Selective oxidation of 5-methylcytosine by tet-family proteins

Abstract

The present invention provides for novel methods for regulating and detecting the cytosine methylation status of DNA. The invention is based upon identification of a novel and surprising catalytic activity for the family of TET proteins, namely TET1, TET2, TET3, and CXXC4. The novel activity is related to the enzymes being capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation.

1 - 96 . (canceled) 
     
     
         97 . A method comprising:
 (a) obtaining an isolated cell; and   (b) regulating activity of a TET enzyme in said isolated cell, wherein said regulating comprises contacting said isolated cell with a TET inhibitor, wherein said contacting causes a decreased conversion of 5-methylated cytosine to 5-hydroxymethylated cytosine in said isolated cell,   wherein said TET enzyme comprises a TET1 enzyme, a TET2 enzyme, a TET3 enzyme, a CXXC4 enzyme, or any combination thereof.   
     
     
         98 . The method of  claim 97 , wherein said isolated cell is a mammalian cell. 
     
     
         99 . The method of  claim 97 , wherein said decreased conversion comprises decreasing a level of 5-hydroxymethylated cytosine in said isolated cell after said contacting compared to a level of 5-hydroxymethylated cytosine in said isolated cell prior to said contacting. 
     
     
         100 . The method of  claim 97 , wherein said isolated cell is a lymphocyte. 
     
     
         101 . The method of  claim 100 , wherein said lymphocyte is a T cell. 
     
     
         102 . The method of  claim 97 , wherein said isolated cell is a genetically modified cell. 
     
     
         103 . The method of  claim 102 , wherein said genetically modified cell comprises foreign DNA. 
     
     
         104 . The method of  claim 97 , wherein said isolated cell is obtained from a subject. 
     
     
         105 . The method of  claim 104 , wherein said subject has cancer or is suspected of having cancer. 
     
     
         106 . The method of  claim 104 , wherein said subject has cancer. 
     
     
         107 . The method of  claim 104 , further comprising administering said isolated cell to said subject. 
     
     
         108 . The method of  claim 106 , wherein said cancer is leukemia. 
     
     
         109 . The method of  claim 108 , wherein said leukemia is acute myeloid leukemia. 
     
     
         110 . The method of  claim 97 , wherein said TET inhibitor comprises a small molecule inhibitor, a competitive inhibitor, an antibody or antigen-binding fragment thereof, or a nucleic acid that inhibits TET1, TET2, TET3, or CXXC4. 
     
     
         111 . The method of  claim 110 , wherein said TET inhibitor is the small molecule inhibitor. 
     
     
         112 . The method of  claim 110 , wherein said TET inhibitor is the competitive inhibitor. 
     
     
         113 . The method of  claim 110 , wherein said TET inhibitor is the antibody or antigen-binding fragment thereof. 
     
     
         114 . The method of  claim 110 , wherein said TET inhibitor is the nucleic acid that inhibits TET1, TET2, TET3, or CXXC4. 
     
     
         115 . The method of  claim 115 , wherein said nucleic acid comprises RNA or DNA. 
     
     
         116 . The method of  claim 116 , wherein said nucleic acid comprises RNA. 
     
     
         117 . The method of  claim 117 , wherein said RNA is siRNA. 
     
     
         118 . The method of  claim 118 , wherein said siRNA is a TET1 siRNA, a TET2 siRNA, or a TET3 siRNA. 
     
     
         119 . The method of  claim 98 , wherein said mammal is a human. 
     
     
         120 . The method of  claim 97 , wherein said TET enzyme is the TET1 enzyme. 
     
     
         121 . The method of  claim 97 , wherein said TET enzyme is the TET2 enzyme. 
     
     
         122 . The method of  claim 97 , wherein said TET enzyme is the TET3 enzyme.