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-9447452-B2
Publication Date
2016-09-20
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 novel catalytic activity for the family of TET proteins, namely TET1, TET2, TET3, and CXXC4. This novel enzymatic activity enables these enzymes to convert the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine through hydroxylation.
The invention addresses the problem that active DNA demethylation mechanisms were unknown, despite their known importance in embryogenesis, stem cell pluripotency, differentiation, and cancer. DNA methylation is highly aberrant in cancer, and control of DNA demethylation pathways may improve therapeutic interventions, including improving the efficiency of reprogramming somatic cells to pluripotent stem cells and enhancing stem cell therapies.
Claims Coverage
The patent includes one independent claim directed to a comprehensive kit that integrates several components for detecting and purifying methylcytosine and 5-hydroxymethylcytosine. The main inventive features involve the enzymatic conversion and detection of these cytosine derivatives.
Kit comprising TET family enzymes for conversion of methylcytosine to hydroxymethylcytosine
The kit includes one or more catalytically active TET family enzymes, functional derivatives, or catalytic fragments capable of converting methylcytosine residues into 5-hydroxymethylcytosine in nucleic acids to enable subsequent analysis.
Use of bacteriophage-derived glucosyltransferases for enzymatic modification
The kit contains enzymes encoded by bacteriophages of the 'T even' family, specifically alpha-glucosyltransferases, beta-glucosyltransferases, and beta-glucosyl-alpha-glucosyl-transferases, which enzymatically add glucose or glucose derivatives to 5-hydroxymethylcytosine for labeling.
Inclusion of glucose or glucose-derivative donor substrates
The kit comprises glucose or glucose-derivative donor substrates such as uridine diphosphate glucose (UDPG), optionally radiolabeled with 14C or 3H, facilitating enzymatic tagging of modified cytosine residues.
Proteins for detecting glucose or glucose-derivative modifications
The kit includes one or more proteins such as lectins, antibodies or antigen-binding fragments, or enzymes that specifically recognize glucose-modified 5-hydroxymethylcytosine residues, optionally enhanced by tags like biotin, beads, gold particles, or fluorescent molecules.
Provision of standard DNA purification and assay reagents with instructions
The kit further provides standard DNA purification columns, buffers, substrate solutions, packaging materials, and instructions necessary for performing the enzymatic modification, detection, and purification processes.
The patent claims focus on a multifunctional kit that combines catalytically active TET family enzymes and bacteriophage-derived glucosyltransferases along with detection proteins and assay components to enable enzymatic conversion, labeling, detection, and purification of methylcytosine and 5-hydroxymethylcytosine in nucleic acids.
Stated Advantages
Provides novel tools for regulating the DNA methylation status of mammalian cells with implications in reprogramming somatic cells, stem cell therapy, and cancer treatment.
Enables improved detection and mapping of 5-hydroxymethylcytosine, which was previously undetectable using conventional methylation assays.
Offers methods that can increase the efficiency and/or rate of generating induced pluripotent stem (iPS) cells from somatic cells.
Improves efficiency of cloning mammals by nuclear transfer through hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine.
Provides novel diagnostic and therapeutic approaches for diseases associated with aberrant DNA methylation, including myeloid cancers.
Documented Applications
Generating stable human regulatory Foxp3+ T cells by delivering catalytically active TET family enzymes to human T cells.
Improving the efficiency and rate of producing induced pluripotent stem (iPS) cells from adult somatic cells by combining TET family enzymes with known reprogramming factors.
Enhancing the efficiency of mammalian cloning by nuclear transfer or nuclear transplantation via hydroxylation of 5-methylcytosine.
Diagnosing and treating myeloid cancers such as myeloproliferative disorder (MPD), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), systemic mastocytosis, and chronic myelomonocytic leukemia (CMML) using TET family activity assessments and modulators.
Detecting and mapping 5-methylcytosine and 5-hydroxymethylcytosine residues in genomes for research, clinical diagnostics, and drug screening.
Screening for signaling pathways and agents that modulate TET family enzymatic activity for therapeutic applications.
Immunohistochemical and chromatin immunoprecipitation (ChIP) applications to detect 5-hydroxymethylcytosine in cells and tissues.
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