WO1995028492A1 - Elements de regulation de l'adn sensibles aux cytokines - Google Patents

Elements de regulation de l'adn sensibles aux cytokines Download PDF

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WO1995028492A1
WO1995028492A1 PCT/US1995/004511 US9504511W WO9528492A1 WO 1995028492 A1 WO1995028492 A1 WO 1995028492A1 US 9504511 W US9504511 W US 9504511W WO 9528492 A1 WO9528492 A1 WO 9528492A1
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protein
seq
nucleic acid
gene
sequence
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PCT/US1995/004511
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I. Peter Lamb
H. Martin Seidel
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Ligand Pharmaceuticals Incorporated
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Priority claimed from US08/410,780 external-priority patent/US5707803A/en
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Priority to AU22876/95A priority Critical patent/AU681025B2/en
Priority to EP95916343A priority patent/EP0755453A1/fr
Priority to JP7527067A priority patent/JPH09511908A/ja
Publication of WO1995028492A1 publication Critical patent/WO1995028492A1/fr

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to oligonucleotide sequences that bind regulatory proteins that affect transcription in response to various molecules, such as cytokines, to
  • DNA constructs comprising the oligonucleotide sequences, cells transfected with the DNA constructs, and to methods of using the same to provide for the controlled expression of heterologous genes, for the detection and recovery of new regulatory proteins, and for measuring the ability of compounds to act as agonist and antagonists of gene transcription.
  • extracellular signaling molecules such as polypeptide ligands
  • cytokines and growth factors which comprise a large and diverse family of soluble polypeptides that control the growth, differentiation and function of mammalian cells, bind to specific cell surface receptors, that in some way transduce signals that elicit a specific phenotypic response.
  • IFNs ⁇ and ⁇ act as a primary non-specific defense against viral infections.
  • S. Petska and J.A. Laneer.56 Annu. Rev. Biochem.. 727 (1987,. IFN7 (type II) has anti-viral properties but also plays a major role in regulation of the immune response.
  • Type I and type II IFNs bind to distinct cell surface receptors and cause rapid alterations in gene expression. Auget, 55 Cell. 273; Uze, 60 Cell. 225; and G.C. Sen and P. Lengyel, 267 J. Biol. Chem.. 5017 (1992).
  • ISREs interferon- ⁇ stimulated response elements
  • ISGF3 is a complex of 4 binding proteins, called p48, p84 (STATl ⁇ ), p91 (STATl ⁇ ) and pi 13 (STAT2). Recently, cDNAs encoding the proteins that constitute ISGF3 have been isolated and characterized. X-Y Fu et al., 89 Proc. Natl. Acad. Sci..7840 (1992); C.
  • p48 is the DNA binding component of ISGF3 and has homology to myb. Veals, 12 Mol. Cell. Biol.. 3315.
  • p84 and p91 are probably alternatively spliced products of the same gene and are related to pi 13. X-Y Fu, 89 Proc. Natl. Acad. Sci.. 7840 and Schindler, 89 Proc. Natl. Acad. Sci..7836.
  • p84, p91 and pi 13 are novel proteins that contain SH2 and SH3 domains and are found in the cytoplasm of untreated cells. Schindler, 257 Science. 809 and X.Y. Fu, 70 Cell, 323-335 (1992). Thus, IFN ⁇ treatment of cells results in rapid tyrosine phosphorylation of p84, p91 and pi 13, causing them to associate and form a heteromeric complex with p48 to form ISGF3, which then translocates to the nucleus and binds to ISREs, stimulating transcription. Id.; Dale, 86 Proc. Natl. Acad. Sci.. 1203 and Kessler, 4 Genes Dev.. 1753.
  • Interleukin-6 plays a major role in the induction of the acute phase response in hepatocytes.
  • the acute phase response is characterized by the dramatic transcriptional upregulation of a distinct set of genes, termed acute phase response genes. P.C. Heinrich et al, 265 Biochem. J.. 621-636 (1990).
  • Studies of the promoter regions of these genes have identified specific DNA sequences that are required for induction of acute phase response genes by IL-6. See D.R. Kunz et al., 17 Nuc. Acids Res..
  • APREs acute phase response elements
  • STAT3 is a latent transcription factor that is activated to bind DNA by rapid tyrosine phosphorylation.
  • IL-6 also activates STATl ⁇ .
  • heterodimers of STATl ⁇ and STAT3 form in addition to homodimers of STATl ⁇ and homodimers of STAT3. Sadowski et al., 261 Science. 1739-1744 (1993); Raz et al., 269 J. Biol. Chem.. 24391-24395 (1994) and Zhong et al., 264 Science. 95- 98 (1994).
  • IFN7 and IL-6 activate ST AT proteins that can bind to similar sequences (GAS/APREs), they regulate distinct sets of genes. This suggests that there is specificity with respect to the response elements in some of these genes, such that they respond only to one of these cytokines.
  • many cytokines other than IFN 7 and IL-6 cause the rapid activation of GAS-binding protein complexes. See O. Silvennionen et al., 261 Science. 1736 (1993); H.B. Sadowski et al., 261 Science. 1739 (1993); A.C. Lamer et al., 261 Science. 1730 (1993); D. Finbloom et al., 14 Mol. Cell. Biol..
  • the present invention is directed to oligonucleotide sequences comprising DNA regulatory elements that bind, either directly or indirectly, to activated transcriptional regulatory proteins, preferably ST AT proteins, in response to signaling molecules, including cytokines such as interferon gamma (IFN7), interleukin 4 (IL-4), interleukin 6 (IL-6), and granulocyte-macrophage colony stimulating factor (GM-CSF).
  • cytokines such as interferon gamma (IFN7), interleukin 4 (IL-4), interleukin 6 (IL-6), and granulocyte-macrophage colony stimulating factor (GM-CSF).
  • IFN7 interferon gamma
  • IL-4 interleukin 4
  • IL-6 interleukin 6
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • the regulatory elements of the present invention can be used in transcriptional assays to discover agonists or antagonists of a signaling molecule, such as IFN ⁇ , and its cognate transcriptional regulatory protein, STATl ⁇ .
  • the present invention provides oligonucleotide sequences comprising regulatory elements of the nucleotide sequence TATTCCTGGAAGT (SEQ ID NO. 1), TATTCCGGTAAGT (SEQ ID NO. 2), TCTTCCTGTAAGT (SEQ ID NO. 3), TATTCTTGTAAGT (SEQ ID NO. 4), TATTCCTGTTAGT (SEQ ID NO. 5), TATTCCCGTAAGT (SEQ ID NO. 6), TATTCCTATAAGT (SEQ ID NO.
