Methods for genome annotation

Abstract

The present disclosure provides methods and compositions for identifying transcribed regions in a genome. The methods include the use of exogenous molecules such as zinc finger proteins which are capable of binding to and modulating transcription, followed by assay for one or more selected phenotypes.

1 . A method for identifying a transcribed sequence in a region of interest in a genome; wherein the method comprises: 
 (a) contacting a cell with an exogenous molecule comprising a transcriptional regulatory domain, wherein the exogenous molecule binds within the region of interest; and    (b) assaying the cell for at least one selected phenotype;    wherein, if one or more of the selected phenotypes are observed, the region of interest contains a transcribed sequence.    
     
     
         2 . The method of  claim 1 , wherein the transcribed sequence encodes a protein.  
     
     
         3 . The method of  claim 1 , wherein the transcribed sequence is an RNA selected from the group consisting of structural RNA, regulatory RNA, enzymatic RNA, antisense RNA, ribozyme, ribosomal RNA and transfer RNA.  
     
     
         4 . The method of  claim 1 , wherein the exogenous molecule is a zinc finger protein.  
     
     
         5 . The method of  claim 1 , wherein the exogenous molecule binds within the transcribed sequence.  
     
     
         6 . The method of  claim 1 , wherein the exogenous molecule binds outside of the transcribed sequence.  
     
     
         7 . The method of  claim 1 , wherein the transcriptional regulatory domain is an activation domain.  
     
     
         8 . The method of  claim 7 , wherein the activation domain is selected from the group consisting of VP16, p65 and functional fragments thereof.  
     
     
         9 . The method of  claim 1 , wherein the transcriptional regulatory domain is a repression domain.  
     
     
         10 . The method of  claim 9 , wherein the repression domain is selected from the group consisting of KRAB, v-erbA and functional fragments thereof.  
     
     
         11 . The method of  claim 1 , wherein the transcriptional regulatory domain is a bifunctional domain (BFD), wherein the activity of the bifunctional domain is dependent upon interaction of the BFD with a second molecule.  
     
     
         12 . The method of  claim 11 , wherein the BFD is selected from the group consisting of thyroid hormone receptor, retinoic acid receptor, estrogen receptor, glucocorticoid receptor and functional fragments thereof.  
     
     
         13 . The method of  claim 11 , wherein the second molecule is a protein.  
     
     
         14 . The method of  claim 11 , wherein the second molecule is a small molecule.  
     
     
         15 . The method of  claim 14 , wherein the small molecule is selected from the group consisting of 3,5,3′-triiodo-L-thyronine (T3), all-trans-retinoic acid, estradiol, tamoxifen, 4-hydroxy-tamoxifen, RU-486 and dexamethasone.  
     
     
         16 . The method of  claim 1 , wherein the cell is an animal cell.  
     
     
         17 . The method of  claim 16  wherein the cell is a human cell.  
     
     
         18 . The method of  claim 1 , wherein the cell is a plant cell.  
     
     
         19 . The method of  claim 1 , wherein the phenotype is a change in a property selected from the group consisting of cell growth, cell cycle control, cellular physiology and cellular response to a pathogen.  
     
     
         20 . The method of  claim 1 , wherein the phenotype is expression of a RNA molecule.  
     
     
         21 . The method of  claim 1 , wherein the phenotype is an alteration in the transcriptional program of the cell.  
     
     
         22 . The method of  claim 1 , wherein the cell is infected with a virus.  
     
     
         23 . The method of  claim 22 , wherein the genome is a viral genome.