Labeling hydroxymethylated residues
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-10337053-B2
Publication Date
2019-07-02
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 catalytic activity for the family of TET proteins, including TET1, TET2, TET3, and CXXC4. These enzymes are capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation, enabling regulation and detection of cytosine methylation status of DNA.
The problem being solved is the lack of identified molecular entities responsible for active DNA demethylation, which is a key mechanism in mammalian development, differentiation, cancer, and aging. Existing methods could not detect or map 5-hydroxymethylcytosine residues specifically, and no proteins with the proposed DNA demethylation activity were reliably identified prior to this invention.
The invention provides new methods and reagents to promote reprogramming of somatic cells into pluripotent stem cells by increasing the efficiency and rate of generation of induced pluripotent stem cells (iPS). It also provides methods for modulating pluripotency and cellular differentiation status through regulated expression of TET family enzymes and the presence of 5-hydroxymethylcytosine.
Furthermore, the invention offers novel methods for diagnosing and treating myeloid cancers by detecting TET family mutations using methods such as bisulfite treatment of nucleic acids and antibody-based detection of cytosine methylene sulfonate. Novel techniques for labeling and detecting 5-hydroxymethylcytosine are disclosed, including enzymatic tagging and antibody-based detection, overcoming previous limitations in distinguishing methylated cytosine derivatives.
Claims Coverage
The patent includes a set of independent claims focused on methods for labeling, detecting, and sequencing hydroxymethylated residues in mammalian nucleic acids by enzymatic glycosylation and subsequent sequencing.
Method for covalent labeling of 5-hydroxymethylcytosine in nucleic acids
A method comprising covalent labeling of a hydroxyl group on a hydroxymethylated residue in mammalian nucleic acids using alpha-glucosyltransferase, beta-glucosyltransferase, or beta-glucosyl-alpha-glucosyl-transferase to generate labeled 5-hydroxymethylcytosine residues.
Sequencing labeled mammalian nucleic acid containing hydroxymethylated residues
Sequencing of mammalian nucleic acids comprising labeled hydroxymethylated residues, including high-throughput sequencing methods.
Use of glycosyltransferases for labeling 5-hydroxymethylcytosine
Employing glucosyltransferases, including beta-glucosyltransferase, to glycosylate 5-hydroxymethylcytosine, optionally with modified glucose moieties.
Distinguishing 5-methylcytosine and 5-hydroxymethylcytosine residues in nucleic acids
Methods including distinguishing methylated cytosine residues such as 5-methylcytosine from 5-hydroxymethylcytosine residues after labeling and sequencing.
The independent claims cover methods for covalent enzymatic labeling of 5-hydroxymethylcytosine in mammalian DNA by glucosylation with specific enzymes, and the subsequent sequencing of labeled DNA to detect hydroxymethylation, including distinguishing it from 5-methylcytosine. The claims include embodiments of glycosylating with modified glucose and use of different viral glucosyltransferases for labeling.
Stated Advantages
The invention provides novel reagents and methods to promote efficient reprogramming of somatic cells to pluripotent stem cells, enhancing self-renewal and differentiation control.
It enables stable generation of human regulatory Foxp3+ T cells by modulating the DNA methylation status.
It overcomes the inability of prior reagents to detect 5-hydroxymethylcytosine specifically, offering enzymatic and antibody-based detection methods.
The invention allows improved diagnostic and therapeutic methods for myeloid cancers by detecting TET mutations and hydroxymethylation status.
It facilitates improved cloning efficiency by nuclear transfer through modification of DNA methylation status.
Documented Applications
Improving generation of stable human regulatory Foxp3+ T cells by contacting human T cells with catalytically active TET family enzymes or derivatives.
Improving the efficiency and rate of induced pluripotent stem (iPS) cell production by contacting somatic cells with catalytically active TET family proteins in combination with known pluripotency factors.
Improving efficiency of cloning mammals by nuclear transfer or nuclear transplantation by contacting nuclei with catalytically active TET family enzymes.
Diagnosing and treating individuals with myeloid cancers including myeloproliferative disorders, myelodysplastic syndromes, acute myeloid leukemia, systemic mastocytosis, and chronic myelomonocytic leukemia by detecting TET family mutations and hydroxymethylation status.
Detection, labeling, and isolation of 5-hydroxymethylcytosine in nucleic acids using enzymatic glucosylation, antibody-based detection, and chemical conversion approaches.
Screening for agents that modulate TET family enzymatic activity for therapeutic and research purposes.
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