Inducible methods for repressing gene function

Abstract

Methods for the rapid repression of gene function in eucaryotic cells are disclosed including inducible means for both shutting down a targeted gene's transcription and rapidly removing a targeted gene's polypeptide product.

We claim:  
     
         1 . A method for inducibly repressing the transcription of a target gene expressed from a repressible promoter comprising: 
 providing said target gene expressed from said repressible promoter,    providing a transcriptional repressor expressed from an inducible promoter, which transcriptional repressor represses said repressible promoter, and    providing an inducing agent that induces said inducible promoter in order to express said transcriptional repressor and repress said repressible promoter,    thereby causing the transcriptional repression of said target gene.    
     
     
         2 . The method of  claim 1 , wherein the target gene encodes a chimeric polypeptide comprising ubiquitin and a target polypeptide.  
     
     
         3 . The method of  claim 1 , wherein the repressible promoter is a promoter from a gene selected from the group consisting of ANB1, HEM13, ERG 11, OLE 1, GAL1, GAL10, and TET R .  
     
     
         4 . The method of  claim 1 , wherein the transcriptional repressor is selected from the group consisting of ROX1, Tet repressor, and lacI repressor.  
     
     
         5 . The method of  claim 1 , wherein the inducible promoter is a copper-inducible promoter.  
     
     
         6 . The method of  claim 1 , wherein the inducible promoter is induced by Cu +2 , tetracycline, or a gratuitous inducer of the lac operon.  
     
     
         7 . A method for inducibly degrading a target polypeptide wherein said target polypeptide is provided as a ubiquitin-target polypeptide fusion protein in which a specific amino terminal amino acid residue of said target polypeptide is contiguously joined by a peptide bond to a carboxyl terminal residue of a ubiquitin polypeptide, said method comprising: 
 providing said ubiquitin-target polypeptide fusion protein,    providing a ubiquitin isopeptidase which endoproteolytically cleaves said peptide bond thereby liberating said specific amino terminal amino acid residue of said target polypeptide from said carboxyl terminal residue of said ubiquitin polypeptide,    providing an inducible means for the proteolytic destruction of said target polypeptide possessing said liberated amino terminal amino acid residue, and    inducing said inducible proteolytic means    thereby causing the inducible proteolytic destruction of said target polypeptide possessing said liberated amino terminal amino acid residue.    
     
     
         8 . The method of  claim 7 , wherein the target polypeptide is a TAF.  
     
     
         9 . The method of  claim 7 , wherein the specific amino terminal amino acid residue of said target polypeptide is arginine.  
     
     
         10 . The method of  claim 7 , wherein the specific amino terminal amino acid residue of said target polypeptide is selected from the group consisting of arginine, lysine and histidine.  
     
     
         11 . The method of  claim 7 , wherein the specific amino terminal amino acid residue of said target polypeptide is selected from the group consisting of phenylalanine, tryptophan, tyrosine, leucine, and isoleucine.  
     
     
         12 . The method of  claim 7 , wherein the specific amino terminal amino acid residue of said target polypeptide is selected from the group consisting of aspartate, glutamate, cysteine, asparagine and glutamine.  
     
     
         13 . The method of  claim 12 , wherein the inducible means for the proteolytic destruction of the target polypeptide is an inducible transgene encoding an R-transferase.  
     
     
         14 . The method of  claim 12 , wherein the specific amino terminal amino acid residue of said target polypeptide is glutamine or asparagine and the inducible means for the proteolytic destruction of the target polypeptide is an inducible transgene encoding a deamidase specific for an amino-terminal glutamine or asparagine.  
     
     
         15 . The method of  claim 7 , wherein said inducible proteolytic means is an inducible transgene encoding a component of the N-end rule system for ubiquitin dependent proteolytic destruction.  
     
     
         16 . The method of  claim 15 , wherein said component of the N-end rule system is selected from the group consisting of UBR1, UBC2, NTA1, and ATE1.  
     
     
         17 . The method of  claim 15 , wherein said component of the N-end rule system is selected from the group consisting of mouse UBR1p and human UBR1p.  
     
