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-12018320-B2
Publication Date
2024-06-25
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 the discovery that TET family proteins, including TET1, TET2, TET3, and CXXC4, catalyze the conversion of 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation. This enzymatic activity is unexpected and offers tools for modulating DNA methylation in mammalian cells.
A key problem addressed is the lack of identified enzymes capable of active DNA demethylation in mammals, which is important for development, differentiation, aging, and cancer. DNA methylation patterns are abnormal in cancer, and controlled DNA demethylation mechanisms were unknown. The invention overcomes this gap by identifying TET proteins with hydroxylase activity that modify 5-methylcytosine to 5-hydroxymethylcytosine, providing new possibilities for studying and manipulating DNA methylation and demethylation.
The invention further provides methods to improve reprogramming of somatic cells into pluripotent stem cells, regulate cellular differentiation, perform stem cell therapies, detect and isolate 5-hydroxymethylcytosine in nucleic acids, treat cancers by modulating TET protein activity, and develop assays and kits for these purposes. The hydroxylation activity of the TET family proteins represents a novel mechanism for regulating epigenetic states and is harnessed in various diagnostic and therapeutic methods.
Claims Coverage
The patent includes one independent claim directed to a method for identifying epigenetic modifications on polynucleotides through enzyme-mediated oxidation and detection. The main inventive features relate to the use and enzymatic activity of TET-family proteins and subsequent steps to detect epigenetic modifications.
Enzymatic oxidation of epigenetic modifications
Providing a polynucleotide comprising an epigenetic modification and contacting it with an enzyme or catalytically active fragment to oxidize the epigenetic modification generating an oxidized form.
Use of TET-family enzymes for oxidation
Utilizing active TET family enzymes including TET1, TET2, TET3, and CXXC4 proteins to catalyze the oxidation of 5-methylcytosine to oxidized derivatives through one or more oxidation reactions.
Generation of glycosylated oxidized epigenetic modifications
Contacting the oxidized epigenetic modification with a glucosyltransferase such as beta- or alpha-glucosyltransferase to produce a glycosylated oxidized modification facilitating detection or identification.
Detection of epigenetic modifications via sequencing or imaging
Using sequencing or imaging techniques on the oxidized or derivative polynucleotide to identify the original epigenetic modification.
The claims focus on methods using catalytic activity of TET-family enzymes to oxidize 5-methylcytosine in DNA, subsequent glycosylation, and identifying epigenetic modifications through detection of these oxidized derivatives, enabling improved epigenetic analyses.
Stated Advantages
Provides novel enzymatic tools for converting 5-methylcytosine to 5-hydroxymethylcytosine enabling regulation of DNA methylation status.
Improves efficiency and rate of inducing pluripotent stem cells from somatic cells.
Enables improved methods for generating stable human regulatory Foxp3+ T cells.
Allows for diagnosis, treatment, and monitoring of cancers, including leukemia, by targeting TET family activity.
Offers new diagnostic and research tools to detect and isolate 5-hydroxymethylcytosine in nucleic acids where previously no such reagents existed.
Documented Applications
Reprogramming somatic cells into induced pluripotent stem cells with increased efficiency and rate using TET family enzymes combined with pluripotency factors.
Generating stable human regulatory Foxp3+ T cells by delivering TET family enzymes to human T cells, optionally with cytokines like TGF-β.
Improving cloning efficiency of mammals by nuclear transfer through treating donor nuclei with catalytically active TET family enzymes.
Detecting and mapping of 5-hydroxymethylcytosine in genomic DNA for basic research, clinical diagnostics, and drug screening.
Treatment of cancers, including myeloid cancers and leukemias, by administering modulators (inhibitors or activators) of TET family enzymatic activity.
Methods for screening and identifying TET family modulators as potential agents for anti-cancer therapies.
Use in stem cell therapies to induce pluripotency and differentiation through modulation of DNA methylation via TET family enzymes.
Use of bisulfite treatment combined with antibodies specific for cytosine-5-methylenesulfonate to detect 5-hydroxymethylcytosine in DNA samples.
Covalent tagging of 5-hydroxymethylcytosine in nucleic acids using glucosyltransferases encoded by T-even phages, enabling detection and purification.
Use of kits containing TET family enzymes, glucosyltransferases, and detection reagents for controlling and analyzing DNA methylation and hydroxymethylation.
Interested in licensing this patent?