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.

We claim: 
     
         1 . A method comprising:
 labeling a hydroxyl group on a hydroxymethylated residue in a nucleic acid to generate a labeled hydroxymethylated residue, wherein said nucleic acid is from an extracellular fluid sample; and   sequencing said nucleic acid comprising said labeled hydroxymethylated residue.   
     
     
         2 . The method of  claim 1 , wherein said extracellular fluid sample is from a mammal. 
     
     
         3 . The method of  claim 1 , wherein said nucleic acid is a mammalian nucleic acid. 
     
     
         4 . The method of  claim 1 , wherein said labeling is covalently labeling. 
     
     
         5 . The method of  claim 1 , wherein said hydroxymethylated residue is a 5-hydroxymethylcytosine. 
     
     
         6 . The method of  claim 5 , wherein said labeling comprises glycosylating said 5-hydroxymethylcytosine. 
     
     
         7 . The method of  claim 1 , wherein said nucleic acid further comprises a methylated cytosine residue. 
     
     
         8 . The method of  claim 7 , wherein said methylated cytosine residue is a 5-methylcytosine. 
     
     
         9 . The method of  claim 1 , wherein said sequencing comprises high-throughput sequencing. 
     
     
         10 . The method of  claim 1 , further comprising binding said labeled hydroxymethylated residue to a support. 
     
     
         11 . The method of  claim 10 , wherein said binding occurs prior to said sequencing. 
     
     
         12 . The method of  claim 1 , wherein said labelling comprises associating a label with said hydroxymethylated residue. 
     
     
         13 . The method of  claim 12 , wherein said label comprises a sugar. 
     
     
         14 . The method of  claim 12 , wherein said label comprises a bead. 
     
     
         15 . A composition comprising a nucleic acid from an extracellular fluid sample, wherein said nucleic acid comprises a covalently labeled hydroxyl group on a hydroxymethylated residue. 
     
     
         16 . The composition of  claim 15 , wherein said extracellular fluid sample is from a mammal. 
     
     
         17 . The composition of  claim 15 , wherein said hydroxymethylated residue is a 5-hydroxymethylcytosine. 
     
     
         18 . The composition of  claim 15 , wherein said extracellular fluid sample is isolated from a subject. 
     
     
         19 . The composition of  claim 15 , wherein a label that is covalently associated with said hydroxyl group comprises a sugar. 
     
     
         20 . The composition of  claim 19 , wherein said sugar comprises a modified glucose.