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-10323269-B2
Publication Date
2019-06-18
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 invention discloses novel methods for regulating and detecting the cytosine methylation status of DNA based on a newly identified enzymatic activity for TET family proteins, including TET1, TET2, TET3, and CXXC4. These enzymes catalyze the hydroxylation of the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine, representing a previously unknown catalytic activity.
The invention addresses the problem that DNA methylation and demethylation are critical in mammalian development and various somatic cellular processes including differentiation and aging and become aberrant during tumorigenesis and cancer. Active DNA demethylation mechanisms were unknown, with no identified proteins or enzymes capable of catalyzing this activity. Hence, the invention provides means for modulating DNA methylation dynamics, including active demethylation, via the TET family enzymatic hydroxylation of 5-methylcytosine.
The invention further provides novel assays and reagents for detection of 5-hydroxymethylcytosine, which had been undetectable by previous antibodies or proteins recognizing 5-methylcytosine. These novel methods include use of thin-layer chromatography and antibodies specific to 5-hydroxymethylcytosine or its chemical derivatives. Additionally, the invention describes methods to improve reprogramming of somatic cells into pluripotent stem cells, enhance stem cell therapies, improve cloning efficiency via nuclear transfer, generate stable human regulatory FOXP3+ T cells, and detect or treat malignancies through modulation of TET activity or DNA methylation status.
Claims Coverage
The claims cover 15 inventive features regarding methods using TET family enzymes or fragments thereof for modifying methylated DNA bases and applications thereof.
Using TET family enzymes to generate 5-hydroxymethylcytosine in human T cells
A method comprising contacting or delivering to a human T cell an effective amount of an enzyme or a fragment thereof that oxidizes at least one methylated DNA base to convert 5-methylcytosine to 5-hydroxymethylcytosine.
Delivery of TET family enzymes to isolated purified human CD4+ T cells
Methods specifying isolated human T cells, purified CD4+ T cells, to be contacted with or delivered TET family enzymes or fragments to achieve hydroxylation of methylated DNA bases.
Co-administration with cytokines or growth factors including TGF-ß
Methods further comprising contacting or delivering a composition comprising cytokines, growth factors, or activating reagents, specifically including TGF-ß, to human T cells to improve generation of regulatory T cells.
Use of catalytically active TET family dioxygenases and functional derivatives
The enzyme or fragment used is a purified dioxygenase, specifically catalytically active TET family enzymes, functional TET family derivatives, or catalytically active fragments thereof.
Increasing and stabilizing FOXP3 expression in human T cells
Methods increase and stabilize FOXP3 expression, a key regulatory T cell marker, via contacting or delivering TET family enzymes or fragments.
Methods of improving the generation efficiency of induced pluripotent stem cells
Methods comprising contacting or delivering somatic cells with effective amounts of catalytically active TET family enzymes, derivatives, or fragments to convert 5-methylcytosine to 5-hydroxymethylcytosine and enhance reprogramming efficiency or rate.
Combining TET family enzymes with pluripotency factors Oct-4, Sox2, c-Myc, and Klf4
Methods further comprising inducing iPS cells by delivery of the pluripotency factors Oct-4, Sox2, c-Myc, and Klf4, optionally packaged in viral vectors such as adenoviral, lentiviral, or retroviral vectors.
Methods improving cloning efficiency via nuclear transfer using TET family enzymes
Methods comprising contacting a nucleus isolated during nuclear transfer with effective hydroxylation-inducing amounts of catalytically active TET family enzymes, derivatives, or fragments to improve cloning efficiency.
Methods stabilizing or improving TET-mediated DNA hydroxylation to modulate stem cell therapies and cancer treatment
Use of TET family enzymes, derivatives, or modulators to alter 5-methylcytosine hydroxylation in cells for applications including stem cell therapies, generating stable regulatory T cells, detection, diagnosis, and treatment of cancers.
