Nucleic acid binding of multi-zinc finger transcription factors

Abstract

A method of identifying transcription factors comprising providing cells with a nucleic acid sequence at least comprising a sequence CACCT (SEQ ID NO: ______ ) as bait for the screening of a library encoding potential transcription factors and performing a specificity test to isolate said factors. Preferably, the bait comprises twice the CACCT (SEQ ID NO: ______ ) sequence, more particularly the bait comprises one of the sequences CACCT-N-CACCT(SEQ ID NO: ______ ), CACCT-N-AGGTG(SEQ ID NO: ______ ), AGGTG-N-CACCT(SEQ ID NO: ______ ), or AGGTG-N-AGGTG (SEQ ID NO: ______ ) wherein N is a spacer sequence. The transcription factors identified using the methods of the invention include separated clusters of zinc fingers, such as, for example, a two-handed zinc finger transcription factor. Also, at least one such zinc finger transcription factor, denominated as SIP1, induces tumor metastasis by down regulation of the expression of E-cadherin. Compounds interfering with SIP1 activity can thus be used to prevent tumor invasion and metastasis.

What is claimed is:  
     
         1 . A process of identifying transcription factors such as activators and/or repressors comprising: 
 providing cells with a nucleic acid sequence at least comprising a sequence CACCT (the first 5 nucleotides of SEQ ID NO: 1), preferably twice a CACCT sequence (the first 5 nucleotides of SEQ ID NO: 1), as bait(s) for the screening of a library encoding potential transcription factors and    performing a specificity test to isolate said transcription factors.    
     
     
         2 . A process of identifying transcription factors such as activators and/or repressors comprising: 
 providing cells with a nucleic acid sequence comprising one of the sequences CACCT-N-CACCT (SEQ ID NO: 1), CACCT-N-AGGTG (SEQ ID NO: 2), AGGTG-N-CACCT (SEQ ID NO: 3), or AGGTG-N-AGGTG (SEQ ID NO: 4) as bait wherein N is a spacer sequence.    
     
     
         3 . A process according to  claim 1  or  claim 2  wherein the transcription factor comprises separated clusters of zinc fingers.  
     
     
         4 . A process according to  claim 1 ,  claim 2 , or  claim 3  wherein the sequence originates from a promoter region.  
     
     
         5 . A process according to  claim 4  wherein the promoter region is selected from the group consisting of Brachyury, a4-integrin, follistatin, and E-cadherin.  
     
     
         6 . A transcription factor produced by the process of  claim 1 ,  claim 2 ,  claim 3 ,  claim 4 , or  claim 5 .  
     
     
         7 . A process for identifying compounds with an interference capability towards transcription factors as defined in  claim 6  by 
 adding a sample comprising a potential compound to be identified to a test system comprising: 
 (i) a nucleotide sequence comprising one of the sequences CACCT-N-CACCT (SEQ ID NO: 1), CACCT-N-AGGTG (SEQ ID NO: 2), AGGTG-N-CACCT (SEQ ID NO: 3), or AGGTG-N-AGGTG (SEQ ID NO: 4) as bait wherein N is a spacer, and (ii) a protein capable to bind said nucleotide sequence,  
 
 incubating said sample in said system for a period of time sufficient to permit interaction of the compound or its derivative or counterpart thereof with said protein,  
 comparing the amount and/or activity of the protein bound to the nucleotide sequence before and after said adding and  
 identification and optionally isolation and/or purification of the compound.  
 
     
     
         8 . The process according to  claim 7  wherein the protein is a Smad-interacting protein.  
     
     
         9 . The process according to  claim 8 , wherein said Smad-interacting protein is SIP 1.  
     
     
         10 . A compound produced by the process of  claim 7 ,  claim 8 , or  claim 9 .  
     
     
         11 . The compound of  claim 10 , wherein said compound modifies regulation of E-cadherin expression by SIP 1.  
     
     
         12 . A pharmaceutical composition to prevent tumor invasion and/or metastasis, said pharmaceutical composition comprising: 
 the compound of  claim 10  or  claim 11  in an amount to prevent tumor invasion and/or metastasis in a subject, and    a pharmaceutically acceptable excipient.    
     
     
         13 . A test kit to perform the process of  claim 7 , said test kit comprising: 
 a nucleotide sequence comprising a sequence selected from the group consisting of CACCT-N-CACCT (SEQ ID NO: 1), CACCT-N-AGGTG (SEQ ID NO: 2), AGGTG-N-CACCT (SEQ ID NO: 3), and AGGTG-N-AGGTG (SEQ ID NO: 4) wherein N is a spacer sequence and    (ii)a protein capable of binding said nucleotide sequence.    
     
     
         14 . A test kit to perform the process of  claim 2 , said test kit comprising: 
 a nucleic acid sequence comprising one of the sequences CACCT-N-CACCT (SEQ ID NO: 1), CACCT-N-AGGTG (SEQ ID NO: 2), AGGTG-N-CACCT (SEQ ID NO: 3), or AGGTG-N-AGGTG (SEQ ID NO: 4), wherein N is a spacer sequence.    
     
     
         15 . A method for detecting an interaction between a first interacting protein and a second interacting protein comprising: 
 providing a suitable host cell with a first fusion protein comprising a first interacting protein fused to a DNA binding domain capable to bind a nucleic acid sequence comprising one of the sequences CACCT-N-CACCT (SEQ ID NO: 1), CACCT-N-AGGTG (SEQ ID NO: 2), AGGTG-N-CACCT (SEQ ID NO: 3), or AGGTG-N-AGGTG (SEQ ID NO: 4) wherein N is a spacer sequence,    providing said suitable host cell with a second fusion protein comprising a second interacting protein fused to a DNA binding domain capable to bind a nucleic acid sequence comprising one of the sequences CACCT-N-CACCT (SEQ ID NO: 1), CACCT-N-AGGTG (SEQ ID NO: 2), AGGTG-N-CACCT (SEQ ID NO: 3) or AGGTG-N-AGGTG (SEQ ID NO: 4) wherein N is a spacer sequence,    subjecting said host cell to conditions under which the first interacting protein and the second interacting protein are brought into close proximity and determining whether a detectable gene present in the host cell and located adjacent to said nucleic acid sequence has been expressed to a greater degree than if expressed in the absence of the interaction between the first and the second interacting protein.    
     
     
         16 . An isolated nucleic acid sequence comprising a sequence selected from the group consisting of CACCT-N-CACCT (SEQ ID NO: 1), CACCT-N-AGGTG (SEQ ID NO: 2), AGGTG-N-CACCT (SEQ ID NO: 3), and AGGTG-N-AGGTG (SEQ ID NO: 4) wherein N is a spacer.  
     
     
         17 . A method of identifying a new target gene, said method comprising: 
 identifying said new target gene using a nucleic acid sequence, said nucleic acid sequence comprising a sequence selected from the group consisting of CACCT (the first five nucleotides of SEQ ID NO: 1), CACCT-N-CACCT (SEQ ID NO: 1), CACCT-N-AGGTG (SEQ ID NO: 2), AGGTG-N-CACCT (SEQ ID NO: 3), and AGGTG-N-AGGTG (SEQ ID NO: 4) wherein N is a spacer.