     
         18 . A method for repressing the function of a target gene expressing a target polypeptide by repressing the transcription of said target gene and repressing the stability of said target polypeptide, said method comprising: 
 inducibly repressing the transcription of said target gene by the method of  claim 1 , or    inducibly repressing the stability of said target polypeptide by the method of  claim 7     thereby repressing the function of said target gene by repressing the transcription of said target gene and repressing the stability of said target polypeptide.    
     
     
         19 . A method for repressing the function of a target gene expressing a target polypeptide by repressing the transcription of said target gene and degrading said target polypeptide, said method comprising: 
 inducibly repressing the transcription of said target gene, and    inducibly degrading said target polypeptide by the method of  claim 7     thereby repressing the function of said target gene by repressing the transcription of said target gene and degrading said target polypeptide.    
     
     
         20 . A method for repressing the function of a target gene expressing a target polypeptide by repressing the transcription of said target gene and degrading said target polypeptide, said method comprising: 
 inducibly repressing the transcription of said target gene, and    inducibly degrading said target polypeptide    thereby repressing the function of said target gene by repressing the transcription of said target gene and degrading said target polypeptide.    
     
     
         21 . A eukaryotic cell containing: 
 a target gene encoding a target polypeptide    an inducible transgene encoding proteolytic means for the degradation of a target polypeptide encoded by a target gene.    
     
     
         22 . The eukaryotic cell of  claim 21 , wherein said inducible transgene comprises a component of the N-end rule proteolytic system.  
     
     
         23 . The eukaryotic cell of  claim 22 , wherein said component of the N-end rule proteolytic system is selected from the group consisting of UBR1, UBC2 and NTA1.  
     
     
         24 . The eukaryotic cell of  claim 21 , in which said target gene is expressed from a repressible promoter.  
     
     
         25 . The eukaryotic cell of  claim 24 , further containing an inducible transcriptional repressor, which transcriptional repressor represses said repressible promoter.  
     
     
         26 . The eukaryotic cell of  claim 25 , wherein said repressible promoter is a promoter from a gene selected from the group consisting of ANB1, HEM13, ERG11, OLE1, GAL1, GAL10, and TET R .  
     
     
         27 . The eukaryotic cell of  claim 25 , wherein said transcriptional repressor is selected from the group consisting of ROX1, Tet repressor, and lacI repressor.  
     
     
         28 . A target gene chimera comprising a ubiquitin polypeptide and a target polypeptide.  
     
     
         29 . The target gene chimera of  claim 28 , further comprising a specific amino terminal amino acid residue of said target polypeptide wherein said specific amino terminal amino acid residue is contiguously joined by a peptide bond to a carboxyl terminal reside of said ubiquitin polypeptide.  
     
     
         30 . The target gene chimera of  claim 29 , in which said specific amino terminal amino acid residue is arginine.  
     
     
         31 . The target gene chimera of  claim 29 , in which said specific amino terminal amino acid residue is selected from the group consisting of arginine, lysine and histidine.  
     
     
         32 . The target gene chimera of  claim 29 , in which said specific amino terminal amino acid residue is selected from the group consisting of phenylalanine, tryptophan, tyrosine, leucine, and isoleucine.  
     
     
         33 . The target gene chimera of  claim 29 , in which said specific amino terminal amino acid residue is selected from the group consisting of aspartate, glutamate, cysteine, asparagine and glutamine.  
     
     
         34 . The target gene chimera of  claim 29 , further comprising an epitope tag.  
     
     
         35 . The target gene chimera of  claim 29 , further comprising a segment of the lacI repressor carboxyl to said specific amino terminal amino acid residue.  
     
     
         36 . The target gene chimera of  claim 29 , further comprising a lysine residue carboxyl to said specific amino terminal amino acid residue.  
     
     
         37 . A eukaryotic cell expressing the target gene chimera of  claim 29 .  
     
     
         38 . The target gene chimera of  claim 29 , wherein said target polypeptide is a transcriptional repressor which represses an inducible promoter.  
     
     
         39 . The method of  claim 1 , wherein the inducible promoter is repressed by a derepressible repressor and wherein said derepressible repressor is further subject to targeted proteolysis by the N-end rule system.