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-12338489-B2
Publication Date
2025-06-24
Expiration Date
2029-09-28
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
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 a surprising catalytic activity of TET family enzymes including TET1, TET2, TET3, and CXXC4. These enzymes are capable of converting 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation, which represents a novel epigenetic modification mechanism.
The problem addressed arises from the vital role DNA methylation and demethylation play in mammalian development, differentiation, aging, and cancer. Prior to the invention, active DNA demethylation mechanisms were unclear, with no identified enzymes capable of converting methylcytosine into demethylated forms. Furthermore, there was a lack of techniques and reagents to specifically detect or map 5-hydroxymethylcytosine in genomes, as it was not recognized by existing methyl-binding proteins or antibodies.
The invention solves these problems by identifying TET family proteins as 20G-Fe(II) oxygenases capable of hydroxylating 5-methylcytosine to 5-hydroxymethylcytosine. This activity explains active DNA demethylation and provides methods to enhance reprogramming of somatic cells into pluripotent stem cells. The invention also provides reagents and methods to detect 5-hydroxymethylcytosine, including antibodies and enzymatic tagging strategies, supporting new approaches for epigenetic analyses and cancer diagnostics.
Claims Coverage
The claims present five main inventive features related to the catalytic conversion of methylated cytosine residues in nucleic acids and their derivatization for detection and analysis.
selective catalytic conversion of 5-methylcytosine to 5-hydroxymethylcytosine by TET family proteins
A method of converting methylated cytosine in an isolated eukaryotic nucleic acid molecule to a modified cytosine residue by contacting the nucleic acid with a polypeptide comprising TET1, TET2, TET3, or CXXC4 polypeptides or catalytically active fragments thereof.
derivatization of modified cytosine residues using glucosyltransferases
Subsequent to catalytic conversion, contacting the isolated nucleic acid molecule with a glucosyltransferase polypeptide, specifically β-glucosyltransferase (BGT) or α-glucosyltransferase (AGT), to derivatize the modified cytosine residue.
detection of methylation status including bisulfite treatment
Detection of methylation status of the isolated nucleic acid based on presence or absence of the modified cytosine residue, optionally involving contacting the nucleic acid with bisulfite to distinguish cytosine modifications.
use of specific TET family polypeptide sequences
Specifying TET family polypeptides and their catalytically active fragments to include those with at least 90% sequence identity to sequences SEQ ID NO:27 (TET1), SEQ ID NO:28 (TET2), and SEQ ID NO:29 (CXXC4), including conservatively substituted variants.
application to samples from subjects including cancer patients
The method applied to isolated nucleic acid molecules obtained from cell or tissue samples, extracellular fluid samples, or from subjects having or suspected of having cancer, facilitating diagnostic and analytical use.
Together, the claims protect methods of direct enzymatic conversion of 5-methylcytosine to 5-hydroxymethylcytosine using TET family enzymes, derivatization of the modified base for detection by glucosyltransferases, and associated methods for detecting methylation status, including applications in samples from subjects and cancer diagnostics.
Stated Advantages
Novel identification of TET family enzymes as hydroxylases converting 5-methylcytosine to 5-hydroxymethylcytosine enables regulation and detection of cytosine methylation status.
Improvement in reprogramming efficiency and rate for generating induced pluripotent stem cells from somatic cells.
Enhanced methods for generating stable human regulatory Foxp3+ T cells by modulating methylation status.
Provision of new reagents and assays for sensitive and specific detection of 5-hydroxymethylcytosine in DNA, overcoming prior limitations.
Potential for improved diagnosis, stratification, and treatment of cancers, especially myeloid malignancies, through monitoring TET activity and DNA hydroxymethylation.
Applications in stem cell therapies and methods to improve efficiency of mammalian cloning by nuclear transfer.
Documented Applications
Regulation of DNA methylation in mammalian cells and modulation of cell differentiation and pluripotency status.
Improving efficiency and rate of induced pluripotent stem cell (iPS) generation from adult somatic cells.
Enhanced generation of stable human regulatory Foxp3+ T cells.
Improvement of cloning efficiency in mammals by nuclear transfer or nuclear transplantation.
Detection, mapping, and quantitative analysis of 5-methylcytosine and 5-hydroxymethylcytosine in genomic DNA.
Diagnostic and prognostic methods for myeloproliferative disorders, myelodysplastic syndromes, leukemias, including acute myeloid leukemia and related hematologic cancers.
Screening methods for modulators of TET family enzyme activity applicable to anti-cancer therapies.
Methods and compositions to enhance stem cell therapies including controlled differentiation into desired cell types.
Interested in licensing this patent?