SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS
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 . A method comprising:
contacting with, or delivering to a somatic cell obtained from a subject, an effective amount of an enzyme or fragment thereof that converts 5-methylcytosine to 5-hydroxymethylcytosine in the somatic cell.
2 . The method of claim 1 , wherein the somatic cell is an adult somatic cell.
3 . The method of claim 1 , wherein the subject is a mammal.
4 . The method of claim 1 , wherein the somatic cell is a fibroblast.
5 . The method of claim 1 , wherein the somatic cell is a regulatory T cell.
6 . The method of claim 5 , wherein the regulatory T cell is a Foxp3+ T cell.
7 . The method of claim 1 , wherein the enzyme or fragment thereof is a recombinant enzyme or a fragment of a recombinant enzyme.
8 . The method of claim 1 , wherein the enzyme or fragment thereof comprises at least a portion of a TET family enzyme or a fragment thereof.
9 . The method of claim 1 , wherein the enzyme or fragment thereof is TET1, TET2, TET3, CXXC4, a fragment of any of these, or any combination thereof.
10 . The method of claim 1 , wherein the enzyme or fragment thereof comprises a nucleic acid sequence having at least about 90% sequence homology to any one of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
11 . The method of claim 1 , further comprises contacting with, or delivering to, the somatic cell, an effective amount of an inhibitor that decreases a conversion of 5-methylcytosine to 5-hydroxymethylcytosine in the somatic cell.
12 . The method of claim 11 , wherein the inhibitor is an antibody.
13 . The method of claim 11 , wherein the inhibitor inhibits a TET3 enzyme.
14 . The method of claim 1 , further comprises inducing expression of one or more genes comprising: Oct-4, Sox2, c-MYC, Klf4, Nanog, Lin-28, or any combination thereof, in the somatic cell, wherein the somatic cell is reprogrammed to an induced pluripotent stem (iPS) cell.
15 . The method of claim 14 , wherein a nucleic acid sequence to induce said expression is associated with a viral vector.
16 . The method of claim 1 , further comprising contacting with, or delivering to the somatic cell retinoic acid.
17 . An isolated and purified induced pluripotent stem (iPS) cell, the isolated and purified iPS cell comprising:
a. induced expression of one or more genes comprising: Oct-4, Sox2, c-MYC, Klf4, Nanog, Lin-28, or any combination thereof, in the somatic cell; and
b. an effective amount of an enzyme or fragment thereof that converts 5-methylcytosine to 5-hydroxymethylcytosine in the iPS cell.
18 . A kit comprising:
a. an enzyme or fragment thereof that converts 5-methylcytosine to 5-hydroxymethylcytosine;
b. one or more nucleic acid sequences that code for a product that induces expression of one or more genes comprising: Oct-4, Sox2, c-MYC, Klf4, Nanog, Lin-28, or any combination thereof, in a cell; and
c. instructions for use.
19 . The kit of claim 18 , further comprising a reagent suitable for detection of a 5-hydroxylmethylcytosine to 5-methylcytosine conversion.
20 . The kit of claim 18 , wherein the reagent is an antibody or binding portion thereof.