  • TATTCCTGTCAGT SEQ ID NO. 8
  • TATACCTGTAAGT SEQ ID NO. 9
  • TATGCCTGTAAGT SEQ ID NO. 10
  • TATTCCTTTAAGT SEQ ID NO. 11
  • TATTCCTCTAAGT SEQ ID NO. 12
  • TATTCCTGCAAGT SEQ ID NO. 13
  • TATTCCTGTACGT SEQ ID NO. 14
  • the present invention also provides a DNA construct comprising the regulatory elements of the oligonucleotide sequences described above operably linked to a promoter, which promoter is operably linked to a heterologous gene, wherein the DNA construct is linked in such a manner that the heterologous gene is under the transcriptional control of the oligonucleotide sequence and promoter. Also provided is a host cell transfected with this DNA construct. The present invention also provides a method for the controlled expression of a heterologous gene of interest comprising culturing the transfected host cells containing an appropriate transcriptional regulatory protein(s) in the presence of a signaling molecule.
  • the signaling molecule in this method comprises a cytokine and the transcriptional regulatory protein comprises a ST AT protein.
  • the present invention further provides a method for detecting the presence of an activated transcriptional regulatory protein, such as a novel STAT protein, in a sample comprising contacting the sample with an oligonucleotide sequence as described above under conditions where the transcriptional regulatory protein is activated and binds with the oligonucleotide sequence to form a complex, and detecting the presence of the complex in the sample. Thereafter, the complex can be separated from the sample, and the transcriptional regulatory protein isolated from the regulatory element.
  • an activated transcriptional regulatory protein such as a novel STAT protein
  • the present invention provides a method for measuring the ability of a compound to act as an agonist of gene transcription comprising (a) contacting the compound with a transfected host cell as described above under conditions in which the heterologous gene is capable of being expressed in response to the compound, and (b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the absence of the compound.
  • the present invention also provides a method for measuring the ability of a compound to act as an antagonist of gene transcription comprising (a) contacting the compound with a transfected host cell as described above in the presence of a predetermined amount of a signaling molecule under conditions in which the heterologous gene is expressed in response to the signaling molecule, and (b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the presence of the signaling molecule, but the absence of the compound.
  • the heterologous gene may be any appropriate reporter gene such as the gene for luciferase, chloramphenicol acetyl transferase, green fluorescent protein or ⁇ - galactosidase.
  • the present invention provides a method for selectively measuring the ability of a compound to agonize or antagonize the induction of STAT heterodimers comprising (a) contacting the compound with a host cell according to claim 19 under conditions in which the heterologous gene is capable of being expressed in response to the compound, wherein the host cell is transfected with a DNA construct comprising a single copy of an oligonucleotide sequence comprising a regulatory element that that is capable of selectively binding to an activated STAT heterodimer, and (b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the absence of the compound.
  • Oligonucleotide or "DNA” molecule or sequence refers to a molecule comprised of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C), in either single-stranded form or a double-stranded helix, and comprises or includes a "regulatory element” according to the present invention, as that term is defined herein.
  • the exact size, strandedness and orientation i.e. 3' to 5', or 5' to 3 1 ) will depend upon many factors, which, in turn, depend upon the ultimate function and use of the oligonucleotides of the present invention.
  • oligonucleotide or "DNA” includes double-stranded DNA found in linear DNA molecules or fragments, viruses, plasmids, vectors, chromosomes or synthetically derived DNA.
  • particular double-stranded DNA sequences may be described according to the normal convention of giving only the sequence in the 5' to 3' direction.
  • Regulatory element refers to a deoxyribonucleotide sequence comprising the whole, or a portion of, an oligonucleotide sequence to which an activated transcriptional regulatory protein, or a complex comprising one or more activated transcriptional regulatory proteins, binds so as to transcriptionally modulate the expression of an associated gene or genes, including heterologous genes.
  • Signaling molecule refers to an extracellular polypeptide, oligosaccaride or other non-peptidyl molecule, in either a free or bound form, that interacts with a receptor at or near the surface of a cell. This interaction in turn triggers an intracellular pathway which includes the activation of one or more transcriptional regulatory proteins that bind to a regulatory element, thereby transcriptionally modulating the expression of an associated gene or genes.
  • signaling molecule includes naturally occurring molecules, such as cytokines, peptidyl and non- peptidyl hormones, antibodies, cell-surface antigens, or synthetic mimics of any of these signaling molecules, or compounds that mimic the action of any of these signaling molecules.
  • Cytokines refer to a diverse grouping of soluble polypeptides, including growth factors and hormones, that control the growth, differentiation and function of cells in such a manner as to ultimately elicit a phenotypic response in an organism.
  • Preferred cytokines useful with the regulatory elements and associated methods of the present invention include IFN7, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, EL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, Epo, Tpo, GM-CSF, Oncostatin M, growth hormone, G-CSF, LIF, EGF, CNTF and PDGF.
  • Transcriptional regulatory protein refers to cytoplasmic or nuclear proteins that, when activated, bind the regulatory elements/oligonucleotide sequences of the present invention either directly, or indirectly through a complex of transcriptional regulatory proteins or other adapter proteins, to transcriptionally modulate the activity of an associated gene or genes.
  • transcriptional regulatory proteins can bind directly to the DNA regulatory elements of the present invention, or can bind indirectly to the regulatory elements by binding to another protein, which in turn binds to or is bound to a DNA regulatory element of the present invention. See e.g.. S.A. Veals et al., 13 Molec. Cell. Biol.. 196-206 (1993).
  • transcriptional regulatory proteins include, but are not limited to, those proteins referred to in the art as STAT proteins (Z. Zhong et al., 264 Science. 95) STF proteins (C. Schindler et al., 13 EMBO J.. 1350 (1994)), Mammary Gland-Specific Nuclear Factor (M. Schmidt-Ney et al., 6 Mol. Endochronol.. 1988 (1992); Wakao, et al., 13 EMBO J. 2182-2191 (1994); and H. Wakao et al., 267 J. Biol. Chem.. 16365 (1992)), APRF (Wegenka, 13 Mol. Cell Bio..
  • Transcriptionally modulate the expression of an associated gene or genes means to change the rate of transcription of such gene or genes.
