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WO1997033900A1 - Proteine fixant e2a - Google Patents

Proteine fixant e2a Download PDF

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Publication number
WO1997033900A1
WO1997033900A1 PCT/US1997/004117 US9704117W WO9733900A1 WO 1997033900 A1 WO1997033900 A1 WO 1997033900A1 US 9704117 W US9704117 W US 9704117W WO 9733900 A1 WO9733900 A1 WO 9733900A1
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Mu-En Lee
Edgar Haber
Wilson O. Endege
Matthew D. Layne
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President And Fellows Of Harvard College
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to regulation of gene expression and differentiation in vascular smooth muscle cells.
  • E12 and E47 which are alternatively spliced products of the E2A gene (Sun et al . , Mol. Cell. Biol. 11:5603-11, 1991), belong to a growing family of transcription factors characterized by a highly conserved helix-loop-helix (HLH) motif (Kadesch, Cell. Growth Differ. 4:49-55, 1993; Kadesch, Immunol. Today 13:31-6, 1992) .
  • HLH helix-loop-helix
  • E2A proteins are widely expressed.
  • E47 forms homodimers, while both E12 and E47 heterodimerize with tissue-specific HLH factors (Hsu et al . , Proc. Natl. Acad. Sci. USA 91:3181-5, 1994; Johnson et al . , Proc. Natl. Acad. Sci. USA 89:3596-600, 1992; Shirakata, Genes & Dev. 7:2456-70, 1993; Sun et al . , Mol. Cell. Biol. 11:5603-11, 1991; Shirakata, EMBO J. 14:1766-72, 1995).
  • most HLH proteins Immediately adjacent to the HLH domain, most HLH proteins contain a basic region that allows homodimers or heterodi ers to bind the CANNTG consensus sequence, which is known as the E-box
  • Idl, Id2, Id3, and Id4 members of the Id family (Idl, Id2, Id3, and Id4) and the Drosophila extramacrochaetae (emc) protein do not contain the basic DNA-binding region. Heterodimers of Id and other HLH proteins do not bind DNA, and thus Id proteins function as inhibitors of DNA binding. In vitro, Idl and Id2 have higher affinity for E2A proteins than MyoD and they may dissociate functional MyoD-E2A heterodimers (Benezra et al . , Cell 61:49-59, 1990; Sun et al . , Mol. Cell. Biol. 11:5603-11, 1991).
  • E2A proteins are involved in regulating growth and differentiation of many different cell types.
  • E2A proteins have important roles in transcriptional activation of pancreatic and immunoglobulin genes (Cordle et al . , Mol. Cell. Biol. 11:2881-6, 1991; Henthorn et al . , Science 247:467-70, 1990; Murre et al . f Mol. Cell. Biol. 11:1156-60, 1991; Nelson et al . , Genes & Dev. 4:1035-43, 1990). They are essential for B cell development, as deletion of the E2A gene by homologous recombination prevents B cell differentiation in mice (Bain et al .
  • E2A proteins may be important in the differentiation of hematopoietic and neural tissue, respectively (Hsu et al . , Proc. Natl. Acad. Sci. USA 91:3181-5, 1994; Johnson et al . , Proc. Natl. Acad. Sci. USA 89:3596-600, 1992;
  • induction of myogenesis can be achieved by overexpression of myogenic factors in C3H10T1/2 embryonic fibroblasts or simply by removal of growth factors in the culture medium of C2C12 myoblasts (Olson and Klein, Genes & Dev. 8:1-8, 1994; Tapscott et al., Science 242:405-11, 1988; Weintraub, Cell 75:1241-4, 1993).
  • E2A proteins in conjunction with skeletal muscle-specific HLH proteins, are required for terminal differentiation and cell cycle withdrawal of skeletal muscle cells. Although in most instances the effect of E2A proteins on differentiation and growth inhibition appears to require heterodimerization with tissue-specific HLH proteins (Jan and Jan, Cell 75:827-30, 1993; Olson and Klein, Genes & Dev.
  • the vascular smooth muscle cell in mature animals is a highly specialized cell type whose principal function is contraction (Owens, Physiol. Rev. 75:487-517, 1995; Schwartz et al . , Physiol. Rev. 70:1177-1209, 1990).
  • the vascular smooth muscle cell is not terminally differentiated.
  • the adult vascular smooth muscle cell proliferates at an extremely low rate, it can undergo rapid changes to a highly proliferative phenotype in response to stimuli such as oxidized low density lipoprotein, homocysteine, and mechanical and immunological injuries (Libby and Hansson, Lab. Invest. 64:5-15, 1991; Munro and Cotran, Lab.
  • E2A-BP is herein defined as a protein which (1) has at least 70% sequence identity with SEQ ID NO:16, (2) binds to both E12 and E47, under physiological conditions, and (3) inhibits binding of E47 homodimer to an E-box probe consisting Of: 5'-GATCTACACCTGCTGCCTCCCAACACCTGCTGCCTCCC AACACCTGCTGCCTCCCAACACCTGCTGAGCT-3' (SEQ ID NO:3).
  • an E2A-BP polypeptide may be introduced into a vascular smooth muscle cell, in order to promote growth of the cell.
  • the sequence of the E2A-BP of the invention is preferably at least 80% identical, more preferably at least 90% identical, and most preferably at least 95% identical to SEQ ID NO:16. It can have the sequence of a naturally occurring protein from, e.g., a human, mouse (SEQ ID NO: 18) , rat, hamster, chicken, goat, horse, pig, cow, monkey, or ape, or can have one or more amino acid deletions, additions, or substitutions. It may be identical to SEQ ID NO:16; a truncated form of E2A-BP, e.g., the sequence of
  • the polypeptide of the invention may, for example, decrease the level of expression of myogenin and/or myosin heavy chain, and decrease the formation of multinuclear myotubes by these cells.
  • the polypeptide of the invention inhibits binding of E47-MyoD heterodimer to the E-box probe, but binds poorly or not at all to MyoD or to the E-box probe itself.
  • the invention also features an antibody (monoclonal or polyclonal) , which specifically binds to E2A-BP and can be made using standard methods.
  • substantially pure polypeptide is meant a polypeptide which is separated from those components (e.g., proteins and other naturally-occurring organic molecules) , which accompany it in its natural state.
  • components e.g., proteins and other naturally-occurring organic molecules
  • substantially pure polypeptides include recombinant polypeptides derived from a eukaryote, but produced in E. coli or another prokaryote, or in a eukaryote other than that from which the polypeptide was originally derived.
  • the polypeptide is substantially pure when it constitutes at least 60%, by weight, of the protein in the preparation.
  • the protein in the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, E2A-BP polypeptide.
  • a substantially pure E2A-BP polypeptide may be obtained by, for example, extraction from a natural source (e.g., a vascular smooth muscle), expression of a recombinant nucleic acid encoding an E2A-BP polypeptide; in vitro translation; or chemical synthesis of the protein. Purity of the polypeptide can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • the invention also features isolated nucleic acids (DNA, RNA, or combinations or modifications thereof) that encode E2A-BP, as is defined above.
  • the isolated nucleic acid may contain the nucleotide sequence of SEQ ID NO:15, or a portion thereof, e.g., SEQ ID NO:2, or may contain the nucleotide sequence of SEQ ID NO:17.
  • the isolated nucleic acid may hybridize under high stringency conditions to a probe having a nucleotide sequence complementary to SEQ ID NO:15 or SEQ ID NO:17, or a portion thereof.
  • the invention also includes all degenerate variants of sequences which encode E2A-BP.
  • an E2A-BP-encoding nucleic acid molecule of the invention may be introduced into a vascular smooth muscle cell, using gene therapy methods, in order to promote growth of the cell.
  • isolated nucleic acid is meant a nucleic acid that is free of the genes which, in the naturally- occurring genome of the organism from which the DNA is derived, flank the gene of interest.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote at a site other than its natural site, or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
  • It also includes a recombinant DNA which is part of a hybrid gene encoding an additional polypeptide sequence, such as a polypeptide sequence which facilitates purification (e.g., glutathione-S- transferase (GST) ) .
