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 for improving the generation of stable human Foxp3+ T cells, the method comprising contacting with, or delivering to, a human T cell an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET family catalytically active fragment thereof, or combination thereof.
2 . The method of claim 1 , wherein the human T cell is a purified human CD4+ T cell.
3 . The method of claim 1 , further comprising contacting with, or delivering to, the human T cell a composition comprising at least one cytokine, growth factor, activating reagent, or combination thereof.
4 . The method of claim 3 , wherein said composition comprises TGF-β.
5 . A method for improving efficiency or rate with which an induced pluripotent stem (iPS) cell is produced from an adult somatic cell, the method comprising contacting with, or delivering to a somatic cell, an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active thereof, or combination thereof.
6 . The method of claim 5 , wherein the catalytically active TET family enzyme is TET1 or TET2.
7 . The method of claim 5 , further comprising contacting with, or delivering to, the somatic cell, an effective amount of a TET family inhibitor.
8 . The method of claim 7 , wherein the TET family inhibitor is a TET3 inhibitor.
9 . The method of claim 5 , further comprising contacting the somatic cell with or delivering to the somatic cell a combination of nucleic acid sequences encoding Oct-4, Sox2, c-MYC, and Klf4.
10 . The method of claim 5 , wherein the combination of nucleic acid sequences encoding Oct-4, Sox2, c-MYC, and Klf4 are delivered in a viral vector.
11 . The method of claim 5 , wherein the somatic cell is a fibroblast.
12 . A method for improving efficiency of cloning a mammal by nuclear transfer or nuclear transplantation, the method comprising contacting a nucleus extracted from a cell to be cloned with an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, during a nuclear transfer protocol.
13 . The method of claim 12 , wherein the catalytically active TET family enzyme is TET1 or TET2.
14 . The method of claim 12 , further comprising contacting the nucleus from the cell to be cloned with a TET family inhibitor.
15 . The method of claim 14 , wherein the TET family inhibitor is a TET3 inhibitor.
16 . A kit for the detection and purification of methylcytosine and 5-hydroxymethylcytosine, the kit comprising:
(a) one or more catalytically active TET family enzymes, functional TET family derivatives, or TET catalytically active fragments thereof for the conversion of methylcytosine to 5-hydroxymethylcytosine;
(b) one or more enzymes encoded by bacteriophages of the “T even” family;
(c) one or more glucose or glucose-derivative donor substrates;
(d) one or more proteins to detect glucose or glucose-derivative modified nucleotides;
(e) standard DNA purification columns, buffers, and substrate solutions; and
(f) packaging materials and instructions therein to use said kits.
17 . The kit of claim 16 , wherein the enzyme encoded by bacteriophages of the “T even” family is selected from the group consisting of alpha-glucosyltransferases, beta-glucosyltransferases, and beta-glucosyl-alpha-glucosyl-transferases.
18 . The kit of claim 17 , wherein the alpha-glucosyltransferase is encoded by a bacteriophage selected from the group consisting of T2, T4, and T6 bacteriophages.
19 . The kit of claim 17 , wherein the beta-glucosyltransferase is encoded by a bacteriophage selected from T4 bacteriophages.
20 . The kit of claim 17 , wherein the beta-glucosyl-alpha-glucosyl-transferase is encoded by a bacteriophage selected from the group consisting of T2 and T6 bacteriophages.
21 . The kit of claim 16 , wherein the glucose or glucose-derivative donor substrate is uridine diphosphate glucose (UDPG).
22 . The kit of claim 21 , wherein the glucose or glucose-derivative donor substrate is radiolabeled.
23 . The kit of claim 22 , wherein the uridine diphosphate glucose is radiolabeled with 14C or 3H.
24 . The kit of claim 16 , wherein the protein that detects glucose or glucose-derivative modified nucleotides is selected from a group comprising a lectin, an antibody or antigen-binding fragment thereof, or an enzyme.
25 . The kit of claim 16 , wherein the protein recognizes only the glucose or glucose-derivative.
26 . The kit of claim 16 , wherein the protein recognizes the glucose or glucose-derivative only in the context of 5-hydroxymethylcytosine.
27 . The kit of claim 24 , wherein the antibody or antigen-binding fragment thereof is modified with at least one tag.
28 . The kit of claim 27 , wherein the tag is a biotin molecule, a bead, a gold particle, or a fluorescent molecule.
29 . The kit of claim 24 , wherein the enzyme is a hexokinase or a beta-glucosyl-alpha-glucosyl-transferase.
30 . The kit of claim 24 , wherein the lectin is Musa acuminata lectin (BanLec).