Selective oxidation of 5-methylcytosine by TET-family proteins
Inventors
Rao, Anjana • Tahiliani, Mamta • Koh, Kian Peng • Agarwal, Suneet • Iyer, Aravind
Assignees
Boston Childrens Hospital • US Department of Health and Human Services
Publication Number
US-10612076-B2
Publication Date
2020-04-07
Expiration Date
2029-09-28
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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.
Core Innovation
The invention provides novel methods for regulating and detecting the cytosine methylation status of DNA based on the identification of a previously unknown catalytic activity of the family of TET proteins, which includes TET1, TET2, TET3, and CXXC4. This catalytic activity enables these enzymes to convert the cytosine nucleotide 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) by hydroxylation.
This novel TET family enzymatic activity transforms 5-methylcytosine to 5-hydroxymethylcytosine, which impacts the DNA methylation status in mammalian genomes, influencing epigenetic gene regulation processes such as pluripotency and cellular differentiation. The invention also provides methods employing TET enzymes or derivatives for reprogramming somatic cells into pluripotent stem cells and for modulating differentiation status, as well as for generating stable human regulatory Foxp3+ T cells.
The problem addressed relates to the fundamental unknown molecular mechanism of active DNA demethylation in mammalian cells, which plays vital roles in embryogenesis, development, differentiation, aging, and is aberrant in tumorigenesis and cancer. Prior mechanisms for DNA demethylation were limited to passive replication-dependent demethylation or speculative, unreliable active processes without identified molecular catalysts. There was also a lack of specific methods and reagents to detect and distinguish 5-hydroxymethylcytosine from 5-methylcytosine and unmethylated cytosine in DNA.
Claims Coverage
The patent contains multiple inventive features centered on regulating cytosine methylation via TET family enzymes and detecting modified cytosine residues, with independent claims covering methods of regulating methylation in adult cells, especially T cells, and the enzymatic hydroxylation process.
Regulating cytosine methylation status in adult cells
A method of regulating methylation status of one or more cytosine bases in an isolated and purified adult cell by contacting the cell with an effective amount of an enzyme or inhibitor that converts or inhibits the conversion of 5-methylcytosine to 5-hydroxymethylcytosine or further oxidation products, or a combination thereof.
Differentiation of naive T cells to stable Foxp3+ regulatory T cells
Methods whereby regulation of methylation in naive T cells, including purified human CD4+ T cells, via TET family enzymes results in differentiation to stable human Foxp3+ regulatory T cells, optionally in combination with cytokines like TGF-β.
Improving induced pluripotent stem cell (iPS) production
Methods for enhancing the efficiency or rate of iPS cell production from somatic cells by delivery of catalytically active TET family enzymes or derivatives, optionally combined with expression of pluripotency factors (Oct-4, Sox2, c-MYC, Klf4) and/or TET family inhibitors such as TET3-specific inhibitors.
Enhancing cloning efficiency by nuclear transfer
Methods for improving mammalian cloning efficiency by contacting a nucleus with catalytically active TET family enzymes or derivatives during nuclear transfer protocols, optionally along with TET family inhibitors.
Detecting 5-hydroxymethylcytosine in biological samples using specific antibodies or proteins
Methods of detecting 5-hydroxymethylcytosine in biological samples by contacting with detectably labeled antibodies, antigen-binding fragments, intrabodies, or proteins specifically binding 5-hydroxymethylcytosine and detecting the bound label.
Kits for modulating gene transcription and detection of 5-hydroxymethylcytosine
Kits comprising catalytically active TET family enzymes or derivatives and, optionally, cytokines, pluripotency nucleic acids (Oct-4, Sox2, c-MYC, Klf4), and detection reagents such as antibodies specific for 5-hydroxymethylcytosine to modulate gene transcription and generate stable regulatory T cells or enhance iPS generation.
Methods for enhancing stem cell therapies
Methods for improving stem cell therapies by contacting or delivering stem cells with catalytically active TET family enzymes, functional derivatives, or derivatives thereof, optionally encoded by nucleic acids.
Methods for cancer treatment using TET family modulators
Methods for treating individuals with or at risk for cancer by administering agents that modulate hydroxylase activity of TET family enzymes, including inhibitors or activators, specifically for leukemias such as acute myeloid leukemia involving TET1 translocation.
Screening methods for identifying TET family enzyme modulators
Assays involving cells expressing TET family enzymes or derivatives contacted with test molecules to determine changes in 5-hydroxymethylcytosine levels for identifying modulators that inhibit or activate TET enzyme activity.
Methods for detecting and tagging 5-hydroxymethylcytosine via glucose conjugation
Methods to covalently tag 5-hydroxymethylcytosine residues in nucleic acids by enzymatic addition of glucose moieties using phage-derived alpha-, beta-, or beta-alpha-glucosyltransferases and detecting or isolating the tagged DNA using specific proteins, antibodies, lectins, or enzymatic reactions.
Detecting 5-hydroxymethylcytosine via bisulfite treatment and cytosine-5-methylenesulfonate antibodies
Methods comprising treating nucleic acid with bisulfite converting 5-hydroxymethylcytosine to cytosine-5-methylenesulfonate and detecting this adduct with specific antibodies or proteins to quantify or isolate hydroxymethylated DNA.
The claims cover a comprehensive suite of methods and kits that utilize the novel catalytic activity of TET family enzymes to regulate cytosine methylation status in adult and somatic cells, enhance stem cell reprogramming and therapeutic applications, detect and tag 5-hydroxymethylcytosine residues using enzymatic and immunoassay approaches, and provide therapeutic and diagnostic interventions, particularly for immunological and cancer-related conditions.
Stated Advantages
Novel enzymatic tools for regulating DNA methylation status in mammalian cells.
Improved efficiency and rate of induced pluripotent stem cell generation.
Methods for generating stable human regulatory Foxp3+ T cells.
New reagents and approaches for detecting and distinguishing 5-hydroxymethylcytosine in genomic DNA.
Potential to improve cloning efficiency by nuclear transfer.
Applications in cancer diagnosis and treatment by targeting TET family enzymes and their activities.
Documented Applications
Regulation and detection of cytosine methylation status in DNA.
Promoting reprogramming of somatic cells into pluripotent stem cells, including enhancing iPS cell generation efficiency.
Generation of stable human regulatory Foxp3+ T cells for immune therapies.
Improvement of cloning efficiency in mammals by nuclear transfer.
Diagnosis and treatment of myeloid cancers such as myeloproliferative disorders, myelodysplastic syndromes, acute myeloid leukemia, systemic mastocytosis, and chronic myelomonocytic leukemia.
High-throughput screening for TET family enzyme modulators for therapeutic applications.
Detection and isolation of 5-hydroxymethylcytosine in biological samples using enzymatic tagging and antibody-based methods.
Stem cell therapy treatments involving modulation of pluripotency and differentiation by administering TET family enzymes or derivatives.
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