  • GST glutathione-S- transferase
  • the invention also features an isolated nucleic acid (DNA, RNA, or combinations or modifications thereof) having at least 50% sequence identity (preferably at least 70%, more preferably at least 80%, more preferably 90%, and most preferably at least 95%) to SEQ ID NO:15, and encoding E2A-BP, as defined above.
  • the percent sequence identity of one DNA to another is determined by standard means, e.g., by the Sequence Analysis Software Package developed by the Genetics Computer Group (University of Wisconsin Biotechnology Center, Madison, WI) (or an equivalent program; see, e.g., Ausubel et al . , supra) , employing the default parameters thereof.
  • Hybridization is carried out using standard techniques, such as those described in Ausubel et al . (Current Protocols in Molecular Biology, John Wiley & Sons, 1989) .
  • “High stringency” refers to nucleic acid hybridization and wash conditions characterized by high temperature and low salt concentration, e.g. , wash conditions of 65°C at a salt concentration of approximately 0.1 X SSC.
  • “Low” to “moderate” stringency refers to DNA hybridization and wash conditions characterized by low temperature and high salt concentration, e .g. , wash conditions of less than 60°C at a salt concentration of at least 1.0 X SSC.
  • high stringency conditions may include hybridization at about 42°C, and about 50% formamide; a first wash at about 65°C, about 2X SSC, and 1% SDS; followed by a second wash at about 65°C and about 0.1% x SSC.
  • Lower stringency conditions suitable for detecting DNA sequences having about 50% sequence identity to an E2A-BP gene are detected by, for example, hybridization at about 42°C in the absence of formamide; a first wash at about 42°C, about 6X SSC, and about 1% SDS; and a second wash at about 50°C, about 6X SSC, and about 1% SDS.
  • an isolated nucleic acid such as a DNA, containing an E2A-BP promoter, e.g., a human E2A-BP promoter.
  • promoter is meant a DNA sequence sufficient to direct transcription of a coding sequence to which it is linked. Promoters may be constitutive or inducible, and may be coupled to other regulatory sequences or "elements" which render promoter-dependent gene expression cell-type specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5' or 3' region of the native gene, or within an intron.
  • an E2A-BP promoter is capable of directing gene expression in vascular smooth muscle cells.
  • An E2A-BP promoter of the invention may be operably linked to the coding sequence of E2A-BP, or may be operably linked to a sequence which encodes a heterologous polypeptide, e . g. , a growth inhibitor, such as retinoblastoma, an inhibitor of cyclins (e.g., p21, p57, pl8, or pl7) , or a vasodilator, such as cNOS or a prostacyclin.
  • the E2A-BP promoter may also be operably linked to a segment of DNA which is transcribed into an RNA that is antisense to an MRNA naturally produced in a vascular smooth muscle cell.
  • RNAs that are antisense to MRNAS encoding proteins such as E2A-BP, heparin-binding epidermal growth factor (HBEGF) , and platelet-derived growth factor (PDGF)
  • E2A-BP promoter a coding sequence and a regulatory sequence(s) (i.e., a promoter) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s) .
  • the invention also features an isolated, single- stranded nucleic acid consisting of a nucleotide sequence which is antisense to at least a portion of a naturally occurring E2A-BP sense strand, e.g., E2A-BP mRNA (an "antisense oligonucleotide”) .
  • the antisense oligonucleotide may consist of DNA, RNA, or combinations or modifications thereof. For example, stabilized analogues of deoxyribonucleotides (see below) may be used.
  • the antisense oligonucleotide may be produced by standard in vitro methods, e .g. , by standard chemical synthesis (see, e .g. , Ausubel et al .
  • the antisense oligonucleotides described above may be introduced into a cell, such as a vascular smooth muscle cell, in order to inhibit expression of E2A-BP in the cell.
  • antisense RNA oligonucleotides may also be produced in vivo by transcription of a nucleic acid introduced into a cell.
  • a nucleic acid may have a sequence containing (a) an expression control sequence (i.e., a promoter, such as the E2A-BP promoter) which permits expression in a vascular smooth muscle cell (e.g., a human vascular smooth muscle cell) , operably linked to (b) a sequence which is transcribed into an RNA antisense to at least a 10, preferably at least a 14, or more preferably at least a 20 (e.g., at least a 30) nucleotide sequence of a target mRNA.
  • an antisense RNA may also be formulated to be complementary to all of a specific mRNA sequence, e.g., an E2A-BP mRNA sequence.
  • the invention also features a method of inhibiting proliferation of a vascular smooth muscle cell by introducing into the cell a compound (e.g., a small organic compound, a peptide having a sequence corresponding to the E2A-BP binding site on E2A, a peptide having a sequence corresponding to the E2A binding site on E2A-BP, or an antibody specific for either E2A-BP or E2A) , which inhibits binding between E2A-BP and an E2A transcription factor, such as E12 or E47.
  • a compound e.g., a small organic compound, a peptide having a sequence corresponding to the E2A-BP binding site on E2A, a peptide having a sequence corresponding to the E2A binding site on E2A-BP, or an antibody specific for either E2A-BP or E2A
  • Such compounds may be identified using any of several screening methods. For example, in one screening method, E2A can be contacted with E2A-BP in the presence of a candidate compound, and the level of E2A-BP binding to E2A can then be determined. A decrease in the level of binding in the presence of the compound, compared to the level of binding in the absence of compound, may be used as an indication of the ability of the candidate compound to inhibit E2A-BP/E2A binding. In another screening method, a complex containing E2A and E2A-BP can be contacted with a candidate compound. Whether the candidate compound decreases the binding of E2A-BP to E2A in the complex can be determined as an indication of the ability of the candidate compound to inhibit E2A-BP/E2A binding.
  • a cell that expresses E2A-BP and E2A can be cultured in the presence of a candidate compound.
  • the level of expression of an E2A-regulated gene in the cell can then be determined.
  • An increase in the level of expression of the gene in the presence of the compound, compared to the level of expression in the absence of the compound, is an indication of the ability of the candidate compound to inhibit E2A-BP/E2A binding. Additional variations of these methods are described below.
  • the invention also features a genetically altered non-human mammal, e.g., a mouse, whose muscle cells produce altered levels or forms of a functional E2A-BP gene product.
  • the levels of the E2A-BP gene product in the genetically altered mammal can be increased or decreased.
  • genetically altered mammal is meant a mammal in which the genomic DNA sequence has been manipulated in some way.
  • the genetically altered mammal may be a knockout in which the endogenous E2A-BP sequences have been deleted or otherwise altered to decrease or change the pattern of expression.
  • the genetically altered mammal may be transgenic, retaining endogenous E2A-BP coding sequences and having exogenous E2A-BP sequences as well.
  • the transgenic mammal may express E2A-BP sequences from another species, may overexpress E2A-BP gene product, or may express E2A-BP in tissues and at developmental stages other than those in which E2A-BP is normally expressed.
  • the nucleated cells of genetically altered mammal not producing a functional endogenous E2A-BP gene product may be engineered to encode human E2A-BP polypeptide, and to express functional human E2A-Bp polypeptide, or, alternatively, E2A-BP from another heterologous species.
  • Fig. IA is a schematic representation of a partial amino acid sequence of human E2A-BP (SEQ ID NO:l) containing 768 amino acids.
  • the sequences homologous to the two carboxypeptidase signature domains l and 2 are underlined.
  • the nuclear localization signal, KRIR is in bold.
  • An acidic domain, rich in glutamic acid residues, is in italics.
  • R404, the first amino acid of the original cDNA clone isolated by interaction cloning, is marked by a dagger.
  • IB is a schematic representation of a comparison of two E2A-BP sequences (SEQ ID NO:4 and SEQ ID NO:5), which are the E2A-BP sequences that are homologous and to the sequences of signature domains 1 (SEQ ID NO:6) and 2 (SEQ ID NO:7) in carboxypeptidase E (CPE) .
  • the putative zinc binding amino acids are in bold.
  • the histidine and glutamic acid residues in signature 1 are conserved in E2A-BP, but the histidine in signature 2 is replaced by asparagine.
  • Fig. 2 is a schematic representation of a partial nucleotide sequence of a human cDNA (SEQ ID NO:2), which encodes E2A-BP, as well as the corresponding amino acid sequence (SEQ ID NO:l), in single letter code.