  • STAT protein refers to those transcriptional regulatory proteins designated as “Signal Transducers and Activators of Transcription” (STAT) by Dr. J.E. Darnell of Rockefeller University. See Zhong, 264 Science 95.
  • STAT proteins include the p91 (STATl ⁇ ), p84 (STATl ⁇ ), pi 13 (STAT2) proteins and the STAT-associated p48 family of proteins. S.A. Veals et al., 12 Mol. Cell. Biol.. 3315 (1992). Further, STAT proteins also include a binding protein designated as STAT3 (Zhong, et al., 91 Proc. Natl. Acad. Sci.. 4806-4810 (1994); Zhong, 264 Science 95).
  • STAT4 binding protein designated as STAT4 (Id.).
  • MGF is now renamed STAT 5 (Gouilleux et al., 13 EMBO J. 4361-4369 (1994)), and IL-4-STAT is referred to as STAT6 by some investigators (Hile et al., 11 Trends Genet.. 69 (1995)). Also included are substantially homologous analogs and allelic variations of all of the above STAT proteins.
  • Activate means that one or more transcriptional regulatory proteins within a cell are modified post- translationally, or are constituitively active, such that they can bind directly or indirectly to DNA regulatory elements/oligonucleotide sequences of the present invention in a sequence-specific manner.
  • This modification will typically comprises phosphorylation of the. transcriptional regulatory proteins via a variety of mechanisms, including, but not limited to activation by various protein kinases. See, e.g.. (Shuai, 258 Science. 1808 and P. Cohen, 17 TIBS.408 (1992)).
  • DNA construct refers to any genetic element, including, but not limited to, plasmids, vectors, chromosomes and viruses, that incorporate the oligonucleotide sequences of the present invention.
  • the DNA construct can be a vector comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule.
  • Promoter refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with SI nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of
  • RNA polymerase RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA” boxes and “CCAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • Gene refers to a nucleic acid molecule, the sequence of which includes all the information required for the normal regulated production of a particular protein.
  • a "heterologous" region of a DNA construct i.e. a heterologous gene
  • a heterologous gene is an identifiable segment of DNA within a larger DNA construct that is not found in association with the other genetic components of the construct in nature.
  • the heterologous gene encodes a mammalian gene
  • the gene will usually be flanked by a promoter that does not flank the structural genomic DNA in the genome of the source organism.
  • a promoter of a DNA construct including an oligonucleotide sequence according to the present invention, is "operably linked" to a heterologous gene when the presence of the promoter influences transcription from the heterologous gene, including genes for reporter sequences such as luciferase, chloramphenicol acetyl transferase, ⁇ -galactosidase and secreted placental alkaline phosphatase.
  • reporter sequences such as luciferase, chloramphenicol acetyl transferase, ⁇ -galactosidase and secreted placental alkaline phosphatase.
  • Operably linked sequences may also include two segments that are transcribed onto the same
  • RNA transcript RNA transcripts.
  • two sequences such as a promoter and a "reporter sequence” are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence.
  • reporter sequence operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence.
  • two sequences In order to be "operably linked” it is not necessary that two sequences be immediately adjacent to one another.
  • a host cell has been "transfected" by exogenous or heterologous DNA
  • the transfecting DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transfecting DNA may be maintained on an episomal element such as a plasmid.
  • a stablely transfected cell is one in which the transfecting DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transfecting DNA.
  • “Host cell” refers to a cell line that expresses, either normally or after transfection of the requisite cDNAs, the relevant receptor components for a given signaling molecule, signaling (e.g., kinase) proteins, transcriptional regulatory proteins, and accessory factors such that, upon binding of the signaling molecule to the cell surface, transcriptional regulatory protein-mediated gene transcription is affected.
  • signaling e.g., kinase
  • transcriptional regulatory protein-mediated gene transcription is affected.
  • the host cell line is responsive to cytokines, such that the host cell line expresses, either normally or after transfection of the requisite cDNAs, the relevant cytokine receptor components, JAK proteins, STAT proteins, and accessory factors such that, upon cytokine binding to the cell surface, STAT-mediated gene transcription is affected.
  • HG. 1 is a summary of the oligonucleotide competition data for complexes containing transcriptional regulatory proteins activated by IFN7, IL-6, GM- CSF and IL-4.
  • the name of each oligonucleotide, followed by its sequence, is shown on the left.
  • the mutated nucleotide is underlined and shown in bold type.
  • the ability of each sequence to compete with the Ly6E GAS element (termed the "wild type, WT" sequence) for binding to transcriptional regulatory proteins activated by either IFN-7, IL-6, GM-CSF or IL-4 is shown by the number in the appropriate column;
  • FIG. 2 is an example of an oligonucleotide competition experiment.
  • Nuclear extracts from cells treated with either IFN-7, IL-6, IL-4 or GM-CSF were incubated with the indicated amounts of oligonucleotide 8T (SEQ ID NO. 11) prior to the addition of radiolabeled Ly6E GAS element probe. Complexes were then separated on a non-denaturing polyacrylamide gel, which was then exposed to X-ray film. A reproduction of the resulting autoradiogram is shown. A and B indicate the positions of the complexes of transcriptional regulatory proteins that correspond to STATl ⁇ and STAT3 respectively;
  • HG. 3 is a graph showing transcriptional induction mediated by a single Ly6E GAS element and selected regulatory element-containing oligonucleotide sequences of the present invention in response to cytokines.
  • Cells were transfected with reporter plasmids containing a single copy of the inventive regulatory elements or the wild type Ly6E GAS element driving expression of luciferase.
  • Cells were treated with either IFN-7, IL-6, LIF or OSM as indicated, and induction of transcription over untreated cells determined as described in the Examples.
  • the TK luc control reporter lacks a regulatory element;
  • FIG.4 is a graph showing transcriptional induction mediated by multiple regulatory elements in response to cytokines.
  • FIG. 5 is a comparison of the in vitro binding affinities of the Ly6E GAS element and the 7C, 8 A, 4G, and 8T oligonucleotides containing regulatory elements of the present invention for STATl ⁇ , with the transcriptional induction in response to IFN-7 from constructs containing 4 copies of the same elements.
  • the first column lists the regulatory elements studied.
  • the next column indicates the ability of each element to bind to STATl ⁇ in vitro (data taken from FIG. 1).
