CCCTC-Binding Factor Variants

Abstract

Described herein are engineered CCCTC-binding factor (CTCF) variants that can bind to mutant CTCF binding sequences and method of using the same.

1 . An engineered CCCTC-binding factor (CTCF) variant comprising at least one amino acid residue in at least one zinc finger that differs in sequence from the amino acid sequence of a wild-type CTCF, wherein the engineered CTCF variant binds to a mutant CTCF binding sequence (CBS) with a higher affinity than wild-type CTCF, the mutant CBS comprising at least one nucleotide base that differs in sequence from the nucleotide sequence of a consensus CBS, wherein the at least one amino acid residue that differs in sequence from the amino acid sequence of the wild-type CTCF is selected from the group consisting of amino acid residues at position(s) −1, +1, +2, +3, +5, and +6 of any of ZF7, ZF6, ZF5, ZF4, and ZF3 of the engineered CTCF variant. 
     
     
         2 . The engineered CTCF variant of  claim 1 , wherein the mutant CBS has a Thymine (T), Adenine (A), or Guanine (G) residue at position 2 of the consensus CBS motif, the engineered CTCF comprising an amino acid residue threonine, asparagine, or histidine at ZF7 position +3. 
     
     
         3 . The engineered CTCF variant of  claim 1 , wherein the mutant CBS has a G residue at position 2 of the consensus CBS motif, the engineered CTCF comprising the amino acid sequence DHLQT, EHLNV, AHLQV, EHLRE, DHLQV, EHLKV, EHLVV, DHLRT, or DHLAT at ZF7 positions +2 to +6. 
     
     
         4 . (canceled) 
     
     
         5 . The engineered CTCF variant of  claim 1 , wherein the mutant CBS has a T, or A residue at position 5 of the consensus CBS motif, the engineered CTCF at ZF6 positions +2 to +6 comprising:
 the amino acid sequence NAMKR, GNMAR, EGMTR, SNMVR, or NAMRG; wherein the mutant CBS has a T residue at position 5 of the consensus CBS motif; or   the amino acid sequence EHMGR, DHMNR, THMKR, EHMRR, or THMNR, wherein the mutant CBS has a G residue at position 5 of the consensus CBS motif.   
     
     
         6 . The engineered CTCF variant of  claim 1 , wherein the mutant CBS has a T, or C residue at position 6 of the consensus CBS motif, the engineered CTCF at ZF6 positions −1 to +3 comprising:
 the amino acid sequence MNES or HRES, wherein the mutant CBS has a T residue at position 6 of the consensus CBS motif; or 
 the amino acid sequence RPDT, RTDI, or RHDT, wherein the mutant CBS has a G residue at position 6 of the consensus CBS motif. 
 
     
     
         7 . The engineered CTCF variant of  claim 1 , wherein the mutant CBS has a C, A, or T residue at position 7 of the consensus CBS motif, the engineered CTCF at ZF5 positions +2 to +6 comprising:
 the amino acid sequence HGLKV, HRLKE, HALKV, SRLKE, or DGLRV, wherein the mutant CBS has a T residue at position 7 of the consensus CBS motif;   the amino acid sequence HTLKV, or HGLKV, wherein the mutant CBS has an A residue at position 7 of the consensus CBS motif; or   the amino acid sequence SRLKE, HRLKE or NRLKE, wherein the mutant CBS has a C residue at position 7 of the consensus CBS motif.   
     
     
         8 . The engineered CTCF variant of  claim 1 , wherein the mutant CBS has a C, A, or T residue at position 8 of the consensus CBS motif, the engineered CTCF at ZF5 positions +2 to +6 comprising:
 the amino acid sequence ATLKR, QALRR, GGLVR, or HGLIR, wherein the mutant CBS has a T residue at position 8 of the consensus CBS motif;   the amino acid sequence ANLSR, TGLTR, HGLVR, or GGLTR, wherein the mutant CBS has an A residue at position 8 of the consensus CBS motif;   the amino acid sequence HTLRR, TVLKR, ADLKR, or HGLRR, wherein the mutant CBS has a C residue at position 8 of the consensus CBS motif.   
     
     
         9 . The engineered CTCF variant of  claim 1 , wherein the mutant CBS has a T, A, or C residue at position 10 of the consensus CBS motif, the engineered CTCF at ZF4 positions +2 to +6 comprising:
 the amino acid sequence AHLRK, wherein the mutant CBS has a T residue at position 10 of the consensus CBS motif;   the amino acid sequence AKLRV, EKLRI, or AKLRI, wherein the mutant CBS has an A residue at position 10 of the consensus CBS motif; or   the amino acid sequence TKLKV, wherein the mutant CBS has a C residue at position 10 of the consensus CBS motif.   
     
     
         10 . The engineered CTCF variant of  claim 1 , wherein the mutant CBS has a T, A, or C residue at position 11 of the consensus CBS motif, the engineered CTCF at ZF4 positions +2 to +6 comprising:
 the amino acid sequence ATLRR or RRLDR, wherein the mutant CBS has a T residue at position 11 of the consensus CBS motif;   the amino acid sequence TNLRR, ANLRR, or GNLTR, wherein the mutant CBS has an A residue at position 11 of the consensus CBS motif; or   the amino acid sequence AMLKR, HMLTR, AMLRR, or TMLRR, wherein the mutant CBS has a C residue at position 11 of the consensus CBS motif.   
     
