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-9115386-B2
Publication Date
2015-08-25
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 present invention provides novel methods for regulating and detecting the cytosine methylation status of DNA, based upon the identification of a novel catalytic activity for the family of TET proteins, namely TET1, TET2, TET3, and CXXC4. These enzymes are capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation.
This discovery offers new approaches to modulate pluripotency and cellular differentiation status by altering DNA methylation patterns. The TET family enzymes are shown to be highly expressed in embryonic and induced pluripotent stem cells, and their activity correlates with the presence of 5-hydroxymethylcytosine and changes in pluripotency. The invention thus proposes the use of these enzymes and their derivatives to enhance reprogramming efficiency, generate stable regulatory T cells, and improve cloning techniques via nuclear transfer.
Importantly, the invention addresses the long-standing problem in the field regarding active DNA demethylation, for which molecular mechanisms were previously unknown. Conventional methods for detecting 5-methylcytosine do not differentiate it from 5-hydroxymethylcytosine, and no enzymes capable of active demethylation had been identified. By identifying TET proteins as hydroxylases converting 5-methylcytosine to 5-hydroxymethylcytosine, and by developing novel detection methods specific for 5-hydroxymethylcytosine, the invention provides critical tools to study and manipulate DNA methylation in development, disease, and cancer.
Claims Coverage
The patent includes one independent claim about a kit integrating reagents for detecting 5-hydroxymethylcytosine in mammalian DNA samples and instructions for DNA extraction.
Kit for detecting naturally occurring 5-hydroxymethylcytosine
The kit comprises (1) an antibody or binding portion thereof that binds to the naturally occurring 5-hydroxymethylcytosine nucleotide, (2) a catalytically active TET family enzyme, its functional derivative, a catalytically active fragment thereof, or combinations thereof; and (3) packaging materials and instructions to extract a DNA sample from the biological sample.
The claim discloses an integrated kit comprising specific antibodies against 5-hydroxymethylcytosine and one or more TET family enzymes or related derivatives, together with DNA sample preparation instructions, enabling detection of naturally occurring 5-hydroxymethylcytosine in mammalian genomic DNA.
Stated Advantages
Provides novel methods and reagents for regulating DNA methylation status by enzymatic hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine.
Enables improved efficiency and rate for generation of induced pluripotent stem cells from somatic cells.
Improves generation of stable human regulatory Foxp3+ T cells relevant for immunological tolerance.
Enhances methods for improving cloning efficiency by nuclear transfer through catalytically active TET family enzymes.
Introduces new detection methodologies for 5-hydroxymethylcytosine, overcoming limitations of previous methylation analysis techniques.
Allows diagnosis, monitoring, and treatment of cancers, including myeloid malignancies, linked to aberrant methylation and TET enzyme activity.
Documented Applications
Reprogramming of somatic cells to improve generation of induced pluripotent stem (iPS) cells.
Generation of stable human regulatory Foxp3+ T cells for immunological applications.
Improvement of cloning mammals by nuclear transfer or nuclear transplantation protocols.
Methods for diagnosing and treating myeloid cancers such as myeloproliferative disorders, myelodysplastic syndromes, acute myeloid leukemia, systemic mastocytosis, and chronic myelomonocytic leukemia.
Detection and mapping of 5-hydroxymethylcytosine in genomic DNA for epigenetic and methylation studies.
Screening for agents that modulate TET family enzymatic activity for anti-cancer therapeutic development.
Enhancement of stem cell therapies by increasing pluripotency and directing stem cell differentiation using TET family proteins.
Interested in licensing this patent?