  • the last column shows the fold induction of luciferase activity in response to IFN- ⁇ in cells transfected with reporter plasmids contaimng four copies of the listed regulatory elements; and
  • FIG.6 is a series of gel panels showing tyrosine phosphorylation of STATl ⁇ and STAT3 in response to cytokines.
  • Cells were treated with either IFN- ⁇ , LIF, IL-6 or OSM for the indicated time, and lysates prepared. Lysates were immunoprecipitated with either a STAT1 antisera (top panels) or a STAT3 antisera (bottom panels). After resolution on SDS-polyacrylamide gels and blotting, proteins were detected with antisera directed against either phosphotyrosine, STAT1 or STAT3 as indicated at the left of FIG 6.
  • a STAT1 antisera top panels
  • STAT3 antisera bottom panels
  • the present inventors have discovered a series of DNA regulatory elements (i.e. response elements) that in response to various signaling molecules, bind, either directly or indirectly, to activated transcriptional regulatory proteins, and accordingly, transcriptionally modulate the expression of one or more genes operably linked with such regulatory elements.
  • response elements DNA regulatory elements
  • selected point mutations in the Ly6E GAS response element can transform an element from one that binds with a variety of cytokine-induced, activated transcriptional regulatory proteins to a regulatory element that selectively binds just one type or class of cytokine-induced, activated transcriptional regulatory proteins.
  • sequence TATACCTGTAAGT SEQ ID NO.
  • nucleotide sequences such as TATTCCTGGAAGT (SEQ ID NO. 1) yield a regulatory element that can mediate transcriptional induction in response to a variety of different cytokine-induced transcriptional regulatory proteins, including the STATl ⁇ and STAT3 proteins, as well as those transcriptional regulatory protein(s) that are activated by the IL-4, IL- 13 and GM-CSF cytokines.
  • the regulatory elements according to the present invention are selected from the nucleotide sequences TATTCCTGGAAGT (SEQ ID NO. 1), TATTCCGGTAAGT (SEQ ID NO. 2), TCTTCCTGTAAGT (SEQ ID NO. 3), TATTCTTGTAAGT (SEQ ID NO.4), TATTCCTGTTAGT (SEQ ID NO.5), TATTCCCGTAAGT (SEQ ID NO. 6), TATTCCTATAAGT (SEQ ID NO. 7),
  • SEQ ID NOs 9-14 comprise STATl ⁇ protein selective regulatory elements
  • SEQ ID NOs 1-5 comprise regulatory elements that bind a variety of transcriptional regulatory proteins
  • SEQ ID NOs 6-8 comprise regulatory elements that selectively bind the STATl ⁇ and STAT3 transcriptional regulatory proteins.
  • these regulatory elements alone, or with additional flanking nucleotide sequences, can form various oligonucleotide sequences according to the present invention.
  • nucleotide sequences comprise between 13 and 200 nucleotides, including the regulatory elements of the present invention.
  • sequences in excess of 200 nucleotides that contain the regulatory elements of the present invention, that are capable of binding activated transcriptional regulatory proteins, and of transcriptionally modulating the expression of one or more genes thereby are considered to be within the scope of the present invention.
  • the oligonucleotide sequences of the present invention can also comprise multimers of two or more "units" of the basic regulatory elements.
  • such multimer oligonucleotide sequences can, as a practical matter, contain from about 2 to 15 units of the same or different regulatory elements according to the present invention.
  • Such multimeric oligonucleotide sequences are useful as probes for detecting, isolating and/or purifying transcriptional regulatory proteins.
  • a multimer of the regulatory elements can enhance the expression of the gene from the DNA construct in response to various cytokines or other signaling molecules.
  • signaling molecules activate transcriptional regulatory proteins that bind directly or indirectly to the regulatory elements/oligonucleotide sequences of the present invention.
  • Nonlimiting examples of such signaling molecules include polypeptides such as cytokines and antibodies, and cell-surface antigens, oligosaccarides typically found at or near the surface of cell, non-peptidyl molecules such as TUBag4 (P. Constant et al., 264 Science, 267 (1994)) and synthetic mimics any of these molecules, in both their free and bound forms.
  • the present invention includes cell to cell or cell to substrate transcriptional regulatory protein activation via signaling molecules bound to or near the surface of a cell or other substrate.
  • the signaling molecules according to the present invention comprise cytokines that activate transcriptional regulatory proteins that bind to the regulatory elements/oligonucleotide sequences of the present invention.
  • cytokines include, but are not limited to, Interleukins 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13 and 15 (IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13 and IL- 15), granulocyte-macrophage colony stimulating factor (GM-CSF), granuloctyte colony stimulating factor (G-CSF), colony stimulating factor 1 (CSF-1), interferons alpha, beta, and gamma (IFN ⁇ , IFN ⁇ , IFN ⁇ ), epidermal growth factor (EGF), platelet derived growth factor (PDGF), leukemia inhibitory factor (LIF), Oncostatin-M, nerve growth factor (NGF), ciliary neurotrophic factor (CNTF
  • Particularly preferred cytokines according to the present invention include, but are not limited to, IFN ⁇ , IL-4, IL-6, GM-CSF, Oncostatin-M, G-CSF, LIF, EGF, PDGF, Epo, Tpo and CNTF.
  • the regulatory elements and/or oligonucleotide sequences of the present invention will also prove useful in detecting, isolating and purifying new transcriptional regulatory proteins that display binding specificity to the regulatory elements/ oligonucleotide sequences of the present invention. Further, it is contemplated that these regulatory elements/oligonucleotide sequences will prove particularly useful in the discovery of novel STAT proteins or STAT-related transcriptional regulatory proteins.
  • DNA regulatory element binding activity may also be stimulated in vitro by treating a cytoplasmic extract, supplemented with cell membranes, with a signaling molecule, such as cytokine.
  • an antibody specific for a transcriptional regulatory protein can be used to specifically interfere with the binding of the regulatory element of the present invention to the activated transcriptional regulatory protein, thereby assisting in the identification of the transcriptional regulatory protein.
  • an unknown transcriptional regulatory protein identified or purified using the regulatory elements/oligonucleotide sequences of the present invention can be used to immunize animals to prepare an antibody specific for the transcriptional regulatory protein using methods well known in the art. See, e.g.. E. Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the disclosure of which is herein incorporated by reference.
  • the regulatory elements/nucleotide sequences of the present invention thus can serve as a "probe", similar to those used in a variety of nucleic acid detection systems well known in the art, except that the probes of the present invention are used to detect proteins, rather than a nucleic acid sequences, which specifically bind to the regulatory elements/oligonucleotide sequences of the present invention.