     
         11 . The engineered CTCF variant of  claim 1 , wherein the mutant CBS has a T, A, or C residue at position 13 of the consensus CBS motif, the engineered CTCF at ZF3 positions +2 to +6 comprising:
 the amino acid sequence QQLIV, SQLIV, QQLLV, GELVV, or QQLLI, wherein the mutant CBS has a T residue at position 13 of the consensus CBS motif;   the amino acid sequence GQLIV, GQLTV, GKLVT, TELII or QGLLV, wherein the mutant CBS has an A residue at position 13 of the consensus CBS motif; or   the amino acid sequence QQLLT, GQLLT, GELLT, or QQLLI, wherein the mutant CBS has a C residue at position 13 of the consensus CBS motif.   
     
     
         12 . The engineered CTCF variant if  claim 1 , wherein the mutant CBS has A, T, and T residues at positions 2, 6, 7, and 10 of the consensus CBS motif, respectively, the engineered CTCF comprising:
 (i) the amino acid sequence AKLKK, AKLRK, AHLRV, AKLRV, or SKLRL at ZF4 positions +2 to +6 of the engineered CTCF;   (ii) the amino acid sequence ERLRV, NRLKV, SRLKE, or NRLKV at ZF5 positions +2 to +6 of the engineered CTCF;   (iii) the amino acid sequence RPDT, RTET, or RADV at ZF6 positions −1 to +3 of the engineered CTCF; and   (iv) the amino acid sequence DNLLA, SNLLV, DNLMA, or DNLRV at ZF7 positions +2 to +6 of the engineered CTCF.   
     
     
         13 . The engineered CTCF variant of  claim 1 , wherein the mutant CBS has G, G, T, and T residues at positions 2, 6, 7, and 10 of the consensus CBS motif, respectively, the engineered CTCF comprising:
 (i) the amino acid sequence GHLKK, AHLRK, or GKLRI at ZF4 positions +2 to +6 of the engineered CTCF;   (ii) the amino acid sequence SRLKE, DALRR, DGLKR, or TRLRE at ZF5 positions +2 to +6 of the engineered CTCF;   (iii) the amino acid sequence at RPDTMKR or RTENMKM at ZF6 positions −1 to +6 of the engineered CTCF; and   (iv) the amino acid sequence EHLKV, DHLLA, or HHLDV at ZF7 positions +2 to +6 of the engineered CTCF.   
     
     
         14 . The engineered CTCF variant of  claim 1 , wherein the mutant CBS has A, and A residues at positions 2, 5, and 11 of the consensus CBS motif, respectively, the engineered CTCF comprising:
 (i) the amino acid sequence SNLRR, GNLVR, GNLRR, GNLKR, ANLRR, NNLRR, or TNLRR at ZF4 positions +2 to +6 of the engineered CTCF;   (ii) the amino acid sequence EHMKR, EHMRR, THMKR, EHMNR, or EHMAR at ZF6 positions +2 to +6 of the engineered CTCF; and   (iii) the amino acid sequence DNLLT, DNLLV, DNLQT, DNLLA, DNLAT, DNLQA, DNLMA, or DNLMT at ZF7 positions +2 to +6 of the engineered CTCF.   
     
     
         15 . (canceled) 
     
     
         16 . The engineered CTCF variant of  claim 1 , wherein the mutant CBS that has T, and T residues at positions 6, 7, and 10 of the consensus CBS motif, respectively, the engineered CTCF comprising:
 (i) the amino acid sequence GHLKK, AHLKK, TKLRL, TKLKL, GHLRK, THLKK, or AHLRK at ZF4 positions +2 to +6 of the engineered CTCF;   (ii) the amino acid sequence TRLKE or SRLKE at ZF5 positions +2 to +6 of the engineered CTCF; and   (iii) the amino acid sequence RADN, RHDT, RRDT, RPDT, RTSS, or RNDT at ZF6 positions −1 to +3 of the engineered CTCF.   
     
     
         17 . The engineered CTCF variant of  claim 1 , wherein the engineered CTCF variant interacts with cohesion to mediate the formation of an enhancer-promoter loop to modulate gene expression. 
     
     
         18 . A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an engineered CTCF variant according to  claim 1 . 
     
     
         19 . The method of  claim 18 , wherein the subject has cancer. 
     
     
         20 . A method of activating or repressing expression of a gene under the control of a mutant CBS of  claim 1 , the gene being aberrantly expressed under the control of the mutant CBS, the method comprising contacting the mutant CBS with an engineered CTCF according to any one of  claims 1 - 17 , thereby regulating the expression of the gene. 
     
     
         21 . The method of  claim 20 , wherein the engineered CTCF activates or represses expression of the gene by interacting with cohesion to mediate the formation of an enhancer-promoter loop. 
     
     
         22 . A pharmaceutical composition comprising an engineered CTCF variant according to  claim 1 . 
     
     
         23 . A gene expression system for regulation of a gene, the system comprising a nucleic acid encoding an engineered CTCF variant according to  claim 1 . 
     
     
         24 . A method of altering the structure of chromatin comprising contacting an engineered CTCF variant according to  claim 1  with a mutant CBS to form a binding complex, such that the structure of the chromatin is altered. 
     
     
         25 . A method of modulating expression of a gene that is under the control of a CBS bearing one or more mutations, the method comprising contacting the CBS bearing one or more mutations with an engineered CTCF variant according to  claim 1 . 
     
     
         26 . A kit comprising an engineered CTCF variant according to  claim 1  and instructions for use in a method described herein.