SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS
First Claim
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.
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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.
81 Citations
96 Claims
- 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.
- 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.
- 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.
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16. A method for detecting a 5-hydroxymethylcytosine nucleotide in a biological sample, the method comprising contacting a biological sample with a detectably labeled antibody or a binding portion thereof, labeled intrabody, or labeled protein, that specifically binds to 5-hydroxymethylcytosine, and detecting the amount of bound label, wherein the presence of the bound label is indicative of the 5-methylcytosine being converted to 5-hydroxymethylcytosine.
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17. A kit for modulating gene transcription via hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine, the kit comprising the following separate components:
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(a) at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, or nucleic acid molecule that comprises a sequence encoding at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment, or combination thereof in an appropriate buffer or solution; and (b) packaging materials and instructions therein to use said kit to hydroxylate 5-methylcytosine to 5-hydroxymethylcytosine, for the purpose of modulating gene transcription. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24)
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25. A method for improving stem cell therapies, the method comprising contacting with, or delivering to, a stem 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 fragment thereof, or combination thereof, or at least one nucleic acid molecule that comprises a sequence encoding a catalytically active TET family enzyme, a functional TET family derivative, TET catalytically active fragment thereof, or combination thereof.
- 26. A method for treating an individual affected with or at risk for cancer, the method comprising administering to an individual affected with, or at risk for, cancer, an effective amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment, or a combination thereof, involved in transforming 5-methylcytosine into 5-hydroxymethylcytosine.
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31. A method for screening for an agent with TET family enzyme modulating activity, the method comprising the steps of:
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a) providing a cell comprising at least one TET family enzyme, functional TET family derivative, TET catalytically active fragment, recombinant TET family enzyme thereof, or combination thereof; b) contacting said cell with a test agent, thereby creating a test sample; and c) detecting the level of 5-hydroxymethylated cytosine in the cell in the test sample with the level expressed in a control sample; and (d) determining whether or not the test agent increases or decreases the level of 5-hydroxymethylated cytosine, wherein a statistically significant decrease in the level of 5-hydroxymethylated cytosine indicates the test agent is an inhibitor, and a statistically significant increase in the level of 5-hydroxymethylated cytosine indicates the test agent is an activator.
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- 35. A method for covalent tagging of 5-hydroxymethylcytosine in a nucleic acid, the method comprising contacting a nucleic acid molecule with an enzyme that adds one or more glucose molecules to a 5-hydroxymethylcytosine residue to generate glucosylated-5-hydroxymethylcytosine or gentibiose-containing-5-hydroxymethylcytosine, wherein the enzyme is an alpha-glucosyltransferase, beta-glucosyltransferase, or a beta-glucosyl-alpha-glucosyl-transferase.
- 39. A method for detecting 5-hydroxymethylcytosine in a nucleic acid, the method comprising contacting a nucleic acid with an enzyme that utilizes labeled glucose or glucose-derivative donor substrates to add one or more labeled glucose molecules or glucose-derivatives to a 5-hydroxymethylcytosine residue to generate glucosylated-5-hydroxymethylcytosine or gentibiose-containing-5-hydroxymethylcytosine, wherein the enzyme is an alpha-glucosyltransferase, a beta-glucosyltransferase, or a beta-glucosyl-alpha-glucosyl-transferase.
- 55. A method for detecting 5-hydroxymethylcytosine in a nucleic acid, the method comprising contacting a nucleic acid with an enzyme and utilizing glucose or glucose-derivative donor substrates that trap covalent enzyme-DNA intermediates to detect 5-hydroxymethylcytosine residues, wherein the enzyme is an alpha-glucosyltransferase, a beta-glucosyltransferase, or a beta-glucosyl-alpha-glucosyl-transferase.
- 65. A method to detect 5-hydroxymethylcytosine in a nucleic acid, the assay comprising contacting a nucleic acid with sodium hydrogen sulfite to convert a 5-hydroxymethylcytosine in a nucleic acid to cytosine-5-methylsulfonate, and contacting the sodium hydrogen sulfite contacted nucleic acid with a protein specific for cytosine-5-methylsulfonate.
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72. A kit for the detection and purification of methylcytosine and 5-hydroxymethylcytosine for use in downstream applications, the kit comprising:
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(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. - View Dependent Claims (73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87)
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88. A method for diagnosing a myelodysplastic syndrome, a myeloproliferative disorder, acute myelogenous leukemia, systemic mastocytosis, or chronic myelomonocytic leukemia in an individual in need thereof, the method comprising the steps of
(i) determining a level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in a tissue or a cell sample from an individual in need thereof, and (ii) comparing the level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in the tissue or cell sample from the individual with a level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in a tissue or cell sample from a normal control, wherein a difference in the level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, between the sample from the individual in need and the control sample is indicative of the individual having a myelodysplastic syndrome, a myeloproliferative disorder, acute myelogenous leukemia, systemic mastocytosis, or chronic myelomonocytic leukemia.
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90. A method for monitoring progression of or an effect of a therapy on a a myelodysplastic syndrome, a myeloproliferative disorder, acute myelogenous leukemia, systemic mastocytosis, or chronic myelomonocytic leukemia, the method comprising the steps of
(i) determining a level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in a tissue or cell sample from an individual in need thereof to establish a baseline level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in the tissue or cell sample; (ii) determining a level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in a tissue or cell sample from the individual at least one time following the establishment of the baseline level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof in the tissue or cell sample thereby establishing at least one follow-up level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, wherein a difference in the follow-up level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, relative to the baseline level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in the tissue or cell sample is indicative of the progression or effect of a therapy on a myelodysplastic syndrome, a myeloproliferative disorder, acute myelogenous leukemia, systemic mastocytosis, or chronic myelomonocytic leukemia in the individual.
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92. A kit for the detection and purification of 5-hydroxymethylcytosine, the kit comprising:
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(a) at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof for the conversion of methylcytosine to 5-hydroxymethylcytosine; (b) sodium bisulfite; (c) one or more proteins to detect sodium bisulfite treated nucleotides; (e) standard DNA purification columns, buffers, and substrate solutions; and (f) packaging materials and instructions therein to use said kits. - View Dependent Claims (93, 94, 95)
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96. The kit of claim 130, wherein the tag is selected from the group consisting of a biotin molecule, a bead, a gold particle, or a fluorescent molecule.
Specification