  • Figs. 3A and 3B are schematic representations of the nucleotide sequences of portions of a rat E2A-BP cDNA.
  • Fig. 3A shows a portion of the sequence of the sense strand (SEQ ID NO:8) and
  • Fig. 3B shows a portion of the sequence of the antisense strand (SEQ ID NO:9).
  • Fig. 4 is a schematic representation of the nucleotide sequence of a full-length human E2A-BP cDNA (SEQ ID NO:15) .
  • Fig. 5 is a schematic representation of the amino acid sequence (SEQ ID:NO 16) encoded by the full-length human cDNA (SEQ ID NO:15).
  • the open reading frame of the full-length human E2A-BP contains 845 amino acids and has a predicted molecular weight of 96 kDa.
  • Fig. 6 is a schematic representation of the nucleotide sequence of the full-length murine cDNA (SEQ ID NO:17) .
  • Fig. 7 is a schematic representation of the amino acid sequence (SEQ ID NO:18) encoded by the full-length murine cDNA (SEQ ID NO:17).
  • the open reading frame of the full-length urine E2A-BP contains 1128 amino acids and has a predicted molecular weight of 128 kDa.
  • Naturally occurring E2A-BP is a nuclear protein that is expressed in vascular smooth muscle cells. It binds to, and modulates the activities of, E2A transcription factors.
  • the invention features nucleic acids that encode E2A-BP, as well as the E2A-BP polypeptides themselves. Also within the invention are the E2A-BP promoter, therapeutic methods employing E2A-BP nucleic acids or polypeptides, and methods for identifying compounds which modulate E2A-BP activity.
  • E2A-BP Nucleic Acids
  • SEQ ID NO:15 The nucleotide sequences of a full length cDNA encoding human E2A-BP (SEQ ID NO:15) and of a partial human cDNA encoding human E2A-BP (SEQ ID NO:2) are shown in Figs. 4 and 2, respectively.
  • the invention includes all degenerate variants of the coding sequence of SEQ ID NO:15 or SEQ ID NO:2, as well as any isolated nucleic acid having a nucleotide sequence that encodes any other E2A-BP (as defined above) .
  • the nucleotide sequence of a full-length mouse E2A-BP cDNA (SEQ ID NO:17) is shown in Fig. 6.
  • the invention includes all degenerate variants of the coding sequence of SEQ ID NO:17.
  • Figs. 3A and 3B The nucleotide sequence of a cDNA encoding a portion of rat E2A-BP (SEQ ID NO:8 and SEQ ID NO:9; see description of Figs. 3A and 3B, above) is shown in Figs. 3A and 3B.
  • the rat cDNA was isolated by PCR using primers derived from the human E2A- BP cDNA sequence.
  • Nucleic acids encoding naturally occurring E2A-BP polypeptides from species in addition to human and rat are included in the invention, and may be obtained using standard methods, such as the PCR method used to isolate the rat clone.
  • the invention also includes nucleic acids which encode E2A-BP polypeptides having substitutions and/or deletions of single and/or multiple amino acids.
  • nucleic acids can be generated using standard methods, and the polypeptides that they encode can easily be screened for E2A-BP activity, as described below.
  • Plasmids encoding full-length human E2A-BP or mouse E2A-BP cDNAs, as well as E. coli strains containing the above plasmids, were deposited with the American Type Culture Collection (ATCC, Rockville, Maryland) , under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure on March 12, 1997.
  • the E. coli strains are INV ⁇ F' (Invitrogen)
  • the cDNAS are cloned in the vector pCR2.1 (Invitrogen).
  • E2A-BP nucleic acids may be used in gene therapy and antisense methods for treating vascular diseases.
  • E2A-BP nucleic acids may be used in methods for producing E2A-BP polypeptides.
  • E2A-BP Polypeptides may be used in gene therapy and antisense methods for treating vascular diseases.
  • E2A-BP nucleic acids may be used in methods for producing E2A-BP polypeptides.
  • the amino acid sequences of full-length human E2A-BP (SEQ ID NO:16) and a partial human E2A-BP amino acid sequence (SEQ ID NO:l) are shown in Figs. 5 and IA, respectively.
  • the amino acid sequence of full-length mouse E2A-BP (SEQ ID NO:18) is shown in Fig. 7.
  • polypeptides which (1) have at least 70% sequence identity with SEQ ID NO:l, SEQ ID NO:16, or SEQ ID NO:18; (2) bind to both E12 and E47, under physiological conditions; and (3) inhibit binding of E47 homodi er to an E-box probe consisting of: 5'- GATCTACACCTGCTGCCTCCCAACACCTGCTGCCTCCCAACACCTGCTGCCTCCC AACACCTGCTGAGCT-3' (SEQ ID NO:3), are included in the invention.
  • Polypeptides according to the invention may be produced by transformation of a suitable host cell with all or part of an E2A-BP-encoding cDNA fragment (e.g., the cDNA described above) in a suitable expression vehicle.
  • a suitable host cell with all or part of an E2A-BP-encoding cDNA fragment (e.g., the cDNA described above) in a suitable expression vehicle.
  • E2A-BP-encoding cDNA fragment e.g., the cDNA described above
  • coli or in a eukaryotic host (e.g., yeast, such as Saccharomyces cerevisiae; insect cells, such as Sf-9 cells; or mammalian cells, such as COS-1, NIH-3T3, and JEG3 cells).
  • yeast such as Saccharomyces cerevisiae
  • insect cells such as Sf-9 cells
  • mammalian cells such as COS-1, NIH-3T3, and JEG3 cells.
  • expression vehicles may be chosen from, e.g., those described in Cloning Vectors '. A Laboratory Manual (P.H. Pouwels et al . , 1985, Supp. 1987) and by Ausubel et al . supra .
  • an expression system which may be used is a mouse 3T3 fibroblast host cell transfected with a pMAMneo expression vector (Clonetech, Palo Alto, CA) .
  • pMAMneo provides: an RSV-LTR enhancer linked to a dexamethasone-inducible MMTV-LTR promotor, an SV40 origin of replication, which allows replication in mammalian systems, a selectable neomycin gene, and SV40 splicing and polyadenylation sites.
  • DNA encoding an E2A-BP polypeptide can be inserted into the pMAMneo vector in an orientation designed to allow expression. The recombinant E2A-BP could then be isolated as described below.
  • Other host cells which may be used in conjunction with pMAMneo, or similar expression systems, include COS cells and CHO cells (ATCC Accession Nos. CRL 1650 and CCL 61, respectively) .
  • E2A-BP polypeptides may also be produced in stably-transfected mammalian cell lines.
  • a number of vectors suitable for stable transfection of mammalian cells are available to the public, e.g., see Pouwels et al . (supra) ; methods for constructing such cell lines are well known in the art (see, e .g. , Ausubel et al . , supra) .
  • cDNA encoding E2A-BP is cloned into an expression vector which includes the dihydrofolate reductase (DHFR) gene.
  • DHFR dihydrofolate reductase
  • Integration of the plasmid and, therefore, the E2A-BP-encoding gene into the host cell chromosome is selected for by inclusion of 0.01-300 ⁇ ,M ethotrexate in the cell culture medium (see, e.g.,
  • DHFR-deficient CHO cell line e.g., CHO DHFR ' cells, ATCC Accession No. CRL 9096
  • CHO DHFR ' cells ATCC Accession No. CRL 9096
  • E2A-BP polypeptide Once an E2A-BP polypeptide is expressed, it may be isolated using standard methods, such as affinity chromatography. For example, E2A or an antibody against E2A-BP may be attached to a column and used to isolate the E2A-BP polypeptide. Lysis and fractionation of E2A- BP-harboring cells prior to affinity chromatography may be performed by standard methods (see, e .g. , Ausubel et al . , supra) . Once isolated, the recombinant protein can, if desired, be further purified, e.g.
  • E2A-BP polypeptides can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis , 2nd ed. , 1984, The Pierce Chemical Co. , Rockford, IL) .
  • E2A-BP polypeptides may be used in therapeutic methods to promote vascular smooth muscle cell growth in, e.g., wound healing (see below).
