Detection of 5-hydroxymethylcytosine by glycosylation
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-10508301-B2
Publication Date
2019-12-17
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 present invention provides novel methods for regulating and detecting the cytosine methylation status of DNA. It is based on the identification of a novel and surprising catalytic activity for the family of TET proteins, including TET1, TET2, TET3, and CXXC4. This activity relates to these enzymes being capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation.
DNA methylation plays vital roles in development, differentiation, and aging and is known to become aberrant during tumorigenesis and cancer. Active DNA demethylation by enzymatic mechanisms has been postulated but the molecular basis was unknown. The invention addresses this by discovering that TET family enzymes mediate conversion of 5-methylcytosine to 5-hydroxymethylcytosine, providing tools for regulating the DNA methylation status of mammalian cells, and methods for detecting and isolating 5-hydroxymethylcytosine in nucleic acids.
The invention also provides methods to improve reprogramming of somatic cells into pluripotent stem cells, enhancing efficiency of induced pluripotent stem (iPS) cell generation, and modulating pluripotency and differentiation by delivery or expression of catalytically active TET family enzymes or derivatives. Furthermore, methods for generation of stable human regulatory Foxp3+ T cells via administration of TET family enzymes or fragments are disclosed. Additionally, the invention includes diagnostic and therapeutic methods for myeloid cancers based on detecting hydroxymethylation status and use of TET enzyme modulators.
Claims Coverage
The patent contains one independent claim centered on a method involving labeled glucose-mediated detection of 5-hydroxymethylcytosine in mammalian nucleic acids. Several main inventive features are described therein.
Detection of 5-hydroxymethylcytosine using labeled glucose and beta-glucosyltransferase
A method of contacting a mammalian nucleic acid sequence with an enzyme comprising a beta-glucosyltransferase that utilizes a labeled glucose or glucose-derivative donor substrate, specifically uridine diphosphate glucose (UDPG), to add a labeled glucose molecule or derivative to 5-hydroxymethylcytosine, generating a labeled glucosylated-5-hydroxymethylcytosine.
Use of labeled glucose for detection and characterization of 5-hydroxymethylcytosine
Further comprising detection of the 5-hydroxymethylcytosine after labeling, including sequencing of the nucleic acid and distinguishing 5-hydroxymethylcytosine from 5-methylcytosine.
Use of glucose-recognizing moieties for detecting labeled 5-hydroxymethylcytosine
The method can include contacting the nucleic acid with a moiety that recognizes glucose or labeled glucose derivatives, wherein the moiety can be a protein such as an antibody or fragment thereof, including versions modified with tags like biotin or fluorescent labels.
The claims cover a method for enzymatic labeling and detection of 5-hydroxymethylcytosine in mammalian nucleic acids using labeled glucose donor substrates and beta-glucosyltransferase enzymes, along with subsequent detection and discrimination methods. The inventive features focus on enzymatic labeling and specific detection of hydroxymethylated cytosine.
Stated Advantages
Provides novel tools for regulating DNA methylation status in mammalian cells.
Enhances efficiency and rate of reprogramming somatic cells into pluripotent stem cells.
Enables generation of stable human regulatory Foxp3+ T cells.
Provides new methods for diagnosis and treatment of myeloid cancers based on detecting hydroxymethylation status.
Facilitates improved stem cell therapies by modulating pluripotency and differentiation using TET family enzymes.
Allows direct detection and mapping of 5-hydroxymethylcytosine, overcoming limitations of previous detection methods.
Enables screening for modulators of TET family enzyme activity for therapeutic applications, including cancer treatment.
Documented Applications
Regulating and detecting cytosine methylation and hydroxymethylation status of DNA in research and clinical settings.
Improving reprogramming of somatic cells into induced pluripotent stem cells by delivery of TET family enzymes in combination with pluripotency factors.
Enhancing the generation of stable human regulatory Foxp3+ T cells via administration of TET family enzymes and cytokine compositions.
Improving the efficiency of mammalian cloning by nuclear transfer via hydroxylating TET family enzymes applied to donor nuclei.
Methods of diagnosing and treating myeloid cancers such as myelodysplastic syndromes, myeloproliferative disorders, acute myeloid leukemia, systemic mastocytosis, and chronic myelomonocytic leukemia based on TET enzyme function and hydroxymethylcytosine detection.
Screening for agents modulating TET family enzyme activity to develop anti-cancer therapeutics.
Methods and kits for covalent tagging, detection, and purification of methylcytosine and 5-hydroxymethylcytosine in nucleic acids for genomic analyses.
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