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-12291742-B2
Publication Date
2025-05-06
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, TEM3, 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 of the TET family of enzymes, including TET1, TET2, TET3, and CXXC4, which is capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation. This enzymatic conversion represents a previously unknown mechanism for regulating and detecting cytosine methylation status in DNA.
DNA methylation and demethylation are crucial for mammalian development, cell differentiation, aging, and play important roles in diseases such as cancer. While passive DNA demethylation mechanisms are understood, active DNA demethylation had no identified molecular basis. The invention addresses this gap by identifying TET proteins as enzymes mediating active DNA demethylation through hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine, which has implications for DNA methylation regulation and gene expression.
The invention also discloses novel methods and reagents for utilizing the TET family proteins and their derivatives to promote somatic cell reprogramming into pluripotent stem cells, to modulate pluripotency and differentiation, to generate stable human regulatory FOXP3+ T cells, and to improve cloning efficiency by nuclear transfer. Furthermore, it provides novel detection methods for 5-hydroxymethylcytosine, including antibody-based and biochemical techniques, overcoming prior limitations due to lack of specific recognition for modified cytosines.
Claims Coverage
The patent contains one independent claim detailing a method involving enzymatic conversion of 5-methylcytosine to an oxidized derivative and subsequent nucleic acid sequencing. The claim covers the use of isolated TET family enzymes, their fragments, or variants, and optionally includes steps of sugar residue addition or targeting specific TET polypeptides and nucleic acid molecules.
Enzymatic conversion of 5-methylcytosine to an oxidized derivative
The method comprises contacting nucleic acid containing cytosine and 5-methylcytosine with an isolated enzyme comprising a TET polypeptide, a catalytically active fragment thereof, or variant, in an effective amount to convert 5-methylcytosine to an oxidized derivative.
Subsequent conversion and sequencing of nucleic acid
After enzymatic oxidation, the method includes converting cytosine to uracil and conducting a sequencing reaction to obtain the nucleic acid sequence, allowing distinction of modified cytosines.
Use of purified dioxygenase enzymes
The isolated enzymes used include dioxygenases, which may be recombinantly expressed.
Use of specific TET polypeptides and fragments with defined sequence homology
The TET polypeptides include those with at least 90% sequence identity to SEQ ID NOs: 27, 28, or 29, including TET2 and its catalytically active fragments containing a defined catalytic motif (SEQ ID NO: 1).
Optional sugar residue addition to oxidized derivatives
The method optionally includes adding a sugar residue, such as glucose or glucose-derivative donor substrates, to the oxidized 5-methylcytosine, catalyzed by alpha-glucosyltransferase or beta-glucosyltransferase enzymes encoded by bacteriophages.
Application to eukaryotic and somatic genomic DNA
The nucleic acid targeted can be isolated eukaryotic genomic DNA, including from somatic cells.
The independent claim covers a method employing TET family enzymes or their active fragments or variants to enzymatically convert 5-methylcytosine in nucleic acids to oxidized derivatives, with optional addition of sugar residues, followed by bisulfite conversion and sequencing to detect methylation patterns. The claim also specifies use of particular TET sequences and glycosyltransferase enzymes for labeling oxidized cytosines, enabling detection and analysis of DNA methylation and hydroxymethylation status.
Stated Advantages
The invention provides novel reagents and methods to regulate DNA methylation status, thereby improving the efficiency and rate of somatic cell reprogramming to induced pluripotent stem cells.
It enables generation of stable human regulatory FOXP3+ T cells by modulating 5-methylcytosine hydroxylation, which was previously difficult to achieve.
The discovery allows novel diagnostic and therapeutic strategies for cancers associated with DNA methylation aberrations, including myeloid malignancies.
Provides specific and sensitive methods for detecting and mapping 5-hydroxymethylcytosine in DNA, overcoming prior limitations due to lack of suitable detection reagents.
Offers improved methods to enhance cloning efficiency by nuclear transfer by treating nuclei with catalytically active TET proteins.
Documented Applications
Reprogramming of somatic cells to induced pluripotent stem cells with increased efficiency and rate by delivery of active TET family enzymes and pluripotency factors.
Generation of stable human regulatory FOXP3+ T cells by contacting human T cells with active TET family enzymes and cytokine compositions such as TGF-β.
Enhancement of mammalian cloning efficiency by nuclear transfer via treatment of isolated nuclei with catalytically active TET family enzymes.
Detection and mapping of 5-hydroxymethylcytosine residues in genomic DNA using enzyme-mediated conversion and antibody-based detection methods.
Methods for diagnosis, prognosis, and treatment of cancers, especially myeloid malignancies, by assessing and modulating levels and activity of TET enzymes and DNA hydroxymethylation status.
Screening methods for identifying modulators of TET family enzymes to develop anti-cancer therapies.
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