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-10731204-B2
Publication Date
2020-08-04
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 identifies a novel and surprising catalytic activity for the family of TET proteins (TET1, TET2, TET3, and CXXC4) related to their ability to hydroxylate the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine in DNA. This enzymatic activity serves as a tool for regulating and detecting the cytosine methylation status of mammalian DNA.
The problem being solved relates to the understanding and manipulation of DNA methylation and demethylation processes, which are crucial in mammalian development, cellular differentiation, aging, and tumorigenesis. While passive DNA demethylation is well understood, an enzyme capable of active demethylation had not been identified prior to this invention. Moreover, methods to detect and study the novel 5-hydroxymethylcytosine nucleotide were lacking, hindering epigenetic studies, diagnostics, and therapeutic developments.
The invention provides novel methods and reagents for somatic cell reprogramming into pluripotent cells by utilizing TET family enzymes to increase the efficiency and rate of induced pluripotent stem cell (iPS) generation, and modulate pluripotency and cellular differentiation. It also describes methods for generating stable human regulatory Foxp3+ T cells by converting 5-methylcytosine to 5-hydroxymethylcytosine, thus addressing the need for stable epigenetic modification in T cell lineage commitment.
Additionally, the invention provides diagnostic and therapeutic methods for myeloid cancers and other diseases involving aberrant DNA methylation. It defines novel assays for detecting 5-hydroxymethylcytosine, including antibody-based detection and chemical modification (such as bisulfite treatment) to distinguish this modification from 5-methylcytosine. Kits and screening methods for TET family modulators are also included to facilitate therapeutic targeting.
Claims Coverage
The independent claims describe methods of converting methylated cytosine residues in isolated nucleotide sequences to hydroxymethylated cytosine residues using TET family enzymes, and methods for detecting methylation status based on presence of the modified base. Additional claims cover detection methods including use of glucosyltransferase labeling and sequencing, and claims extend to the compositions of enzymes involved.
Method of converting methylated cytosine to hydroxymethylcytosine using TET family enzymes
A method involving contacting an isolated nucleotide sequence with an enzyme or catalytically active fragment comprising TET1, TET2, TET3, or CXXC4 to convert methylated cytosine residues to hydroxymethylated cytosine residues.
Detection of methylation status based on presence of hydroxymethylated base
Methods for detecting methylation status of an isolated nucleotide sequence by assessing presence or absence of hydroxymethylated cytosine, including bisulfite treatment or sequencing.
Labeling hydroxymethylated cytosine using glucosyltransferase with labeled glucose substrates
Detection methods comprising contacting the isolated nucleotide sequence with a glucosyltransferase protein (beta-glucosyltransferase or alpha-glucosyltransferase) that adds a labeled glucose or glucose derivative to hydroxymethylated cytosine, facilitating detection.
Use of TET enzymes on clinical or biological samples
Methods applying TET family enzymes or fragments to DNA from cells or tissues, including samples from subjects with or suspected of having cancer, to convert methylcytosine to hydroxymethylcytosine for diagnostic purposes.
Indirect labeling of hydroxymethylated cytosine residues
Methods including indirect labeling of hydroxymethylated cytosine residues with fluorophores or biotinyl groups for detection.
The claims cover the enzymatic conversion of methylated cytosine to hydroxymethylated cytosine using TET family enzymes and catalytically active fragments, and related detection methods based on chemical or enzymatic labeling. The scope extends to clinical sample analysis and indirect labeling techniques, providing tools for epigenetic analysis and potential diagnostic applications.
Stated Advantages
Provides novel methods to regulate and detect DNA cytosine methylation status via TET family protein-mediated hydroxylation.
Enhances efficiency and rate of induced pluripotent stem cell generation from somatic cells.
Facilitates generation of stable human regulatory Foxp3+ T cells through epigenetic modification.
Allows improved diagnosis and treatment of myeloid cancers by detecting hydroxymethylation status and modulating TET activity.
Enables specific detection and isolation of 5-hydroxymethylcytosine for research, diagnostic, and therapeutic purposes.
Documented Applications
Reprogramming of somatic cells into pluripotent stem cells, improving iPS cell generation efficiency and rate.
Generating stable human regulatory Foxp3+ T cells for immunological applications.
Improving the efficiency of cloning mammals via nuclear transfer or transplantation.
Diagnostic and therapeutic applications for myeloid cancers including myeloproliferative disorders, acute myeloid leukemia, systemic mastocytosis, and chronic myelomonocytic leukemia.
Detection and mapping of 5-hydroxymethylcytosine in DNA for epigenetic research and cancer diagnostics.
Screening for TET family enzyme modulators for potential cancer therapy.
Enhancement of stem cell therapies to induce differentiation into desired cell types for tissue replacement.
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