  • the sensitivity of such a nucleic acid detection assay can be increased by altering the manner in which a signal is detected by an observer.
  • assay sensitivity can be increased through the use of labeled oligonucleotide sequences using a wide variety of detectable labels, including, without limitation, enzyme labels, radioisotopic labels, fluorescent labels, and modified bases. See, e.g., U.S. Patent Nos. 4,581,333, 4,358,535, 4,446,237, 4,582,789, and 4,563,417, as well as European Patent Application Nos. EP 144914 and EP 119448, the disclosures of which are herein incorporated by reference.
  • DNA probes according to the present invention preferably include the regulatory elements alone, or as part of a longer oligonucleotide sequence of the present invention, labeled with a detectable label, such as a radioisotope, an enzyme, a fluorescent label, a chemical label, or a modified base.
  • a detectable label such as a radioisotope, an enzyme, a fluorescent label, a chemical label, or a modified base.
  • the present invention provides a method for detecting the presence of novel transcriptional regulatory proteins in a sample.
  • samples are preferably biological samples, including, but not limited to, cells, cell culture supernatant, cell or tissue extracts, or particular fractions thereof, and other biological fluids such as blood, sera, urine, saliva, etc.
  • Binding of the probe containing the regulatory elements/ oligonucleotide sequences of the present invention to a transcriptional regulatory protein in the sample may be detected by any appropriate means known in the art. For example, direct or indirect, or competitive binding assays may be used. In such assays, association of the labeled probe with the proteinaceous material of the sample is then detected.
  • the oligonucleotide sequence is modified by the incorporation of a radioactivity labeled nucleotide.
  • the novel transcriptional regulatory protein can be separated and purified from the probe-protein complex by any of a variety of techniques well known to those of skill in the art. For example, such isolation and purification can be based on affinity chromatography, which relies on the interaction of the protein to be purified with an immobilized ligand.
  • affinity chromatography which relies on the interaction of the protein to be purified with an immobilized ligand.
  • a regulatory element and/or oligonucleotide sequence of the present invention immobilized on a support would serve as the immobilized ligand, which in turn would be used to isolate and purify a novel transcriptional regulatory protein from a sample.
  • the regulatory element/oligonucleotide sequence of the present invention is immobilized on a solid support or carrier.
  • solid phase carrier or support refers to any support capable of binding the oligonucleotide sequences/DNA regulatory elements of the present invention.
  • Well known supports, or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • purification steps prior to affinity separation may also include one or more additional methods, such as ammonium sulfate precipitation, size exclusion chromatography (gel filtration), ion exchange chromatography, differential precipitation and the like, all well known in the art.
  • additional methods such as ammonium sulfate precipitation, size exclusion chromatography (gel filtration), ion exchange chromatography, differential precipitation and the like, all well known in the art.
  • HIC hydrophobic interaction chromatography
  • Elution from HIC supports can be effected by alterations in solvent, pH, ionic strength, or by the addition of chaotropic agents or organic modifiers, such as ethylene glycol.
  • the regulatory elements/oligonucleotide sequences of the present invention may be included in a recombinant DNA construct which contains a regulatory element/oligonucleotide sequence operably linked to a promoter and a heterologous gene.
  • the heterologous gene comprises a reporter sequence, such as the gene for luciferase.
  • a recombinant DNA construct such as a reporter plasmid according to the present invention, can be constructed using conventional molecular biology, microbiology, and recombinant DNA techniques well known to those of skill in the art.
  • Promoter sequences useful in DNA constructs according to the present invention include all prokaryotic, eukaryotic or viral promoters capable of driving transcription of a heterologous gene of interest in combination with a regulatory element of the present invention, when transfected into an appropriate host cell.
  • Suitable prokaryotic promoters include, but are not limited to, promoters recognized by the T4, T3, Sp6, and T7 polymerases, the P R and P L promoters of bacteriophage ⁇ , the transcriptional regulatory protein, recA, heat shock, and lacZ promoters of E. coli. the ⁇
  • Preferred eukaryotic promoters include the yeast cyc-1 promoter, the promoter of the mouse metallothionein I gene, the thymidine kinase promoter of the Herpes simplex virus, the SV40 early promoter, and the yeast gal-4 gene promoter. See Guarante et al., 78 Proc. Natl. Acad. Sci. USA. 2199-2203 (1981), D. Hamer et al., 1 J. Mol. Appl. Gen.. 273-288 (1982), S.
  • a DNA construct according to the present invention utilizes the thymidine kinase gene promoter of the Herpes simplex virus.
  • the third component of the recombinant DNA or construct molecules of the present invention is a "heterologous gene" which may be composed of any set of nucleotides regardless of sequence.
  • heterologous genes include the structural genes for luciferase, ⁇ -galactosidase, chloramphenicol acetyl transferase, secreted placental alkaline phosphatase, human growth hormone, tPA and interferon.
  • the heterologous gene comprises a reporter gene whose product is used to assess regulation of transcription via a promoter and a regulatory element/oligonucleotide sequence of the present invention.
  • the expression of this "reporter sequence” results in the formation of a reporter product (e.g., protein) which is readily detectable.
  • the reporter sequence will preferably be selected such that the reporter molecule will have a physical and chemical characteristics which will permit or facilitate its identification or detection by means well known in the art. In one embodiment, the presence of the reporter molecule will be detected through the use of an antibody or an antibody fragment, capable of specific binding to the reporter molecule.
  • a reporter such as ⁇ -galactosidase or luciferase can be assayed enzymatically or immunologically.
  • a preferred reporter molecule is LUC, well known in the art. See, e.g.,
  • a plasmid containing the recombinant DNA molecule of the present invention is introduced into mammalian cells, which are then grown to, at or near confluency.
  • any host cell capable of activating one or more transcriptional regulatory proteins in response to an appropriate signaling molecule or molecules can be transfected with the DNA constructs of the present invention.
  • host cells comprise mammalian cells, such as HepG2 and U937 cells (available from ATCC, Rockville, Maryland).
  • the reporter cells are treated with a compound or sample suspected of containing a signaling molecule capable of inducing or activating a transcriptional regulatory protein, for example, a medium that has been in contact with cells suspected of producing a signaUng molecule, such as a cytokine.