  • E2A-BP polypeptides, or E2A-BP polypeptide fragments e .g. , ⁇ E2A-BP
  • E2A-BP polypeptides, or E2A-BP polypeptide fragments may also be used in methods for generating antibodies to E2A-BP, which may be used, e .g. , in E2A-BP purification methods.
  • the E2A-BP promoter which directs gene expression in vascular smooth muscle cells, is also included in the invention.
  • the E2A-BP promoter can be identified using standard methods of molecular biology (see, e.g., Ausubel et al . , supra) .
  • an E2A-BP cDNA probe can be used to isolate a genomic clone containing the E2A-BP gene from a genomic library.
  • the promoter region can be identified in the genomic clone using standard methods, such as primer extension and/or SI nuclease mapping. Further characterization of the E2A-BP promoter may be achieved by comparing the sequence located 5' to the E2A- BP coding sequence with known promoter element sequences.
  • a construct that includes E2A-BP promoter sequences which confer vascular smooth muscle cell- specific expression to a reporter gene to which the sequences are operably linked can be progressively deleted, by 5', 3', and/or nested deletions, until the ability of the promoter to induce transcription of the reporter gene in transfected cells is reduced.
  • other sequences such as 3' untranslated and intronic sequences, may be analyzed for effects on promoter activity.
  • the E2A- BP promoter may be used in gene therapy methods to direct vascular smooth muscle cell-specific expression of the E2A-BP gene, genes encoding heterologous polypeptides, or DNA sequences encoding antisense transcripts (e.g., transcripts antisense to E2A-BP RNA, or RNAs encoding growth promoting proteins, such as heparin-binding epidermal growth factor (HBEGF) and platelet-derived growth factor (PDGF) .
  • antisense inhibition of E2A-BP expression may be achieved by introduction of antisense oligonucleotides directly into vascular smooth muscle cells.
  • the nucleic acids of the invention can be used in gene therapy methods for treatment of vascular diseases, such as arteriosclerosis.
  • a vector containing the E2A-BP gene can be administered to a patient for use in expressing E2A-BP in a vascular smooth muscle cell.
  • the E2A-BP promoter operably linked to the coding sequence of a heterologous gene, i.e., a gene which encodes a protein other than E2A-BP, can be used to express the heterologous gene in vascular smooth muscle cells.
  • An E2A-BP promoter sequence directs transcription of DNA to which it is linked, preferably in vascular smooth muscle cells compared to -non vascular smooth muscle cells.
  • Heterologous genes the expression of which is regulated by E2A-BP promoter sequences in vascular smooth muscle cells include, e.g., sequences encoding t-PA (Pennica et al . , 1982, Nature 301:214); cyclin inhibitors, such as p21, p57, pl8, and pl7 (El-Deiry et al . , 1993, Cell
  • thrombolytic agents may be expressed under the control of the E2A-BP promoter sequences for expression by vascular smooth muscle cells in blood vessels, e.g., vessels occluded by aberrant blood clots.
  • heterologous proteins e.g., proteins which inhibit smooth muscle cell proliferation, e.g., interferon- ⁇ and atrial natriuretic polypeptide, may be specifically expressed in vascular smooth muscle cells to ensure the delivery of these therapeutic peptides to an arteriosclerotic lesion or an area at risk of developing an arteriosclerotic lesion, e .g. , an injured blood vessel.
  • the E2A-BP promoter sequences of the invention may also be used in gene therapy to promote angiogenesis to treat diseases such as peripheral vascular disease or coronary artery disease (Isner et al., 1995, Circulation 91:2687-2692).
  • the DNA of the invention can be operably linked to sequences encoding cellular growth factors which promote angiogenesis, e.g., VEGF, acidic fibroblast growth factor, or basic fibroblast growth factor.
  • Antisense Therapy e.g., VEGF, acidic fibroblast growth factor, or basic fibroblast growth factor.
  • the E2A-BP nucleic acids of the invention may also be used in methods for antisense treatment.
  • Antisense treatment may be carried out by administering to a mammal, such as a human, DNA containing the E2A-BP promoter operably linked to a DNA sequence (an antisense template) , which is transcribed into an antisense RNA.
  • a mammal such as a human
  • DNA containing the E2A-BP promoter operably linked to a DNA sequence (an antisense template) which is transcribed into an antisense RNA.
  • antisense oligonucleotides may be introduced directly into vascular smooth muscle cells.
  • the antisense oligonucleotide may be a short nucleotide sequence (generally at least 10, preferably at least 14, more preferably at least 20 (e.g., at least 30) , and up to 100 or more nucleotides) formulated to be complementary to a portion or all of a specific mRNA sequence. Standard methods relating to antisense technology have been described (see, e.g., Melani et al . , Cancer Res. 51:2897-2901, 1991). Following transcription of a DNA sequence into an antisense RNA, the antisense RNA binds to its target nucleic acid molecule, such as an mRNA molecule, thereby inhibiting expression of the target nucleic acid molecule.
  • target nucleic acid molecule such as an mRNA molecule
  • an antisense sequence complementary to a portion or all of the E2A-BP mRNA could be used to inhibit the expression of E2A-BP, thereby promoting differentiation.
  • Such antisense therapy may be used to treat conditions characterized by proliferation of vascular smooth muscle cells, such as arteriosclerosis, e .g. , restenosis in response to angioplasty.
  • the antisense therapy of the invention may also be used to treat cancer by inhibiting angiogenesis at the site of a solid tumor, as well as other pathogenic conditions which are caused by or exacerbated by angiogenesis, e .g. , inflammatory diseases such as rheumatoid arthritis and diabetic retinopathy.
  • the promoter of the invention can be operably linked to antisense templates which are transcribed into antisense RNA capable of inhibiting the expression of growth promoting proteins, such as HBEGF and PDGF.
  • the antisense oligonucleotides of the invention may be provided exogenously to a target vascular smooth muscle cell.
  • the antisense oligonucleotide may be produced within the cell by transcription of a nucleic acid molecule including a promoter sequence operably linked to a sequence encoding the antisense oligonucleotide.
  • the nucleic acid molecule is contained within a non- replicating linear or circular DNA or RNA molecule, is contained within an autonomously replicating plasmid or viral vector, or is integrated into the host genome. Any vector that can transfect a vascular smooth muscle may be used in this method of the invention.
  • Preferred vectors are viral vectors, including those derived from replication-defective hepatitis viruses (e.g., HBV and HCV) , retroviruses (see, e .g. , WO 89/07136; Rosenberg et al . , N. Eng. J. Med. 323(9) :570-578, 1990), adenovirus (see, e .g. , Morsey et al . , J. Cell. Biochem., Supp. 17E, 1993), adeno-associated virus (Kotin et al . , Proc. Natl. Acad. Sci.
  • viral vectors including those derived from replication-defective hepatitis viruses (e.g., HBV and HCV) , retroviruses (see, e .g. , WO 89/07136; Rosenberg et al . , N. Eng. J. Med. 323(9) :570-578,
  • HSV herpes simplex viruses
  • Methods for constructing expression vectors are well known in the art (see, e.g., Sambrook et al., supra) .
  • Additional suitable gene delivery systems include liposomes, receptor-mediated delivery systems, naked DNA.
  • the invention also includes any other methods which accomplish in vivo transfer of nucleic acids into eukaryotic cells.
  • the nucleic acids may be packaged into liposomes, receptor-mediated delivery systems, non-viral nucleic acid-based vectors, erythrocyte ghosts, or microspheres (e.g., microparticles; see, e.g., U.S. Patent No. 4,789,734; U.S. Patent No. 4,925,673; U.S. Patent No. 3,625,214; Gregoriadis, Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press, 1979)).
  • naked DNA may be administered. Delivery of nucleic acids to a specific site in the body for gene therapy or antisense therapy may also be accomplished using a biolistic delivery system, such as that described by Williams et al .
  • delivery of antisense oligonucleotides may be accomplished by direct injection of the oligonucleotides into target tissues, for example, in a calcium phosphate precipitate or coupled with lipids.
  • the antisense oligonucleotides of the invention may consist of DNA, RNA, or any modifications or combinations thereof.
