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-10533213-B2
Publication Date
2020-01-14
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 present invention identifies a novel catalytic activity of the TET family of proteins (TET1, TET2, TET3, and CXXC4), which is the hydroxylation-mediated conversion of 5-methylcytosine in DNA into 5-hydroxymethylcytosine. This activity underlies novel methods for regulating and detecting the cytosine methylation status of DNA, a process that is vital in various aspects of mammalian development, stem cell pluripotency, differentiation, and cancer.
The problem addressed by the invention arises from the crucial role of DNA methylation and demethylation in gene regulation, development, and tumorigenesis, where aberrant DNA methylation patterns are implicated. While passive demethylation mechanisms are well understood, mechanisms and molecular entities responsible for active DNA demethylation have remained unknown. The invention overcomes this by identifying TET proteins as enzymes capable of active hydroxylation of 5-methylcytosine, providing tools to regulate demethylation pathways and detect modified cytosine residues.
The invention provides methods to improve the reprogramming of somatic cells into induced pluripotent stem (iPS) cells by delivering catalytically active TET proteins or derivatives, thereby increasing the efficiency and rate of iPS generation. Similarly, it enhances cloning efficiency by nuclear transfer. The invention further provides novel methods to generate stable human regulatory FOXP3+ T cells by modulating methylation status via TET activity. Importantly, it offers compositions and methods to detect 5-hydroxymethylcytosine in DNA, which conventional methylation detection methods do not distinguish, enabling improved epigenetic analyses.
Claims Coverage
The claims focus on inventive methods of detecting and modulating 5-methylcytosine and 5-hydroxymethylcytosine involving TET family proteins and related enzymes, detection kits, therapeutic methods, and screening assays, encompassing several independent claims.
Method for detecting 5-methylcytosine via TET-mediated oxidation and modified nucleotide detection
A method comprising oxidation of 5-methylcytosine residues in nucleic acid by contacting with TET1, TET2, TET3, CXXC4, a catalytic fragment thereof, or any combination, creating a modified nucleic acid, and detecting the modified nucleic acid as indicative of 5-methylcytosine presence.
Method for adding glucose residues to 5-hydroxymethylcytosine for detection
Methods including adding glucose molecules or glucose-derivative donor substrates to 5-hydroxymethylcytosine residues using alpha-glucosyltransferases, beta-glucosyltransferases, or beta-glucosyl-alpha-glucosyl-transferases to facilitate identification and sequencing of these residues.
The independent claims comprise methods for enzymatic conversion of 5-methylcytosine to 5-hydroxymethylcytosine using TET family proteins, subsequent labeling with glucose derivatives using bacteriophage glucosyltransferases, and detection of these modified nucleotides, covering diagnostic and analytical techniques for DNA methylation status.
Stated Advantages
Provides novel enzymatic tools to actively convert 5-methylcytosine into 5-hydroxymethylcytosine, enabling regulation and detection of DNA methylation status.
Increases the efficiency and rate of generation of induced pluripotent stem cells from somatic cells.
Improves the efficiency of cloning mammals by nuclear transfer or nuclear transplantation.
Enables improved generation of stable human Foxp3+ regulatory T cells with stable FOXP3 expression.
Facilitates more accurate detection, mapping, and quantification of 5-hydroxymethylcytosine and 5-methylcytosine in genomic DNA, addressing limitations of existing methylation detection methods.
Provides novel methods for diagnosis and treatment of cancers, especially myeloid cancers, via modulation of TET family activity and detection of epigenetic signatures.
Documented Applications
Reprogramming somatic cells into induced pluripotent stem (iPS) cells by delivering catalytically active TET proteins or derivatives combined with pluripotency factors.
Improving efficiency of mammalian cloning by nuclear transfer using TET proteins.
Generating stable human regulatory Foxp3+ T cells by TET-mediated hydroxylation of 5-methylcytosine.
Labeling, detecting, and quantifying 5-hydroxymethylcytosine in DNA for epigenetic studies and diagnostic assessments.
Screening methods to identify modulators of TET protein activity for therapeutic purposes, especially in cancer treatment.
Treatment of myeloid cancers (e.g., myeloproliferative disorders, myelodysplastic syndromes, acute myeloid leukemia, systemic mastocytosis, chronic myelomonocytic leukemia) by modulating TET family activity and measuring 5-hydroxymethylcytosine levels.
Enhancing stem cell therapies by increasing pluripotency or differentiation potential via TET protein delivery.
Diagnostic methods and kits to determine methylation and hydroxymethylation patterns in biological samples for disease prognostics and familial predisposition.
Interested in licensing this patent?