Synthetic transcriptional modulators and uses thereof

Abstract

Novel synthetic transcriptional modulators having at least one selected ligand linked to at least one transcriptional modulating portion are described. The transcriptional modulators of the present invention can include a ligand linked to a chemical moiety. These transcriptional modulators can be used to selectively control gene expression and to identify components of the transcriptional machinery.

We claim:  
     
         1 . A method for identifying a transcriptional modulator, comprising: 
 providing a cell comprising (i) a genetic construct encoding a chimeric protein which comprises at least one ligand-binding domain and a DNA-binding domain which is heterologous thereto, wherein the ligand-binding domain binds to a selected ligand, and (ii) a target gene under the control of at least one transcriptional regulatory element which is recognized by the DNA-binding domain of the chimeric protein;    contacting the cell with a test compound which comprises the selected ligand linked to a test transcriptional modulating portion under conditions which allow transcription to occur; and    detecting any changes in transcriptional activity in the presence of the test compound relative to that detected in the absence of the test compound, wherein a change in the level of transcriptional activity of the target gene detected in the presence of the test compound relative to that detected in the absence of the test compound indicates that the test compound is a transcriptional modulator.    
     
     
         2 . A method for identifying a transcriptional modulator, comprising: 
 providing a reaction mixture under conditions which allow transcription to occur, said reaction mixture including, 
 (i) a chimeric protein which comprises at least one ligand-binding domain and a DNA-binding domain which is heterologous thereto, wherein the ligand-binding domain binds to a selected ligand;  
 (ii) a target gene under the control of at least one transcriptional regulatory element which is recognized by the DNA-binding domain of the chimeric protein;  
 (iii) a cell-free transcription system;  
 (iv) a test compound which comprises the selected ligand linked to a test transcriptional modulating portion; and  
   detecting any changes in transcriptional activity of the target gene in the presence of the test compound relative to that detected in the absence of the test compound,    wherein a change in the level of transcriptional activity of the target gene detected in the presence of the test compound relative to that detected in the absence of the test compound indicates that the test compound is a transcriptional modulator.    
     
     
         3 . A method for identifying a transcriptional modulator from a plurality of test compounds, comprising: 
 providing cells comprising, 
 (i) a genetic construct encoding a chimeric protein which comprises at least one ligand-binding domain and a DNA-binding domain which is heterologous thereto, wherein the ligand-binding domain binds to a selected ligand;  
 (ii) a target gene under the control of at least one transcriptional regulatory element which is recognized by the DNA-binding domain of the chimeric protein;  
   contacting an aliquot of the cells with one or more test compounds, each of which comprises the selected ligand linked to at least one of a plurality of test transcriptional modulating portions under conditions which allow transcription to occur; and    detecting any changes in transcriptional activity of the target gene in the presence of a given test compound relative to that detected in the absence of the test compound,    wherein a change in the level of transcriptional activity of the target gene detected in the presence of the test compound relative to that detected in the absence of the test compound indicates that the test compound is a transcriptional modulator.    
     
     
         4 . The method of  claim 1 , wherein the transcriptional modulating portion is a chemical moiety.  
     
     
         5 . The method of  claim 1 , wherein the transcriptional modulating portion is a proteinaceous domain.  
     
     
         6 . The method of  claim 1 , wherein the chimeric protein is in the nucleus of the cell.  
     
     
         7 . The method of  claim 1 , wherein the chimeric protein is in the cytoplasm of the cell.  
     
     
         8 . The method of  claim 4 , wherein one or more of the test compound has a molecular weight of less than about 3 kD.  
     
     
         9 . The method of  claim 4 , wherein one or more of the test compound has a molecular weight of less than about 1.5 kD.  
     
     
         10 . The method of  claim 4 , wherein the test compound is membrane-permeant.  
     
     
         11 . The method of  claim 4 , wherein the transcriptional modulating portion is a portion suspected of having transcriptional activation or repressor activity.  
     
     
         12 . The method of  claim 4 , wherein the transcriptional modulating portion is a portion suspected of having nuclear import activity.  
     
     
         13 . The method of  claim 4 , wherein the transcriptional modulating portion is a portion suspected of having nuclear export activity.  
     
     
         14 . The method of  claim 4 , wherein the test compound comprises the selected ligand covalently linked to a transcriptional modulating portion.  
     
     
         15 . The method of  claim 4 , wherein the test compound is a member of a combinatorial library.  
     
     
         16 . The method of  claim 4 , wherein the changes in transcriptional activity are detected as variations in observed levels of mRNA, or protein product encoded by the target gene.  
     
     
         17 . The method of  claim 16 , wherein the target gene is selected from the group consisting of a gene encoding a protein conferring resistance to a drug, a gene encoding an enzyme, a gene which rescues an auxotrophic phenotype, and a gene encoding a cell surface antigen.  
     
     
         18 . The method of  claim 17 , wherein the target gene encodes a protein which provides for calorimetric, luminescent or fluorescent detection.  
     
     
         19 . The method of  claim 4 , wherein an increase in target gene activity or expression is indicative of a transcriptional activator.  
     
     
         20 . The method of  claim 4 , wherein a decrease in target gene activity or expression is indicative of a transcriptional repressor.  
     
     
         21 . The method of  claim 4 , wherein the transcriptional modulator is not itself the product of gene transcription or translation.  
     
     
         22 . The method of  claim 4 , wherein the transcriptional modulator is membrane-permeant.  
     
     
         23 . The method of  claim 4 , wherein the transcriptional modulator has a molecular weight of less than about 3 kDa.  
     
     
         24 . The method of  claim 4 , wherein the selected ligand is selected from the group consisting of FK506, FK520, rapamycin, cyclosporin A, tetracycline, steroid ligand, and derivatives thereof which are modified to have a higher binding affinity for the ligand-binding domain compared to unmodified forms.  
     
     
         25 . The method of  claim 4 , wherein the ligand-binding domain of the chimeric protein comprises between about 50 and 350 amino acids.  
     
     
         26 . The method of  claim 4 , wherein the ligand-binding domain of the chimeric protein is less than about 200 amino acid residues in length.  
     
     
         27 . The method of  claim 4 , wherein the ligand-binding domain of the chimeric protein binds to the ligand with an affinity of less than or equal to about 10 −6  M.  
     
     
         28 . The method of  claim 4 , wherein the ligand-binding domain of the chimeric protein comprises an immunophilin domain, a cyclophilin domain, a steroid binding domain, an antibiotic domain, an antibody domain, a dihydrofolate reductase (DHFR) domain, or a DNA gyrase domain.  
     
     
         29 . The method of  claim 4 , wherein the ligand-binding domain comprises an FK506 binding protein (“FKBP”) 12 or a variant thereof which is modified to have a higher binding affinity for the selected ligand compared to an unmodified form.  
     
     
         30 . The method of  claim 29 , wherein the FKBP12 variant comprises up to 10 amino acid substitutions relative to wild-type FKBP.  
     
     
         31 . The method of  claim 29 , wherein the FKBP12 variant comprises one or more substitutions of Tyr 26, Phe 36, Asp 37, Tyr 82 and Phe 99 with a different amino acid.  
     
     
         32 . The method of  claim 29 , wherein the FKBP12 is encoded by a nucleotide sequence capable of selectively hybridizing to a nucleotide sequence encoding an FKBP or a variant thereof.  
     
     
         33 . The method of  claim 4 , wherein the DNA-binding domain is selected from the group consisting of a homeodomain, and a zinc finger domain.