Transcription activator-like effector (tale) - lysine-specific demethylase 1 (lsd1) fusion proteins

Abstract

Fusion proteins comprising a DNA binding domain, e.g., a TAL effector repeat array (TALE) or zinc finger array, and a catalytic domain comprising a sequence that catalyzes histone demethylation, and methods of use thereof.

1 . A fusion protein comprising
 an engineered DNA-binding domain that binds specifically to a preselected target sequence, and   a catalytic domain comprising a sequence that catalyzes histone demethylation.   
     
     
         2 . The fusion protein of  claim 1 , further comprising a linker between the DNA binding domain and the catalytic domain. 
     
     
         3 . The fusion protein of  claim 1 , wherein the DNA-binding domain is or comprises an engineered transcription activator-like (TAL) effector repeat array, zinc finger, triplex-forming oligonucleotide, peptide nucleic acid, or a DNA-binding domain from a homing meganuclease (preferably a catalytically inactive homing meganuclease), or a catalytically inactive Cas9 nuclease. 
     
     
         4 . The fusion protein of  claim 1 , wherein the catalytic domain comprises full length LSD1, or a catalytic domain of LSD1. 
     
     
         5 . The fusion protein of  claim 4 , wherein the catalytic domain comprises amino acids 172-833 of the human LSD1 variant 2. 
     
     
         6 . The fusion protein of  claim 1 , comprising a plurality of catalytic domains, optionally with linkers therebetween. 
     
     
         7 . A method of reducing methylation of histones associated with a selected DNA sequence in a mammalian cell, the method comprising contacting the cell with a fusion protein comprising an engineered DNA-binding domain that binds specifically to a target sequence, wherein the target sequence is within about 10 kb, 5 kb, 2 kb, or 1 kb, 500 bp, 250 bp, 100 bp, 50 bp, 40 bp, 30 bp, or 20 bp, of the selected DNA sequence, and a catalytic domain comprising a sequence that catalyzes histone demethylation. 
     
     
         8 . A method of reducing methylation of histones associated with a selected DNA sequence in a mammalian cell, the method comprising contacting the cell with a nucleic acid encoding a fusion protein comprising an engineered DNA-binding domain that binds specifically to a target sequence, wherein the target sequence is within about 10 kb, 5 kb, 2 kb, 1 kb, 500 bp, 250 bp, 100 bp, 50 bp, 40 bp, 30 bp, or 20 bp, of the selected DNA sequence, and a catalytic domain comprising a sequence that catalyzes histone demethylation. 
     
     
         9 . The method of  claim 7 , wherein the fusion protein further comprises a linker between the DNA binding domain and the catalytic domain. 
     
     
         10 . The method of  claim 7 , wherein the DNA-binding domain is or comprises an engineered transcription activator-like (TAL) effector repeat array, zinc finger, triplex-forming oligonucleotide, peptide nucleic acid, or a DNA-binding domain from a homing meganuclease (preferably a catalytically inactive homing meganuclease), or a catalytically inactive Cas9 nuclease. 
     
     
         11 . The method of  claim 7 , wherein the catalytic domain comprises a full-length LSD1 or a catalytic domain of LSD1. 
     
     
         12 . The method of  claim 11 , wherein the catalytic domain comprises amino acids 172-833 of the human LSD1 variant 2. 
     
     
         13 . The method of  claim 7 , wherein the cell is a human cell. 
     
     
         14 . The method of  claim 7 , wherein the cell is in a living mammal. 
     
     
         15 . The method of  claim 7 , wherein the selected DNA sequence is a sequence of a p14 ARF  gene. 
     
     
         16 . The method of  claim 8 , wherein the fusion protein further comprises a linker between the DNA binding domain and the catalytic domain. 
     
     
         17 . The method of  claim 8 , wherein the DNA-binding domain is or comprises an engineered transcription activator-like (TAL) effector repeat array, zinc finger, triplex-forming oligonucleotide, peptide nucleic acid, or a DNA-binding domain from a homing meganuclease (preferably a catalytically inactive homing meganuclease), or a catalytically inactive Cas9 nuclease. 
     
     
         18 . The method of  claim 8 , wherein the catalytic domain comprises a full-length LSD1 or a catalytic domain of LSD1. 
     
     
         19 . The method of  claim 18 , wherein the catalytic domain comprises amino acids 172-833 of the human LSD1 variant 2. 
     
     
         20 . The method of  claim 8 , wherein the cell is a human cell. 
     
     
         21 . The method of  claim 8 , wherein the cell is in a living mammal. 
     
     
         22 . The method of  claim 8 , wherein the selected DNA sequence is a sequence of a p14 ARF  gene. 
     
     
         23 . The method of  claim 7 , wherein the fusion protein comprises a plurality of catalytic domains, optionally with linkers therebetween.