  • modifications that the oligonucleotides may contain, inter-nucleotide linkages other than phosphodiester bonds, such as phosphorothioate, methylphosphonate, methylphosphodiester, phosphorodithioate, phosphoramidate, phosphotriester, or phosphate ester linkages (Uhlman et al . , Chem. Rev. 90(4) :544-584, 1990; Anticancer Research 10:1169, 1990), may be present in the oligonucleotides, resulting in their increased stability.
  • Oligonucleotide stability may also be increased by incorporating 3'-deoxythymidine or 2'-substituted nucleotides (substituted with, e .g. , alkyl groups) into the oligonucleotides during synthesis, by providing the oligonucleotides as phenylisourea derivatives, or by having other molecules, such as aminoacridine or poly- lysine, linked to the 3' ends of the oligonucleotides (see, e.g., Anticancer Research 10:1169-1182, 1990).
  • RNA and/or DNA nucleotides which make up the oligonucleotides of the invention may be present throughout the oligonucleotide, or in selected regions of the oligonucleotide, e.g., in the 5' and/or 3' ends.
  • the antisense oligonucleotides may also be modified so as to increase their ability to penetrate the target tissue by, e.g., coupling the oligonucleotides to lipophilic compounds.
  • the antisense oligonucleotides of the invention can be made by any method known in the art, including standard chemical synthesis, ligation of constituent oligonucleotides, and transcription of DNA encoding the oligonucleotides, as is mentioned above.
  • E2A-BP is naturally expressed in vascular smooth muscle cells, which are, therefore, the preferred cellular targets for the antisense oligonucleotides of the invention.
  • Targeting of antisense oligonucleotides to vascular smooth muscle cells may be achieved by coupling the oligonucleotides to ligands of vascular smooth muscle cell receptors.
  • oligonucleotides may be targeted to vascular smooth muscle cells by being conjugated to monoclonal antibodies that specifically bind to vascular smooth muscle-specific cell surface proteins.
  • the antisense oligonucleotides of the invention may be used in therapeutic compositions for treating, e.g., vascular diseases.
  • the therapeutic applications of antisense oligonucleotides in general are described, e .g. , in the following review articles: Le Doan et al . , Bull. Cancer 76:849-852, 1989; Dolnick, Biochem. Pharmacol. 40:671-675, 1990; Crooke, Annu. Rev. Pharmacol. Toxicol. 32, 329-376, 1992.
  • the therapeutic compositions of the invention may be used alone or in admixture, or in chemical combination, with one or more materials, including other antisense oligonucleotides or recombinant vectors, materials that increase the biological stability of the oligonucleotides or the recombinant vectors, or materials that increase the ability of the therapeutic compositions to penetrate vascular smooth muscle cells selectively.
  • the therapeutic compositions of the invention may be administered in pharmaceutically acceptable carriers (e .g. , physiological saline), which are selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington 's Pharmaceutical Sciences , a standard reference text in this field, and in the USP/NF.
  • a therapeutically effective amount is an amount of the antisense molecule of the invention which is capable of producing a medically desirable result in a treated animal.
  • dosage for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages will vary, but a preferred dosage for intravenous administration of DNA is approximately IO 6 to IO 22 copies of the DNA molecule.
  • the compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e . g. , intravenously.
  • DNA may also be administered directly to the target site, e .g.
  • E2A-BP/E2A binding Modulation of the growth of vascular smooth muscle cells can be achieved by contacting the cells with a compound that blocks or enhances E2A-BP/E2A binding.
  • a compound that blocks or enhances E2A-BP/E2A binding can be identified by methods ranging from rational drug design to screening of random compounds. The latter method is preferable, as simple and rapid assays for testing such compounds are available. Small organic molecules are desirable candidate compounds for this analysis, as frequently these molecules are capable of passing through the plasma membrane so that they can potentially modulate E2A-BP/E2A binding within the cell.
  • E2A-BP antibodies specific for either E2A-BP or E2A, or alternatively peptides (or peptide imetics) (a) derived from the binding site on E2A-BP, which would block by occupying the binding site on E2A; or (b) derived from the binding site on E2A, which would block by occupying the binding site on E2A- BP.
  • the screening of compounds for the ability to modulate E2A-BP/E2A binding may be carried out using in vitro biochemical assays, cell culture assays, or animal model systems.
  • labeled E2A- BP e.g., E2A-BP labeled with a fluorochrome or a radioisotope
  • a candidate compound is applied to the column before, after, or simultaneously with the labeled E2A-BP, and the amount of labeled protein bound to the column in the presence of the compound is determined by conventional methods.
  • a compound tests positive for inhibiting E2A-BP/E2A binding if the amount of labeled protein bound in the presence of the compound is lower than the amount bound in its absence.
  • a compound tests positive for enhancing E2A-BP/E2A binding if the amount of labeled protein bound in the presence of the compound is greater than the amount bound in its absence.
  • binding of labeled E2A to immobilized E2A-BP is measured. In all of these methods, large numbers of compounds can be screened very rapidly and easily.
  • candidate compounds may also be screened using cell culture assays.
  • Cells expressing E2A-BP and E2A either naturally or after introduction into the cells of genes encoding E2A and/or E2A-BP (e.g., C2C12 cells transfected with E2A-BP, see below) , are cultured in the presence of the candidate compound.
  • the level of E2A-BP/E2A binding in the cell may be inferred using any of several assays.
  • levels of expression of E2A-regulated genes e.g., genes encoding myogenin, myosin heavy chain, or myosin light chain
  • levels of expression of E2A-regulated genes may be determined using, e.g., Northern blot analysis, RNAse protection analysis, immunohistochemistry, or other standard methods (see below) .
  • the ability of a candidate compound to modulate E2A-BP/E2A binding may be evaluated by determining the effect of the candidate compound on cell differentiation (see below) or cell growth, which may be measured by, e .g. , monitoring uptake of [ 3 H]thymidine.
  • Compounds found to inhibit E2A-BP/E2A binding may be used in methods for inhibiting growth of vascular smooth muscle cells in order to, e.g., prevent or treat arteriosclerosis or angiogenesis.
  • Compounds found to enhance E2A-BP/E2A binding may be used in methods to promote proliferation of vascular smooth muscle cells in order to, e.g., promote angiogenesis in wound healing (e .g. , healing of broken bones, burns, diabetic ulcers, or traumatic or surgical wounds) and organ transplantation.
  • wound healing e.g. , healing of broken bones, burns, diabetic ulcers, or traumatic or surgical wounds
  • organ transplantation e.g., vascular endothelial vascular disease
  • such compounds may be used to treat peripheral vascular disease, cerebral vascular disease, hypoxic tissue damage (e.g., hypoxic damage to heart tissue) , or coronary vascular disease.
  • Compounds identified using the above-described methods may also be used to treat patients who have, or have had, transient ischemic attacks, vascular graft surgery, balloon angioplasty, frostbite, gangrene, or poor circulation.
  • the therapeutic compounds identified using the methods of the invention may be administered to a patient by any appropriate method for the particular compound, e . g. , orally, intravenously, parenterally, transdermally, transmucosally, by inhalation, or by surgery or implantation at or near the site where the effect of the compound is desired (e.g., with the compound being in the form of a solid or semi-solid biologically compatible and resorbable matrix) .
  • a salve or transdermal patch that can be directly applied to the skin so that a sufficient quantity of the compound is absorbed to increase vascularization locally may be used.
  • This method would apply most generally to wounds on the skin.
  • Salves containing the compound can be applied topically to induce new blood vessel formation locally, thereby improving oxygenation of the area and hastening wound healing.
  • Therapeutic doses are determined specifically for each compound, most being administered within the range of 0.001 to 100.0 mg/kg body weight, or within a range that is clinically determined to be appropriate by one skilled in the art. Genetically Altered E2A-BP Mammals
  • Genetically altered mammals can be created which have cells that express altered levels of the endogenous functional E2A-BP gene product.
  • the genetically altered mammals may in addition express heterologous E2A-BP gene product derived from a second animal.
  • knock-out mice which do not express the mouse homologue of E2A-BP (SEQ ID NO:17) can be generated.
