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-10443091-B2
Publication Date
2019-10-15
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 TET family proteins, including TET1, TET2, TET3, and CXXC4. These enzymes can convert the DNA nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation, a previously unknown modification in mammalian DNA.
The problem addressed by the invention arises from the vital roles DNA methylation and demethylation play in mammalian development, cellular differentiation, aging, and disease states such as cancer. While passive demethylation mechanisms are understood, active DNA demethylation mechanisms were unknown, and enzymes capable of active demethylation had not been identified. DNA methylation is dynamic, with aberrant regulation implicated in tumorigenesis; thus, identification of enzymes that can modulate DNA methylation status and methods to detect changes therein is important for therapeutic and diagnostic advances.
The invention further provides novel reagents and methods to promote reprogramming of somatic cells into pluripotent stem cells (iPS cells), modulate pluripotency and differentiation, improve cloning efficiency by nuclear transfer, generate stable human regulatory Foxp3+ T cells, and identify or treat myeloid cancers via detection of TET mutations and hydroxymethylation status. It includes methods for detecting 5-hydroxymethylcytosine using antibodies and chemical labeling, as existing antibodies do not recognize this base modification.
Claims Coverage
The patent contains one independent claim covering a method of detecting 5-hydroxymethylcytosine using specific enzymes and substrates. The main inventive features involve selective labeling and detection of 5-hydroxymethylcytosine in nucleic acids.
Detection of 5-hydroxymethylcytosine using covalent trapping with glucosyltransferases and glucose-derivative substrates
A method of detecting 5-hydroxymethylcytosine in a nucleic acid by contacting it with an enzyme comprising alpha-glucosyltransferase, beta-glucosyltransferase, or beta-glucosyl-alpha-glucosyl-transferase, utilizing a glucose donor substrate or glucose-derivative donor substrate that traps covalent enzyme-DNA intermediates to label 5-hydroxymethylcytosine residues.
Use of uridine diphosphate glucose analogs to enhance detection
Employed glucose donor substrates are uridine diphosphate glucose analogs, such as uridine-2-deoxy-2-fluoro-glucose, for improved covalent trapping and detection of 5-hydroxymethylcytosine.
Application to naturally occurring and converted 5-hydroxymethylcytosine
The method applies both to naturally occurring 5-hydroxymethylcytosine and to nucleic acids treated with TET family enzymes to convert 5-methylcytosine to 5-hydroxymethylcytosine.
Use of bacteriophage-encoded glucosyltransferases
The enzymes utilized are encoded by bacteriophages such as T2, T4, and T6 for alpha-glucosyltransferase; T4 for beta-glucosyltransferase; and T2 or T6 for beta-glucosyl-alpha-glucosyl-transferase.
Versatile application in vitro, in cells, or in vivo
The detection method can be applied to nucleic acids in vitro, within cells, or in living organisms.
The claim covers a method for selective enzymatic tagging and detection of 5-hydroxymethylcytosine in nucleic acids using specific glucosyltransferases and glucose analog substrates that facilitate covalent trapping of enzyme-DNA intermediates, enabling sensitive and specific detection of this epigenetic modification.
Stated Advantages
Provides a novel method to regulate and detect cytosine methylation and hydroxymethylation status via TET family enzyme activity.
Enables enhanced reprogramming of somatic cells into induced pluripotent stem cells with greater efficiency and rate.
Improves cloning efficiency by enhancing nuclear reprogramming methods via TET family enzymes.
Offers specific detection tools for 5-hydroxymethylcytosine in DNA, overcoming prior lack of recognition by conventional reagents.
Facilitates generation of stable human regulatory Foxp3+ T cells, enhancing immunotherapy applications.
Supports diagnosis and treatment of cancers, including myeloid malignancies, through detection of TET mutations and modulation of TET activity.
Documented Applications
Regulating and detecting DNA methylation status via enzymatic conversion of 5-methylcytosine to 5-hydroxymethylcytosine.
Reprogramming somatic cells into induced pluripotent stem cells using TET family enzymes.
Improving efficiency of mammalian cloning by nuclear transfer using TET family hydroxylase activity.
Generating stable human regulatory Foxp3+ T cells by TET-mediated DNA modification.
Diagnosing and treating myeloid cancers like myeloproliferative disorders, myelodysplastic syndromes, acute myeloid leukemia, systemic mastocytosis, and chronic myelomonocytic leukemia.
Screening for modulators of TET family enzymatic activity for research and therapeutic interventions.
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