  • a signaling molecule capable of inducing or activating a transcriptional regulatory protein for example, a medium that has been in contact with cells suspected of producing a signaUng molecule, such as a cytokine.
  • the LUC-producing reporter cells are extracted, and the soluble extracts are supplemented with luciferin and ATP. In the presence of these compounds the action of luciferase generates light, which is detected using a luminometer. The amount of light produced is directly related to the amount of luciferase present in the cellular extract.
  • the present invention provides a method for the controlled expression of a gene of interest.
  • a signaling molecule such as cytokine
  • transfected host cells can be used to drive the expression of a heterologous gene to yield defined quantities of a desired product, such as human growth hormone, by any of a variety of cell culture and fermentation techniques well known to those of skill in the art.
  • transfection of the DNA construct into a host cell provides a convenient means for measuring the transcriptional activity of a reporter product in response to a signaling molecule, such as a cytokine or a medium that has been in contact with cells suspected of producing a signaling molecule, such as a cytokine.
  • a signaling molecule such as a cytokine or a medium that has been in contact with cells suspected of producing a signaling molecule, such as a cytokine.
  • LUC synthesis is increased relative to a control lacking the transcriptional regulatory protein.
  • the amount of LUC enzyme produced is an indirect measure of transcription induced by the activated transcriptional regulatory protein binding to the regulatory elements/oligonucleotide sequences of the present invention, which is operably linked to the LUC gene.
  • a preferred host cell such as a HepG2 cell
  • a reporter DNA construct When a preferred host cell, such as a HepG2 cell, it can be utilized in assays to detect agonists and antagonists of signaling molecules that induce gene transcription via activated transcriptional regulatory proteins.
  • agonists or antagonists of gene transcription include compounds that intervene at any point within the signaling pathway from interaction between the signaling molecule and a cell surface receptor through activation of one or more transcriptional regulatory proteins and binding of the same to DNA regulatory elements, the end result of which is modulation of gene transcription.
  • agonists and antagonists of gene transcription also include potentiators of known compounds with such agonist or antagonist properties.
  • Agonists can be detected by contacting the transfected host cell with a compound or mix of compounds and, after a fixed period of time, determining the level of gene expression (e.g., the level of luciferase produced) within the treated cells. This expression level can then be compared to the expression level of the reporter gene in the absence of the compound(s). The difference between the levels of gene expression, if any, indicated whether the compound(s) of interest agonize the activation of intracellular transcriptional regulatory proteins in an analogous fashion to a known agonist signaling molecule, such as a cytokine. Further, the magnitude of the level of reporter product expressed between the treated and untreated cells provides a relative indication of the strength of that compound(s) as an agonist of gene transcription via a transcriptional regulatory protein pathway.
  • the level of gene expression e.g., the level of luciferase produced
  • such a transfected host cell can be used to find antagonists of known agonists, e.g., cytokines such as IFN ⁇ , of gene transcription utilizing host cells transfected with the DNA constructs according to the present invention.
  • the compound or compounds of interest are contacted with the host cell in conjunction with one or more known agonists (e.g., cytokines) held at a fixed concentration.
  • known agonists e.g., cytokines
  • the present invention provides methods to assay for agonists and antagonists of gene transcription utilizing the regulatory elements/oligonucleotides of the present invention in appropriate DNA constructs and transfected host cells.
  • the present invention provides a unique selective assay for agonists and antagonists of STAT heterodimer pathways, including the STATl ⁇ /STAT3 heterodimer pathway.
  • the agonist and antagonist compounds discovered utilizing these methods can serve as pharmaceutical agents in the intervention of various cytokine-induced disease states and conditions, or to ameliorate disease states caused by cytokine deficiency, such as inflammation, infection, anemia, cytopenia and cancerous or precancerous conditions.
  • IL-4 or 10 ng/ml human GM-CSF both from R&D Systems, Minneapolis, MN for 30 minutes when the cells were at a density of 2-5xl0 5 cells/ml.
  • Nuclear extracts were prepared by NP40 lysis as described in H.B. Sadowski and M.Z. Gilman, 362 Nature, 79 (1993), the disclosure of which is herein incorporated by reference. Protein concentrations were measured using the Bradford dye binding assay (BioRad Laboratories, Hercules, CA).
  • the GAS oligonucleotide based on an IFN ⁇ responsive element in the Ly6E gene promoter was formed by annealing oligonucleotides with the sequences:
  • double-stranded probe oligonucleotides incorporating the regulatory elements of the present invention, used in the Electrophoretic Mobility Shift Assays (EMS As) were formed by annealing oligonucleotides with the sequences:
  • nucleotide sequences shown in bold face type correspond to the nucleotide sequences, including their double stranded complement, tested for activity as regulatory elements according to the present invention.
  • oligonucleotides were labeled by filling in the overhanging ends with Klenow fragment (Boehringer Mannheim) in the presence of [ ⁇ 32 P]- dGTP or dATP (Amersham Corporation, Arlington Heights, EL). Oligonucleotides were purchased from National Biosciences (Plymouth MN) or Integrated DNA Technologies (Coralville, IA). EMSAs were performed in 13 mM HEPES buffer (Sigma Chemical, St.
  • FIG. 2 shows the ability of oligonucleotide sequence 8T (SEQ ID NO. 53-54) to compete for specific cytokine-activated transcriptional regulatory protein complexes.
  • SEQ ID NO. 53-54 oligonucleotide sequence 8T
  • the complex being rated is the slowest-migrating IL-6 induced complex whose position is indicated by the "B" in FIG. 2, and which consists of a homodimer of STAT3 (Z. Zhong et al., 264 Science. 95 (1994); Raz, et al., 269 J. Biol. Chem. 24391-24391 (1994)), and thus is not reflective of complexes that contain STATl ⁇ (p91), which is rated in the IFN ⁇ complex.
  • sequences fall into 2 classes; those that bind to die IFN- ⁇ activated protein STATl ⁇ , but not to the proteins activated by IL-6, GM-CSF or IL-4, and those that bind to the IFN- ⁇ activated proteins STATl ⁇ and to the IL-6 activated protein STAT3, but not to the proteins activated by GM-CSF and IL-4.
  • An example of the former class of sequences is sequence 8T (SEQ ID NO.53- 54), and of the latter class is sequence 7C (SEQ ID NO.49-50).
  • sequence 8T The remarkable selectivity of sequence 8T is demonstrated in FIG. 2.