  • Mice from this line can be manipulated further to express the human homologue of E2A-BP (SEQ ID NO:2 and SEQ: ID NO:15), e.g., by introduction of a transgene encoding the human sequence.
  • part or all of the endogenous mouse genomic sequences can be replaced with the corresponding human E2A-BP sequence by homologous reco bination.
  • human homologue may be directed to particular tissues or cell types, e.g., skeletal muscle cells and vascular smooth muscle cells, through the use of tissue- or cell type-specific regulatory elements. Many such elements are known to skilled artisans.
  • Such transgenic mammals represent model systems for the study of conditions or diseases that are caused, exacerbated, or ameliorated by the E2A- BP protein.
  • the cells of a genetically altered mammal may bear genetic information received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as DNA received by microinjection or by infection with recombinant virus.
  • mammals of the invention are those with one or more cells that contain a recombinant DNA molecule.
  • this molecule becomes stably integrated into the mammal's chromosomes, but the use of DNA sequences that replicate extrachromosomally, such as might be engineered into yeast artificial chromosomes, is also contemplated.
  • the mammal is one in which heterologous genetic information has been taken up and integrated into a germ line cell.
  • transgenic mammals typically have the ability to transfer the genetic information to their offspring. If the offspring in fact possess some or all of the genetic information delivered to the parent animal, then they, too, are transgenic mammals.
  • a genetically altered mammal may be any mammal except Homo sapiens. Farm animals (pigs, goats, sheep, cows, horses, rabbits, and the like), rodents (such as rats, guinea pigs, and mice) , and domestic animals (for example, dogs and cats) are within the scope of the present invention.
  • the genetically altered mammals of the present invention are produced by introducing DNA encoding E2A-BP of the invention into single-celled embryos so that the DNA is stably integrated into the DNA of germ-line cells in the mature mammal, and inherited in a Mendelian fashion. It has been possible for many years to introduce heterologous DNA into fertilized mammalian ova.
  • totipotent or pluripotent stem cells can be transfected by microinjection, calcium phosphate-mediated precipitation, liposome fusion, retroviral infection, or other means.
  • the transfected cells are then introduced into an embryo (for example, into the cavity of a blastula) and implanted into a pseudo-pregnant female that is capable of carrying the embryos to term.
  • the transfected, fertilized ova can be implanted directly into the pseudopregnant female.
  • the appropriate DNA is injected into the pronucleus of embryos, at the single cell stage, and the embryos allowed to complete their development within a pseudopregnant female.
  • a recombinant E47 fusion protein (N3-SH[ALA]) , containing the bHLH domain of hamster shPan-1 (amino acids 509-646, with mutations R551A, V552L, and R553A) with a heart muscle kinase recognition sequence and the FLAG epitope, was expressed and purified as described (Blanar et al . , Proc. Natl. Acad. Sci. USA 92:5870-4, 1995; Blanar and Rutter, Science 256:1014-8, 1992).
  • N3-SH[ALA] was phosphorylated by heart muscle kinase in the presence of ⁇ - 32 P-ATP and used to screen a human aorta ⁇ gtll cDNA expression library (Clonetech) by interaction cloning (Blanar et al . , Proc. Natl. Acad. Sci. USA 92:5870-4, 1995; Blanar and Rutter, Science 256:1014-8, 1992).
  • a 1450-bp cDNA clone ( ⁇ E2A-BP) obtained from interaction cloning was radiolabeled by random priming and used to isolate a 2786 bp cDNA clone (E2A-BP) from the same human aorta ⁇ gtll cDNA library. After restriction mapping, the appropriate fragments were subcloned into plasmids (pBluescript SK, Stratagene) . DNA sequencing was performed by using the dideoxy chain termination method and T7 DNA polymerase.
  • Sequencing templates used were (1) alkaline-denatured double- stranded DNA or (2) single-stranded DNA generated by in vitro excision by helper phage virus (Stratagene) (Sambrook et al . , supra) . Both strands of the 2786 bp human E2A-BP cDNA were sequenced at least once. Using reverse transcription PCR and primers encoding human E2A-BP sequences (forward,
  • rat E2A-BP cDNA fragment was amplified from rat aortic smooth muscle RNA as described (Lee et al . , J. Biol. Chem. 266:16188-92, 1991). This rat E2A-BP cDNA was subcloned into plasmid PCRTMII (Invitrogen) and used as a probe for in situ hybridization (see below) . The authenticity of the rat E2A-BP was confirmed by sequencing.
  • Figs. 3A SEQ ID NO:8; a portion of the sequence of the sense strand
  • 3B SEQ ID NO:9; a portion of the sequence of the antisense strand
  • E2A-BP cDNA corresponding to the 4 kb E2A-BP transcript (see below) was isolated by subjecting human aortic RNA to three successive rounds of amplification using 5' RACE. 5' RACE reagents were obtained from Gibco. The cDNA product was subcloned into pCR2.1 (Invitrogen).
  • the sequence of the full-length human cDNA fragment is shown in Fig. 4 (SEQ ID NO:15).
  • the cDNA has an open-reading frame encoding 845 amino acids beginning at the ATG highlighted in bold and terminating at the TGA highlighted in bold.
  • the predicted amino acid sequence is shown in Fig. 5 (SEQ ID NO:16).
  • a full-length mouse cDNA corresponding to the mouse E2A-BP transcript was isolated using 5' RACE with primers designed from the mouse AEBP-cDNA sequence (EMBL nucleotide sequence accession number X80478) .
  • RT-PCR was used to generate a cDNA which was subcloned into pCR2.1 (Invitrogen) and sequenced.
  • the sequence of the full- length mouse cDNA fragment is shown in Fig. 6 (SEQ ID NO:17).
  • the cDNA has an open-reading frame encoding 1128 amino acids beginning at the ATG highlighted in bold and terminating at the TGA highlighted in bold, and generating the amino acid sequence shown in Fig. 7 (SEQ ID NO:17).
  • RNA probe As a control, a sense RNA probe was synthesized using T7 RNA polymerase to transcribe Hindlll-linearized rat E2A-BP cDNA in PCRTMII. The RNA probes were hydrolyzed for 20 minutes at 60°C to generate probes of approximately 100 nucleotides long. Each tissue section was hybridized with 20 million counts-per- minute (cpm) of probe at 50°C overnight. After the hybridization procedure, the sections were washed at 50°C under stringent conditions and dried. The tissue sections were subsequently dipped into emulsion solution (Kodak NTB2) and exposed for 2 to 4 days at 4°C. The sections were counter-stained with hematoxylin-eosin. cell Lines, Cell Culture, and Reagents
  • C2C12 and COS-7 cells were obtained from the American Type Culture Collection (Rockville, Maryland) . All cells were cultured in Dulbecco's modified Eagle medium (DMEM, JRH) supplemented with 10% fetal calf serum (Hyclone) or Serum Plus (JRH) before transfection. C2C12 cells were cultured in either 2% (differentiation medium) or 20% (growth medium) fetal calf serum after transfection.
  • DMEM Dulbecco's modified Eagle medium
  • JRH Dulbecco's modified Eagle medium
  • C2C12 cells were cultured in either 2% (differentiation medium) or 20% (growth medium) fetal calf serum after transfection.
  • the monoclonal antibody for myosin heavy chain was obtained from Sigma and the anti-c-myc 9E10 peptide antibody was obtained from either Oncogene or Santa Cruz.
  • cDNA probes for rat myogenin, mouse myosin light chain, and mouse myosin heavy chain were provided by A.B. Lassar (Harvard Medical School) .
  • Human MyoD, E12, and E47 plasmid constructs were provided by D. Baltimore (Whitehead Institute, MIT) and F.A. Peverali (EMBL, Heidelberg, Germany) .
  • the hybridized filters were washed in 30 mM NaCl, 3 mM sodium citrate, and 0.1% sodium dodecyl sulfate at 55°C, and then were used to expose film or stored on PhosphorImager screens for 68 hours. To correct for differences in RNA loading, the filters were washed in a 50% formamide solution at 80°C and rehybridized with a radiolabeled 28S oligonucleotide probe. The filters were scanned and radioactivity was measured on a Phosphorlmager running the ImageQuant software (Molecular Dynamics, Sunnyvale, CA) . Cellular Localisation of E2A-BP
  • the expression plasmid Myc-E2A-BP/pCR3 was constructed for cellular localization of E2A-BP.
