Methods of Epigenetic Analysis

Abstract

The present invention provides for methods of epigenetic analysis. In some cases, the methods may include obtaining a sample comprising a nucleic acid sequence. In some cases, the nucleic acid sequence may comprise one or more epigenetic marks. The methods may include performing a sequencing. The methods may include distinguishing a hydroxymethylated base from a methylated base.

We 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. 
     
     
         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 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 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. 
     
     
         17 . A kit for modulating gene transcription via hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine, the kit comprising the following separate components:
 (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.   
     
     
         18 . The kit of  claim 17 , further comprising a composition comprising at least one cytokine, growth factor, activating reagent, or combination thereof for the purposes of generating stable human Foxp3+ regulatory T cells. 
     
     
         19 . The kit of  claim 18 , wherein the composition comprises TGF-β. 
     
     
         20 . The kit of  claim 17 , further comprising at least one nucleic acid sequence selected from the group consisting of nucleic acid sequences encoding Oct-4, Sox2, c-MYC, and Klf4, or any combination thereof. 
     
     
         21 . The kit of  claim 20 , wherein the nucleic acid sequences encoding Oct-4, Sox2, c-MYC, and Klf4 are incorporated into a viral vector. 
     
     
         22 . The kit of  claim 17 , further comprising at least one reagent suitable for the detection of 5-hydroxymethylcytosine. 
     
     
         23 . The kit of  claim 22 , wherein the reagent suitable for the detection of 5-hydroxymethylcytosine is an antibody, an antigen-binding portion thereof, an intrabody, or a protein, that specifically binds to 5-hydroxymethylcytosine. 
     
     
         24 . The kit of  claim 22 , wherein the reagent suitable for the detection of 5-hydroxymethylcytosine is an antibody, an antigen-binding portion thereof, an intrabody, or a protein that is specific for cytosine-5-methylsulfonate. 
     
     
         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. 
     
     
         27 . The method of  claim 26 , wherein the agent that specifically modulates hydroxylase activity is an inhibitor. 
     
     
         28 . The method of  claim 26 , wherein the agent that specifically modulates hydroxylase activity is an activator. 
     
     
         29 . The method of  claim 26 , wherein the cancer is a leukemia. 
     
     
         30 . The method of  claim 29 , wherein the leukemia is an acute myeloid leukemia comprising the t(10:11)(q22:q23) Mixed Lineage Leukemia translocation of TET1. 
     
     
         31 . A method for screening for an agent with TET family enzyme modulating activity, the method comprising the steps of:
 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.   
     
     
         32 . The method as in any one of  claims 1 ,  5 ,  12 ,  17 ,  25 ,  26 , and  31 , in which the catalytically active TET family enzyme is selected from the group consisting of TET1, TET2, TET3, and CXXC4. 
     
     
         33 . The method as in any one of  claims 1 ,  5 ,  12 ,  17 ,  25 ,  26 , and  31 , in which the functional TET family derivative comprises SEQ ID NO: 1. 
     
     
         34 . The method as in any one of  claims 1 ,  5 ,  12 ,  17 ,  25 ,  26 , and  31 , in which the TET family catalytically active fragment comprises SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. 
     
     
         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. 
     
     
         36 . The method of  claim 35 , wherein the 5-hydroxymethylcytosine is naturally occurring. 
     
     
         37 . The method of  claim 35 , further comprising the step of first contacting said nucleic acid with at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, thereby converting 5-methylcytosine to hydroxymethylcytosine. 
     
     
         38 . The method of  claim 35 , wherein the nucleic acid is contacted in vitro, in a cell, or in vivo. 
     
     
         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. 
     
     
         40 . The method of  claim 39 , wherein the glucose or glucose-derivative donor substrate is a uridine diphosphate glucose. 
     
     
         41 . The method of  claim 39 , wherein the labeled glucose or glucose-derivative donor substrates is radioactively labeled. 
     
     
         42 . The method of  claim 39 , wherein the 5-hydroxymethylcytosine is naturally occurring. 
     
     
         43 . The method of  claim 39 , further comprising the step of first contacting said nucleic acid with at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, thereby converting 5-methylcytosine to 5-hydroxymethylcytosine. 
     
     
         44 . The method of  claim 39 , wherein the nucleic acid is contacted in vitro, in a cell, or in vivo. 
     
     
         45 . A method for detecting 5-hydroxymethylcytosine in a nucleic acid, the method comprising contacting the covalently tagged 5-hydroxymethylcytosine of  claim 35  with a protein that recognizes a glucose molecule, glucose-derivative or gentibiosyl molecule. 
     
     
         46 . The method of  claim 45 , wherein the protein recognizes only the glucose molecule, glucose-derivative, or gentibiosyl. 
     
     
         47 . The method of  claim 45 , wherein the protein recognizes the glucose molecule, glucose-derivative, or gentibiosyl only in the context of 5-hydroxymethylcytosine. 
     
     
         48 . The method of  claim 45 , wherein the protein is a lectin. 
     
     
         49 . The method of  claim 48 , wherein the lectin is  Musa acuminata  lectin. 
     
     
         50 . The method of  claim 45 , wherein the protein is an antibody or antigen-binding fragment thereof. 
     
     
         51 . The method of  claim 50 , wherein the antibody or antibody fragment thereof is modified with a tag. 
     
     
         52 . The method of  claim 51 , wherein the tag is a biotin molecule a bead, a gold particle, or a fluorescent molecule. 
     
     
         53 . The method of  claim 45 , wherein the protein is an enzyme. 
     
     
         54 . The method of  claim 53 , wherein the enzyme is hexokinase or 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. 
     
     
         56 . The method of  claim 55 , wherein the glucose donor substrate is a uridine diphosphate glucose analog. 
     