  • This sequence competes for binding to the STATl ⁇ complex induced by IFN- ⁇ (indicated by the "A” in FIG. 2), as evidenced by the reduction in the intensity of this band as increasing amounts of 8T are added to the reaction.
  • the STAT3 complex induced by IL-6 (labeled “B” in FIG. 2) and the complexes induced by GM- CSF and IL-4 are unaffected by the presence of this sequence.
  • IL-6 induces a small amount of STATl ⁇ in He ⁇ G2 cells (P. Lamb et al., 83 Blood 2063), and this complex is also reduced in intensity by the addition of sequence 8T.
  • reporter plasmids were constructed by cloning the regulatory elements into a modified pZLUC plasmid that contains the HSV TK promoter from -35 to +10 as described in Shuai et al., 261 Science. 1744-1746 (1993), the disclosure of which is herein incorporated by reference.
  • HepG2 cells were transfected by calcium phosphate coprecipitation. Cells were seeded at 2X10 ⁇ /ml the day before transfection.
  • cytokines were then added directly to the medium (IFN- ⁇ , 5 ng/ml; IL-6, 10 ng/ml; LIF 10 ng/ml and OSM 10 ng/ml) and the cells harvested 5 hours later.
  • Cells were lysed and luciferase and ⁇ -galactosidase activities determined using standard techniques. For each sample the normalized response was determined by dividing relative light units obtained from the luciferase assay with the ⁇ -galactosidase activity in the same lysate as determined using a chromogenic substrate. Each point represents the average normalized response from three experiments.
  • Reporter plasmids constructed as described above, and containing a single copy of the Ly6E GAS element or the 4G (SEQ ID NOs. 29,30), 8T (SEQ ID NOs. 53,54), 6T (SEQ ID NOs. 41,42), 2C (SEQ ID NOs. 19,20), 9G (SEQ ID NOs. 59,60) and 7C (SEQ ID NOs. 49,50) regulatory element-containing oligonucleotide sequences of the present invention were introduced into HepG2 cells and tested for their ability to induce luciferase activity in response to either IFN- ⁇ , IL-6, LIF or OSM.
  • reporter plasmids containing single regulatory elements according to the present invention exhibit surprising selectivity in their cytokine responsiveness.
  • Reporter constructs containing 4 copies of the regulatory elements 4G, 7C, 8T and 8 A, as well as 4 copies of the Ly6E GAS element were assayed for responsiveness to four test cytokines (FIG.4). All constructs except 4x4G and 4x8T were responsive to BL-6 and OSM, and gave much larger inductions than the constructs with the corresponding single elements. The degree of induction was not always related to the ability of the elements to bind to STATs in vitro, however. Thus the 4x7C reporter gives lower inductions than the wild type 4xLy6E reporter, despite binding STATsl ⁇ and 3 better in vitro.
  • constructs containing 4 copies of the 4G and 8T regulatory element- containing oligonucleotide sequences did not give responses to any cytokine tested.
  • Botii of these oligonucleotides contain regulatory elements that bind preferentially to STATl ⁇ in the in vitro binding assay, although they do so less avidly ti an the wild type Ly6E GAS element, or the 7C or 8 A regulatory-element containing oligonucleotide sequences, scoring at a level of 3 as opposed to 4 or 5 (FIG. 1).
  • IFN- ⁇ , IL-6, OSM and LIF The differential ability of IFN- ⁇ , IL-6, OSM and LIF to activate transcription from regulatory element-containing promoters prompted us to characterize the STATs induced by these cytokines in HepG2 cells.
  • Cells were treated with either IFN- ⁇ , IL-6, OSM or LIF, and lysates were immunoprecipitated with either anti-STATl or anti-STAT3 sera (available from Upstate Biotechnology, Inc., New York).
  • the immunoprecipitates were fractionated on SDS-polyacrylamide gels, blotted and proteins detected using a phosphotyrosine antibody. As controls, aliquots of the same immunoprecipitations were blotted and detected witii either anti-STATl or anti-STAT3 sera.
  • IFN-7 induces rapid tyrosine phosphorylation of STAT 1 ⁇ but no detectable phosphorylation of STAT3.
  • LIF induces tyrosine phosphorylation primarily of STAT3, with very low amounts of STATl ⁇ becoming phosphorylated.
  • IL-6 and OSM induce tyrosine phosphorylation of both STATl ⁇ and STAT3, OSM inducing slightly more phosphorylation of both STATs than IL-6.
  • IFN-7 activates a homodimer of STATl ⁇
  • LIF induces primarily a homodimer of STAT3 and IL-6
  • OSM induce homodimers of STATl ⁇ and STAT3 as well as heterodimers of STATl ⁇ and STAT3.
  • the differences in the ability of IFN- ⁇ , BL-6, OSM and LIF to activate transcription from regulatory element containing reporters is therefore accompanied by differences in their ability to activate STATl ⁇ , STAT 3, or both.
  • reporter plasmids containing single regulatory elements are responsive to cytokines that activate two STATs, in this case STATl ⁇ and STAT3, such that they form heterodimers.
  • the heterodimer is able to activate transcription from single regulatory elements, whereas homodimers of STATl ⁇ or STAT3 are not.
  • Reporters containing single regulatory elements, including the elements of the present invention are therefore useful for selectively detecting agonists that mimic the ability of cytokines such as IL-6 to activate both STATl ⁇ and STAT3, allowing heterodimer formation.
  • the DNA constructs are also useful for detecting antagonists of these cytokines.
  • DNA reporter constructs containing multimers of regulatory elements, such as 7C and 8 A, are responsive to cytokines that activate STATl ⁇ homodimers or STATl ⁇ /STAT3 heterodimers, but not STAT3 homodimers.
  • These DNA reporter constructs are therefore useful for selectively detecting agonists that mimic the ability of cytokines, such as IFN ⁇ or IL-6, to activate STATl ⁇ homodimers or STATl ⁇ /STAT3 heterodimers.
  • the DNA constructs are also useful for detecting antagonists of these cytokines.

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Abstract

Séquences d'oligonucléotides comprenant des éléments régulateurs d'ADN comprenant des mutations ponctuelles de l'élément Ly6E GAS qui lient des protéines régulatrices transcriptionnelles activées en réponse à des molécules de signalisation telles que des cytokines. Cette invention concerne également des structures d'ADN comprenant les séquences d'oligonucléotides, des cellules transfectées avec les structures d'ADN et des procédés d'utilisation desdites structures d'ADN et des cellules transfectées pour assurer l'expression régulée de gènes de structure, pour détecter et récupérer des protéines régulatrices de transcription et pour mesurer la capacité de composés à agir comme agonistes ou antagonistes de la transcription génique.
