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 a TET enzyme activity in said isolated cell, wherein said regulating comprises contacting said isolated cell with a TET activator, wherein said contacting causes an increased 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 isolated cell is a stem cell. 
     
     
         100 . The method of  claim 97 , wherein said increased conversion comprises increasing 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. 
     
     
         101 . The method of  claim 97 , wherein said isolated cell is a lymphocyte. 
     
     
         102 . The method of  claim 101 , wherein said lymphocyte is a T cell. 
     
     
         103 . The method of  claim 97 , wherein said isolated cell is a genetically modified cell. 
     
     
         104 . The method of  claim 103 , wherein said genetically modified cell comprises foreign DNA. 
     
     
         105 . The method of  claim 97 , wherein said TET activator comprises ascorbic acid or a salt thereof. 
     
     
         106 . The method of  claim 97 , wherein said TET activator comprises retinoic acid or a salt thereof. 
     
     
         107 . The method of  claim 97 , wherein said TET activator comprises a lipopolysaccharide or a salt thereof. 
     
     
         108 . The method of  claim 97 , wherein said TET activator is a small molecule activator. 
     
     
         109 . The method of  claim 97 , wherein said TET activator is an agonist. 
     
     
         110 . The method of  claim 97 , wherein said TET activator is an antibody or antigen-binding fragment thereof. 
     
     
         111 . The method of  claim 97 , wherein said TET activator is a nucleic acid that activates TET1, TET2, TET3, or CXXC4. 
     
     
         112 . The method of  claim 97 , wherein said isolated cell is obtained from a subject. 
     
     
         113 . The method of  claim 112 , wherein said subject has cancer or is suspected of having cancer. 
     
     
         114 . The method of  claim 112 , wherein said subject has cancer. 
     
     
         115 . The method of  claim 97 , further comprising administering said isolated cell to a subject. 
     
     
         116 . The method of  claim 114 , wherein said cancer is leukemia. 
     
     
         117 . The method of  claim 116 , wherein said leukemia is acute myeloid leukemia. 
     
     
         118 . The method of  claim 97 , wherein said isolated cell is a human cell.