     
         57 . The method of  claim 56 , wherein the uridine diphosphate glucose analog is uridine-2-deoxy-2-fluoro-glucose. 
     
     
         58 . The method of  claim 55 , wherein the 5-hydroxymethylcytosine is naturally occurring. 
     
     
         59 . The method of  claim 55 , further comprising the step of first contacting said nucleic acid with at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, thereby converting 5-methylcytosine to 5-hydroxymethylcytosine. 
     
     
         60 . The method of  claim 55 , wherein the enzyme is tagged. 
     
     
         61 . The method as in any one of  claims 35 ,  39 , and  55 , in which the alpha-glucosyltransferase is encoded by a bacteriophage selected from the group consisting of T2, T4, and T6 bacteriophages. 
     
     
         62 . The method as in any one of  claims 35 ,  39 , and  55 , in which the beta-glucosyltransferase is encoded by a bacteriophage selected from T4 bacteriophages. 
     
     
         63 . The method as in any one of  claims 35 ,  39 , and  55 , in which the beta-glucosyl-alpha-glucosyl-transferase is encoded by a bacteriophage selected from the group consisting of T2 and T6 bacteriophages. 
     
     
         64 . The method of  claim 55 , wherein the nucleic acid is contacted in vitro, in a cell, or in vivo. 
     
     
         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. 
     
     
         66 . The method of  claim 65 , wherein the protein is an antibody, an antigen binding fragment thereof, an enzyme, or an intrabody. 
     
     
         67 . The method of  claim 66 , wherein the antibody comprises an antiserum. 
     
     
         68 . The method of  claim 66 , wherein the antibody, antigen binding fragment thereof, enzyme, or intrabody is modified with a tag. 
     
     
         69 . The method of  claim 68 , wherein the tag is a biotin molecule, a bead, a gold particle, or a fluorescent molecule. 
     
     
         70 . The method of  claim 65 , further comprising isolating the 5-hydroxymethylcytosine containing nucleic acid with the protein specific for cytosine-5-methylsulfonate. 
     
     
         71 . The method of  claim 65 , wherein the nucleic acid is in vitro, a cell, or in vivo. 
     
     
         72 . A kit for the detection and purification of methylcytosine and 5-hydroxymethylcytosine for use in downstream applications, 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.   
     
     
         73 . The kit of  claim 72 , 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. 
     
     
         74 . The kit of  claim 73 , wherein the alpha-glucosyltransferase is encoded by a bacteriophage selected from the group consisting of T2, T4, and T6 bacteriophages. 
     
     
         75 . The kit of  claim 73 , wherein the beta-glucosyltransferase is encoded by a bacteriophage selected from T4 bacteriophages. 
     
     
         76 . The kit of  claim 73 , wherein the beta-glucosyl-alpha-glucosyl-transferase is encoded by a bacteriophage selected from the group consisting of T2 and T6 bacteriophages. 
     
     
         77 . The kit of  claim 72 , wherein the glucose or glucose-derivative donor substrate is uridine diphosphate glucose (UDPG). 
     
     
         78 . The kit of  claim 72 , wherein the glucose or glucose-derivative donor substrate is radiolabeled. 
     
     
         79 . The kit of  claim 77 , wherein the uridine diphosphate glucose is radiolabeled with 14C or 3H. 
     
     
         80 . The kit of  claim 72 , wherein the protein to detect glucose or glucose-derivative modified nucleotides is a lectin, an antibody or an antigen binding fragment thereof, or an enzyme. 
     
     
         81 . The kit of  claim 80 , wherein the protein recognizes only the glucose or glucose-derivative. 
     
     
         82 . The kit of  claim 80 , wherein the protein recognizes the glucose or glucose-derivative only in the context of 5-hydroxymethylcytosine. 
     
     
         83 . The kit of  claim 80 , wherein the antibody or antigen binding fragment thereof is modified with at least one tag. 
     
     
         84 . The kit of  claim 83 , wherein the tag is a biotin molecule, a bead, a gold particle, or a fluorescent molecule. 
     
     
         85 . The kit of  claim 80 , wherein the enzyme is a hexokinase or a beta-glucosyl-alpha-glucosyl-transferase. 
     
     
         86 . The kit of  claim 80 , wherein the lectin is  Musa acuminata  lectin (BanLec). 
     
     
         87 . The kit of  claim 80 , wherein the lectin is modified with a gold particle or a fluorescent tag. 
     
     
         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.   
     
     
         89 . The method of  claim 88 , further comprising a step of comparing the level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in a tissue or cell sample of the individual to the level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in at least one sample from a diseased tissue or a diseased cell, wherein if the level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in the tissue or cell sample from the individual in need is similar to the level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, from at least one of the samples from the diseased tissue or cell then the individual is diagnosed with a myelodysplastic syndrome, a myeloproliferative disorder, acute myelogenous leukemia, systemic mastocytosis, or chronic myelomonocytic leukemia. 
     
     
         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.   
     
     
         91 . The method of  claim 88  or  90 , in which the level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, is assessed using an assay to convert 5-hydroxymethylcytosine to cytosine-5-methylsulfonate. 
     
     
         92 . A kit for the detection and purification of 5-hydroxymethylcytosine, the kit comprising:
 (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.   
     
     
         93 . The kit of  claim 92 , wherein the protein is an antibody, an antigen binding fragment thereof, an intrabody, or an enzyme. 
     
     
         94 . The kit of  claim 92 , wherein the protein is specific for cytosine-5-methylsulfonate. 
     
     
         95 . The kit of  claim 94 , wherein the antibody, antigen binding fragment thereof, intrabody, or enzyme is modified with at least one tag. 
     
     
         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.