PCT/US1995/004511 1994-04-14 1995-04-10 Elements de regulation de l'adn sensibles aux cytokines WO1995028492A1 (fr)

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WO1996030515A1 (fr) * 1995-03-27 1996-10-03 Ligand Pharmaceuticals Incorporated Procedes et reactifs associes pour detecter des modulateurs de l'activite des cytokines
EP0808375A1 (fr) * 1995-02-10 1997-11-26 Center For Blood Research, Inc. Sites de liaison de l'adn et de lil-stat et methode d'identification d'agents de liaison inhibiteurs
FR2775003A1 (fr) * 1998-02-19 1999-08-20 Transgene Sa Methode de criblage de composes capables d'inhiber la fixation entre le facteur de transcription stat1 et le facteur de transcription usf1
WO2001032912A1 (fr) * 1999-11-01 2001-05-10 Sahltech I Göteborg AB Nouvelle utilisation d'un systeme jak-stat
EP1136570A2 (fr) * 2000-03-22 2001-09-26 Kabushiki Kaisha Toshiba Support pour la détection des gènes et son utilisation pour la détection de l'efficacité de la thérapie par interféron
EP1136571A2 (fr) * 2000-03-22 2001-09-26 Kabushiki Kaisha Toshiba Prédiction de réponse de la thérapie par interferons chez un individu infecté par le virus de l' hépatite C par détection des polymorphismes du gène MxA
WO2005001478A1 (fr) * 2003-06-30 2005-01-06 Biovitrum Ab Procedes pour identifier des agents regulant les cytokines

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A. ALTMEYER ET AL.;: "Multiple cytokine interactions regulate Ly-6E antigen expression: cooperative Ly-6E induction by IFNs, TNF, and IL-1 in a T cell lymphoma and in its induction-deficient variants", CELL. IMMUNOL., vol. 138, pages 94 - 107 *
A. BOTHWELL ET AL.;: "Isolation and expression of an IFN-responsive Ly-6C chromosomal gene", J. IMMUNOL., vol. 140, pages 2815 - 2820 *
K.D. KHAN ET AL.;: "Induction of the Ly-6A/E gene by interferon alpha/beta or gamma requires a DNA element to which a tyrosine-phosphorylated 91-kDa protein binds", PROC. NATL. ACAD. SCI. USA, vol. 90, pages 6806 - 6810 *
P. LAMB ET AL.;: "Rapid activation of proteins that interact with the interferon gamma activation site in response to multiple cytokines", BLOOD, vol. 83, 18 April 1994 (1994-04-18), pages 2063 - 2071 *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0808375A1 (fr) * 1995-02-10 1997-11-26 Center For Blood Research, Inc. Sites de liaison de l'adn et de lil-stat et methode d'identification d'agents de liaison inhibiteurs
EP0808375A4 (fr) * 1995-02-10 1999-12-29 Blood Res Center Sites de liaison de l'adn et de lil-stat et methode d'identification d'agents de liaison inhibiteurs
WO1996030515A1 (fr) * 1995-03-27 1996-10-03 Ligand Pharmaceuticals Incorporated Procedes et reactifs associes pour detecter des modulateurs de l'activite des cytokines
US5712094A (en) * 1995-03-27 1998-01-27 Ligand Pharmaceuticals, Inc. Methods for detecting modulators of cytokine action
FR2775003A1 (fr) * 1998-02-19 1999-08-20 Transgene Sa Methode de criblage de composes capables d'inhiber la fixation entre le facteur de transcription stat1 et le facteur de transcription usf1
WO1999042571A1 (fr) * 1998-02-19 1999-08-26 Novimmune S.A. Methode de criblage de composes capables d'inhiber la fixation entre le facteur de transcription stat1 et le facteur de transcription usf1
US6379894B1 (en) 1998-02-19 2002-04-30 Novimaune S.A. Method for screening compounds capable of inhibiting binding between the transcription factor of STAT1 and the transcription factor of USF1
WO2001032912A1 (fr) * 1999-11-01 2001-05-10 Sahltech I Göteborg AB Nouvelle utilisation d'un systeme jak-stat
EP1136571A2 (fr) * 2000-03-22 2001-09-26 Kabushiki Kaisha Toshiba Prédiction de réponse de la thérapie par interferons chez un individu infecté par le virus de l' hépatite C par détection des polymorphismes du gène MxA
WO2001071031A2 (fr) * 2000-03-22 2001-09-27 Kabushiki Kaisha Toshiba Porteur pour detection genique et son utilisation pour detecter la validite d'une therapie interferons
WO2001071007A2 (fr) * 2000-03-22 2001-09-27 Kabushiki Kaisha Toshiba Polymorphisme genetique de la proteine et son utilisation
EP1136570A2 (fr) * 2000-03-22 2001-09-26 Kabushiki Kaisha Toshiba Support pour la détection des gènes et son utilisation pour la détection de l'efficacité de la thérapie par interféron
EP1136570A3 (fr) * 2000-03-22 2002-11-13 Kabushiki Kaisha Toshiba Support pour la détection des gènes et son utilisation pour la détection de l'efficacité de la thérapie par interféron
EP1136571A3 (fr) * 2000-03-22 2002-11-13 Kabushiki Kaisha Toshiba Prédiction de réponse de la thérapie par interferons chez un individu infecté par le virus de l' hépatite C par détection des polymorphismes du gène MxA
WO2001071007A3 (fr) * 2000-03-22 2003-06-12 Toshiba Kk Polymorphisme genetique de la proteine et son utilisation
WO2001071031A3 (fr) * 2000-03-22 2003-09-12 Toshiba Kk Porteur pour detection genique et son utilisation pour detecter la validite d'une therapie interferons
US6667155B2 (en) 2000-03-22 2003-12-23 Kabushiki Kaisha Toshiba Carrier for gene detection and its use for detecting validity of interferon therapy
US7659068B1 (en) 2000-03-22 2010-02-09 Kabushiki Kaisha Toshiba Carrier for gene detection and its use for detecting validity of interferon therapy
WO2005001478A1 (fr) * 2003-06-30 2005-01-06 Biovitrum Ab Procedes pour identifier des agents regulant les cytokines

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