  • the c-Myc peptide tag (EQKLISEED; SEQ ID NO:12) was added in-frame with the E2A-BP open reading frame encoded by SEQ ID NO:2 at the N-terminus using PCR techniques, and cloned into the expression vector pCR3 (Invitrogen) .
  • COS-7 cells were transiently transfected with Myc-E2A-BP/pCRTM3 plasmids using the DEAE-dextran method (Sambrook et al. , supra) . Immunostaining was performed 48 hours after transfection.
  • the transfected cells grown on chamber slides, were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and stained with an anti-c-myc monoclonal antibody (9E10, Oncogene) , followed by a rhodamine-conjugated goat anti-mouse IgG as the secondary antibody. Counter- staining for the nucleus with Hoechst 33258 was performed as recommended by the manufacturer.
  • a DNA fragment containing the partial E2A-BP human open reading frame corresponding to SEQ ID NO:2 was cloned into expression plasmids pcDNA3 and pCRM3 (Invitrogen) in sense or antisense orientations.
  • Stable transformants of C2C12 cells were generated by electroporation, as described previously (Sambrook et al . , supra) . Briefly, 2.5 X IO 6 cells were harvested at 60% confluence and resuspended in 0.8 ml of PBS. The cells were transferred to electroporation cuvettes (0.4 mm, Biorad) and mixed with 20 ⁇ g of plasmid DNA.
  • Stable transfectants were selected in DMEM media supplemented with 0.5 mg/ml of G418 (GIBCO) . Mutagenesis Mutations were introduced into the human E2A-BP cDNA in p-Bluescript vector using the Clonetech site- directed mutagenesis kit. Two conserved residues implicated in metal binding, H-236 and E-239 (as numbered in SEQ ID NO:l), were mutated to glutamine. Two oligonucleotides, mtXbal, 5'-GGCGGCCGCTGTAGAACTAGT-3' (SEQ ID NO:13) and mtSgnl
  • 35 S-labeled proteins were prepared by in vitro transcription and translation (Promega TNT kit) using the - 36 - cDNA plasmid encoding ⁇ E47 (amino acid 561 to 651), E47, E12, MyoD, Id-3, ⁇ E2A-BP (amino acid 404 to 768), mtE2A-BP, and wild type E2A-B.
  • glutathione- S-transferase (GST) fusion proteins GST- ⁇ E47, GST- ⁇ E12 (amino acids 477 to 654), GST-MyoD, GST-Id3 , and GST- ⁇ E2A-BP, were prepared as previously described (Shrivastava et al .
  • a typical binding reaction mixture contained DNA probe at 50,000 cpm, 1 ⁇ g of poly(dl-dC) »poly(dI-dC) , 10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, and 3-5 ml of in vitro translated protein products, in a final volume of 25 ml.
  • the reaction mixture was incubated at room temperature for 30 minutes and analyzed by 5% native polyacrylamide gel electrophoresis in a 0.25 X TBE buffer (22 mM Tris base, 22 mM boric acid, and 0.5 mM EDTA).
  • Cells were harvested in low ionic strength lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 3 mM MgCl 2 , 300 mM sucrose, and 0.1% NP40) as described (Jen et al.. Genes Dev. 6:1466, 1992). Protein lysates were cleared by preincubation with normal rabbit IgG and protein A- agarose (Oncogene) , followed by an overnight incubation with c-Myc-antibody agarose (Santa Cruz) , talon metal affinity resin (Clonetech) , or protein A-agarose.
  • lysis buffer 10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 3 mM MgCl 2 , 300 mM sucrose, and 0.1% NP40
  • E2A-BP bHLH domain of E47 as a probe to screen a human aortic expression library
  • ten interacting clones were isolated; eight clones encoded Id and two clones encoded novel sequences.
  • One of the two novel clones, designated E2A-BP was characterized first.
  • a 1450-bp E2A-BP cDNA identified from the interaction cloning was used to isolate a 2795-bp cDNA clone.
  • E2A-BP lacks an HLH domain, despite its isolation by interaction cloning using the bHLH domain of E47 as a probe.
  • Carboxypeptidases contain two signature domains that are important for binding one atom of zinc (Manser et al . , Biochem. J. 267:517-25, 1990; Reynolds et al. , J. Biol. Chem. 264:20094-9, 1989; Tan et al. , J. Biol. Chem. 264:13165-70, 1989).
  • a histidine and a glutamic acid in signature 1 and an additional histidine in signature 2 are implicated in zinc binding (Fig. IB) .
  • the histidine and glutamic acid in signature l are present in both carboxypeptidase E and E2A-BP.
  • the histidine in signature 2 is present in carboxypeptidase E, but not in E2A-BP.
  • E2A-BP mRNA is Preferentially Expressed in the Aorta
  • E2A-BP mRNA The expression pattern of E2A-BP mRNA was determined in a variety of rat and human organs by Northern blot analysis using a human E2A-BP cDNA probe. For rat organs, a single 4 kb transcript was detected by the E2A-BP probe. The highest expression of E2A-BP was observed in the aorta with its adventitia removed and containing mainly smooth muscle cells. E2A-BP was undetectable in other rat organs, except a low level was detected in the adventitia and esophagus. E2A-BP was also expressed at high levels in human aorta, compared to heart, lung, and skeletal muscle, which have 40, 80, and 100-fold less expression, respectively.
  • E2A-BP is expressed in aortic smooth muscle cells.
  • in situ hybridization was performed.
  • a rat E2A-BP cDNA to generate both sense and antisense cRNA probes.
  • An intense concentration of autoradiographic grains was present after hybridization with the antisense, but not the sense probe, indicating a high level expression of E2A-BP transcript in aortic smooth muscle cells.
  • autoradiographic grains were not detected in skeletal muscle cells hybridized with the E2A-BP antisense probe. Hybridization of the antisense probe to small vessels in skeletal muscle was also detected. This observation is consistent with the low level of E2A-BP expression in skeletal muscle detected by RNA blot analysis.
  • E2A-BP Downregulation of E2A-BP mRNA in Human Adult Skeletal Muscle
  • E2A-BP expression in human fetal and adult skeletal muscle was examined.
  • a high level of E2A-BP mRNA was detected in fetal skeletal muscle cells.
  • E2A-BP mRNA was downregulated markedly in adult skeletal muscle cells that had differentiated terminally.
  • an RNA blot previously hybridized with an E2A-BP probe was rehybridized with a cyclin A probe. Cyclin A mRNA was present at a high level in fetal skeletal muscle but was undetectable in adult skeletal muscle.
  • E2A-BP is a nuclear protein
  • a fusion plasmid was generated which expresses a fusion protein containing a c-myc peptide tag on the N-terminus side of a portion of the E2A-BP cDNA open reading frame corresponding to SEQ ID NO:2.
  • the construct was then transfected into C2C12 and COS-7 cells.
  • the fusion protein was detected by a specific monoclonal antibody (9E10) to the c-myc tag.
  • DNA staining by Hoechst 33258 was used to localize the nucleus.
  • C-myc tagged E2A-BP protein was expressed in the nucleus in both C2C12 and COS-7 cells. This result is consistent with the presence of a nuclear localization signal (Boulikas, J. Cell. Biochem. 55:32-58, 1994), KRIR, at amino acid 599 to 602 of E2A-BP (Fig. IA) .
