 271:18920-18924 (1996).
  • Asp Asp lie Ser Gly Tyr Lys Glu Asn Tyr Thr Arg Trp Leu Cys Tyr
  • Val lie Asn Pro Pro Pro Val Ala Gly Thr lie Ser Tyr Asn Ser Thr 35 40 45 Ser Ser Ser Leu Glu Gin Pro Asn Ala Gly Ser Ser Ser Pro Ala Cys
  • Tyr Ala Asp Glu Tyr Ala lie Gly Tyr Phe Lys Lys Gin Gly Phe Ser
  • Lys Glu lie Lys lie Pro Lys Thr Lys Tyr Val Gly Tyr lie Lys Asp
  • Cys Pro lie Cys Lys Gin Leu lie Ala Leu Cys Cys Tyr His Ala Lys
  • Asp Asp lie Ser Gly Tyr Lys Glu Asn Tyr Thr Arg Trp Leu Cys Tyr
  • Tyr lie Ala Arg Leu Val Phe Asp Pro Lys His Lys Thr Leu Ala Leu 180 185 190 He Lys Asp Gly Arg Val He Gly Gly He Cys Phe Arg Met Phe Pro

Abstract

Cette invention se rapporte à une protéine purifiée appelée P/CAF ayant un poids moléculaire d'environ 93000 daltons, déterminé par électrophorèse sur gel de polyacrylamide au dodécylsulfate de sodium dans des conditions réductrices, qui provoque l'acétylation des histones et qui se fixe également à la protéine cellulaire p300/CBP. Cette invention se rapporte également à un acide nucléique codant cette protéine P/CAF, à un vecteur contenant cet acide nucléique et à un hôte pour ce vecteur, ainsi qu'à un anticorps purifié qui fixe spécifiquement la protéine P/CAF, ainsi qu'à des procédés de criblage de composés qui inhibent ou stimulent la modulation de la transcription et l'activité d'histone-acétyltransférase des protéines P/CAF et p300/CBP.
PCT/US1997/012877 1996-07-23 1997-07-23 Cofacteur de transcription p/caf associe a p300/cbp et utilisations WO1998003652A2 (fr)

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US6562589B1 (en) * 1997-06-17 2003-05-13 The United States Of America As Represented By The Department Of Health And Human Services AIB1, a novel steroid receptor co-activator
AU9550198A (en) 1997-10-21 1999-05-10 University Court Of The University Of Glasgow, The Jmy, a co-activator for p300/cbp, nucleic acid encoding jmy and uses thereof
GB9810562D0 (en) * 1998-05-15 1998-07-15 Cancer Res Campaign Tech Assays ,methods and means for modulating transcription
ATE283915T1 (de) 1998-05-26 2004-12-15 Inst Of Molecul & Cell Biology Polypeptide aus dem creb-bindenden protein und verwandten protein p-300 zur verwendung bei der transkriptionellen regulation
CA2386299A1 (fr) * 1999-10-08 2001-04-19 Merck & Co., Inc. Inhibiteurs d'histone acetyle transferase antiprotozoaire
US7919276B1 (en) * 2000-02-22 2011-04-05 Mount Sinai School Of Medicine ZA loops of bromodomains
EP1136567A1 (fr) 2000-03-24 2001-09-26 Advanced Array Technologies S.A. Procédé et trousse pour l'identification, la détection et/ou la quantification des facteurs de transcription
US7407748B2 (en) 2000-03-24 2008-08-05 Eppendorf Array Technologies S.A. Method and kit for the determination of cellular activation profiles
AU2001270558A1 (en) * 2000-06-27 2002-01-08 Bayer Cropscience Gmbh Histone acetyltransferase inhibitors and their use as insecticides
EP1499714A2 (fr) * 2001-02-05 2005-01-26 Bayer HealthCare AG Regulation de l'histone acetyltransferase humaine
US20070036810A1 (en) * 2003-03-26 2007-02-15 Vadim Iourgenko Cyclic amp response element activator proteins and uses related thereto
EP1660502A2 (fr) 2003-08-28 2006-05-31 Choongwae Pharma Corporation MODULATION DE LA TRANSCRIPTION ACTIVEE PAR ß-CATENINE/TCF
CN110170052B (zh) * 2019-06-21 2020-07-10 复旦大学 Cbp-p300抑制剂在肠道损伤疾病中的应用
CN113736780B (zh) * 2020-05-28 2024-05-28 暨南大学 一种敲除p300基因的BCBL1细胞系及其构建方法与应用

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