Specific modulation of TET enzymes with inhibitors or activators
Administration or screening of agents that inhibit or activate TET family enzyme activity, with specificity to TET1, TET2, TET3, or CXXC4, to modulate DNA methylation status and treat diseases including leukemia and cancers.
Detection of 5-hydroxymethylcytosine using specific antibodies and chemical derivatives
Use of antibodies or antigen-binding fragments specific to 5-hydroxymethylcytosine or cytosine-5-methylsulfonate (bisulfite derivative) for detecting, quantifying, or isolating hydroxymethylated DNA from biological samples.
Assays and kits for detecting and purifying methylcytosine and 5-hydroxymethylcytosine
Kits comprising catalytically active TET family enzymes, phage-derived glucosyltransferases, glucose substrates, proteins recognizing glucosylated nucleotides, and reagents for detection and purification of hydroxymethylated DNA.
Methods for identifying, producing, and using epigenetic markers for diagnostic, prognostic and therapeutic purposes
Methods of determining methylation and hydroxymethylation levels of DNA in patient samples for diagnosis, risk stratification, therapy selection, and monitoring disease progression, especially in hematopoietic malignancies.
Use of phage-derived alpha-glucosyltransferase and beta-glucosyltransferase to label 5-hydroxymethylcytosine
Methods of covalently tagging 5-hydroxymethylcytosine residues in nucleic acids enzymatically to facilitate detection using proteins such as lectins or antibodies that specifically bind glucose- or gentibiosyl-modified bases.
Use of bisulfite treatment to convert 5-hydroxymethylcytosine to cytosine-5-methylsulfonate for detection
Methods of detecting 5-hydroxymethylcytosine in nucleic acids by bisulfite conversion to a methylsulfonate derivative recognized by specific antisera or antibodies for quantification and isolation purposes.
The claims encompass methods of using catalytically active TET family enzymes, derivatives, and fragments to convert 5-methylcytosine to 5-hydroxymethylcytosine for applications including generation of stable regulatory T cells, enhancement of iPS cell production, improving mammalian cloning, detection and diagnosis of methylation status, and treatment of cancers. The claims specify key assay reagents, detection antibodies, and modulators of TET activity for therapeutic modulation and high-throughput screening purposes.
Stated Advantages
Provides novel tools for regulating the DNA methylation status of mammalian cells through enzymatic conversion of 5-methylcytosine to 5-hydroxymethylcytosine.
Enhances efficiency and rate of reprogramming somatic cells into induced pluripotent stem cells.
Enables generation of stable human regulatory FOXP3+ T cells with improved functionality.
Improves efficiency of cloning mammals by nuclear transfer or nuclear transplantation.
Offers improved methods and reagents for detecting and mapping 5-hydroxymethylcytosine, overcoming previous limitations.
Facilitates diagnosis, stratification, and treatment of cancers, especially myeloid malignancies, through detection and modulation of TET enzyme activity and DNA hydroxymethylation status.
Documented Applications
Reprogramming somatic cells into pluripotent stem cells by combining catalytically active TET family enzymes with known pluripotency factors.
Generation of stable human regulatory FOXP3+ T cells by delivering TET family enzymes to human T cells in combination with cytokines such as TGF-ß.
Improving efficiency of cloning mammals by contacting nuclei isolated during nuclear transfer protocols with TET family enzymes.
Diagnostic and prognostic assessment of methylation and hydroxymethylation patterns in diseases including myelodysplastic syndromes, myeloproliferative disorders, acute myeloid leukemia, systemic mastocytosis, and chronic myelomonocytic leukemia.
Treatment of cancers by administration of modulators of TET family enzymatic activity to regulate DNA methylation dynamics.
Use of enzymatic tagging of 5-hydroxymethylcytosine in nucleic acids for detection, purification, and downstream genomic applications including chromatin immunoprecipitation and high-throughput sequencing.
High-throughput screening for TET family enzyme modulators as potential anti-cancer therapeutic agents.
Stem cell therapies enhanced by contacting or delivering catalytically active TET family enzymes to stem cells to increase pluripotency and aid differentiation into desired cell types.
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