  • E2A-BP Binds E12 and E47, but not MyoD and Id3
  • E2A-BP Suppresses Binding of E47 homodimer and E 7-MyoD Heterodi er to the E-box
  • E2A-BP gel mobility shift analysis was performed using an E-box probe consisting of four repeats of the consensus CANNTG sequences in the enhancer of muscle creatine kinase (Lassar et al . , Cell 58:823-31, 1989; Murre et al . , Cell 56:777-83, 1989; Murre et al . , Cell 58:537-44, 1989) and HLH or E2A-BP proteins synthesized by in vitro transcription and translation. The proteins were translated alone or co-translated with other proteins. The effect of E2A-BP on binding of ⁇ E47 homodimers was analyzed. Incubation of the probe with ⁇ E47 resulted in formation of a specific ⁇ E47-E-box complex, which was abolished by incubation with a
  • E47/MyoD heterodimer The binding of E47/MyoD heterodimer to the E-box probe in the presence and absence of E2A-BP was also assessed. Incubation of the E-box probe with full length E47 and MyoD resulted in formation of a specific E47/MyoD-E-box complex, which was abolished by incubation with a 100-fold molar excess of identical nonradiolabeled DNA, but not by incubation with nonidentical DNA. E2A-BP decreased the binding of E47/MyoD heterodimer to the E-box by more than 75%. To determine whether the conserved histidine and glutamic acid residues of signature 1 (Fig.
  • IB are important in inhibiting the binding of E47/MyoD to the E-box, the histidine and glutamic acid residues were mutated to glutamine. Mutation of these two amino acids prevented the inhibition of E47/MyoD-E-box complex formation by E2A-BP.
  • E2A proteins have an important role in regulating differentiation and inhibiting growth of many cell types (Kadesch, Cell. Growth Differ. 4:49-55, 1993; Lassar et al . , Cell 58:823-31, 1989; Olson and Klein, Genes & Dev. 8:1-8, 1994; Peverali et al . , EMBO J. 13:4291-301, 1994), the effect of E2A-BP, which attenuates binding of E2A to DNA, was tested. C2C12 myoblasts were used for these studies because differentiation in these cells is well characterized (Guo et al . , Mol. Cell. Biol. 15:3823-9, 1995; Jen et al .
  • C2C12 myoblasts express skeletal muscle specific genes, such as myogenin, myosin heavy chain (MHC) , and myosin light chain (MLC) and differentiate into multinucleated myotubes (Jen et al . , Genes & Dev. 6:1466-79, 1992).
  • MHC myosin heavy chain
  • MLC myosin light chain
  • C2C12 cells were stably transfected with vectors containing no insert or a full length E2A-BP cDNA, in either sense or antisense orientations. Expression of sense and antisense transcripts was confirmed by Northern blot analysis. Two clones expressing the sense E2A-BP and three clones expressing antisense E2A-BP were selected. Since the responses of these clones were similar, the results of one representative clone from each group are presented. To determine the effect of E2A-BP on the mRNA levels of muscle specific genes, C2C12 clones were cultured in either growth medium for two days or in differentiation medium for one or two days, and total RNA was harvested for Northern blot analysis.
  • C2C12 cells in growth medium expressed MyoD, but not myogenin, MHC, and MLC C2C12 cells in growth medium expressed MyoD, but not myogenin, MHC, and MLC.
  • differentiation medium markedly increased the mRNA levels of myogenin, MHC, and MLC, but did not affect MyoD mRNA levels.
  • differentiation medium failed to induce expression of myogenin, MHC, and MLC mRNA in C2C12 cells transfected with sense E2A-BP.
  • E2A-BP E2A-BP would inhibit expression of MHC
  • cells were immunostained with an anti-MHC primary antibody and a rhodamine-conjugated secondary antibody. The nuclei were labeled by Hoechst 32258. Expression of MHC protein can be easily detected in C2C12 cells transfected with vector alone or antisense E2A-BP, but not in cells transfected with sense E2A-BP.
  • the transfected C2C12 clones were treated with differentiation medium for 4 days. Formation of multinuclear myotubes was detected in C2C12 cells transfected with vector alone, but not in cells transfected with sense E2A-BP. Since C2C12 cells expressed low levels of E2A-BP mRNA, the effect of antisense E2A-BP on myotube expression was also tested. Compared with C2C12 cells transfected with vector alone, transfection of C2C12 cells with antisense E2A-BP accelerated formation of myotubes.
  • E2A-BP Interacts with E2A Proteins in Vivo
  • MOLECULE TYPE Genomic DNA
  • GGAGTGGACC CTACGAAA GTCAAGTTCC CCCCATTGGG ATG GAG TCA CAC CGT 75
  • GAG ATC TCA GAC AAC CCT GGG GAG CAT GAA CTG GGG GAG CCC GAG TTC 847 Glu He Ser A ⁇ p A ⁇ n Pro Gly Glu His Glu Leu Gly Glu Pro Glu Phe 215 220 225
  • CAG CAG CGA CGC CTA CAA CAC CGC CTG CGG CTT CGG GCA CAG ATG CGG 2095 5 Gin Gin Arg Arg Leu Gin His Arg Leu Arg Leu Arg Ala Gin Met Arg 630 635 640 645
  • GAG AAA GAG GAG GAG ATA GCC ACT GGC CAG GCA TTC CCC TTC ACA ACA 2431 Glu Lye Glu Glu Glu He Ala Thr Gly Gin Ala Phe Pro Phe Thr Thr 745 750 755 5 GTA GAG ACC TAC ACA GTG AAC TTT GGG GAC TTC TGAGATCAGC GTCCTACCAA 2484
  • ATTCCCTCGC TCACCCCATC CTCTCTCCCG CCCCTTCCTG GATTCCCTCA CCCGTCTCGA 60
  • TCCCCTCTCC GCCCTTTCCC AGAGACCCAG AGCCCCTGAC CCCCCGCGCC CCTGCTCAGC 120 TGCCTCCTGG CGTTGCTGGC CCTGTGCCCT GGAGGGCGCC CGCAGACGGT GCTGACCGAC 180
  • GCC CGC ACG CCT ACC CAG GAG CAG CTG CTG GCC GCA GCC ATG GCA GCA 2460 Ala Arg Thr Pro Thr Gin Glu Gin Leu Leu Ala Ala Ala Met Ala Ala
  • GAC CAC GCC ATC TTC CGG TGG CTT GCC ATC TCC TTC GCC TCC GCA CAC 2556 A ⁇ p Hie Ala He Phe Arg Trp Leu Ala He Ser Phe Ala Ser Ala Hie 495 500 505 510 CTC ACC TTG ACC GAG CCC TAC CGC GGA GGC TGC CAA GCC CAG GAC TAC 2604 Leu Thr Leu Thr Glu Pro Tyr Arg Gly Gly Cy ⁇ Gin Ala Gin Asp Tyr 515 520 525
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • GAG GCC AAG CAG CCC CGG CCA GAG CCA GAG GAG GAG ACT GAG ATG CCC 729 Glu Ala Lys Gin Pro Arg Pro Glu Pro Glu Glu Glu Thr Glu Met Pro
  • AAA ATC AAG TGC CCA CCT ATT GGG ATG GAG TCA CAC CGC ATT GAG GAC 1209 Lys He Lys Cys Pro Pro He Gly Met Glu Ser His Arg He Glu Asp 375 380 385
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal

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  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

L'invention concerne des acides nucléiques codant E2A-BP, ainsi que les polypeptides E2A-BP eux-mêmes. Sont également décrits le promoteur d'E2A-BP, des méthodes thérapeutiques employant les acides nucléiques ou polypeptides d'E2A-BP, et des méthodes d'identification de composés modulant l'activité d'E2A-BP.
PCT/US1997/004117 1996-03-15 1997-03-14 Proteine fixant e2a WO1997033900A1 (fr)

Applications Claiming Priority (2)

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US1343996P 1996-03-15 1996-03-15
US60/013,439 1996-03-15

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WO1997033900A1 true WO1997033900A1 (fr) 1997-09-18

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0588118A2 (fr) * 1992-08-28 1994-03-23 Hoechst Japan Limited Protéine du type d'une carboxypeptidase apparentée aux os et procédé de sa production

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0588118A2 (fr) * 1992-08-28 1994-03-23 Hoechst Japan Limited Protéine du type d'une carboxypeptidase apparentée aux os et procédé de sa production
US5460951A (en) * 1992-08-28 1995-10-24 Hoechst Japan Limited Bone-related carboxypeptidase-like protein and process for its production

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NATURE, 02 November 1995, Vol. 378, HE et al., "A Eukaryotic Transcriptional Repressor with Carboxypeptidase Activity", pages 92-96. *

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