Methods for distinguishing 5-hydroxymethylcytosine from 5-methylcytosine
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-10767216-B2
Publication Date
2020-09-08
Expiration Date
2029-09-28
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Abstract
Provided herein are methods and kits for distinguishing 5-hydroxymethylcytosine from 5-methylcytosine.
Core Innovation
The invention relates to enzymes with novel hydroxylase activity and methods for their use in labeling and detecting methylated residues. It identifies a novel and surprising catalytic activity of the TET protein family (TET1, TET2, TET3, and CXXC4), capable of converting 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation.
DNA methylation and demethylation are vital for mammalian development, differentiation, aging, and are notably aberrant during tumorigenesis and cancer. DNA methylation primarily occurs at cytosine in CpG dinucleotides and is dynamic during embryogenesis, affecting imprinting, X inactivation, and silencing retroviruses. While passive DNA demethylation mechanisms are known, active demethylation enzymes had not been reliably identified, posing a problem in understanding and controlling DNA methylation status for therapeutic purposes.
The present invention solves this problem by providing novel methods and reagents based on the TET family hydroxylase activity to regulate and detect cytosine methylation status, improve reprogramming of somatic cells to pluripotent stem cells, modulate pluripotency and differentiation, diagnose and treat myeloid cancers, and generate stable human regulatory Foxp3+ T cells. It also introduces novel detection methods for 5-hydroxymethylcytosine, which is not recognized by existing 5-methylcytosine binding proteins or antibodies, thereby addressing a critical gap in studying DNA methylation.
Claims Coverage
The patent contains one independent claim that covers a method for distinguishing 5-hydroxymethylcytosine from 5-methylcytosine in a nucleic acid sequence by labeling and sequencing.
Method for distinguishing 5-hydroxymethylcytosine from 5-methylcytosine
A method comprising obtaining a sample comprising a nucleic acid sequence containing both 5-hydroxymethylcytosine and 5-methylcytosine, labeling the 5-hydroxymethylcytosine, sequencing the nucleic acid sequence, and distinguishing 5-hydroxymethylcytosine from 5-methylcytosine.
Labeling 5-hydroxymethylcytosine by glycosylation
Labeling of 5-hydroxymethylcytosine is performed by glycosylating it, including associating a label with the glucose added during glycosylation.
Conversion of methylated cytosine by dioxygenase enzymes
Contacting the nucleic acid sequence with dioxygenases or catalytic fragments thereof, comprising TET1, TET2, TET3, or CXXC4 proteins (or combinations), to convert methylated cytosine to a modified base.
Optional bisulfite treatment after enzymatic conversion
Following enzymatic conversion, contacting the nucleic acid sequence with sodium bisulfite to facilitate detection.
The independent claim covers a multi-step method utilizing labeling (notably glycosylation) and sequencing techniques to distinguish 5-hydroxymethylcytosine from 5-methylcytosine, incorporating the action of TET family dioxygenases and bisulfite treatment for modified base detection.
Stated Advantages
Provides novel methods and reagents to promote efficient reprogramming of somatic cells into pluripotent stem cells.
Enables modulation of pluripotency and cellular differentiation status via TET family enzyme activity.
Facilitates diagnosis and treatment of myeloid cancers by detecting defects in hydroxymethylation status.
Offers new detection methods for 5-hydroxymethylcytosine that was previously undetectable with standard methylation assays.
Improves the generation of stable human regulatory Foxp3+ T cells for therapeutic use.
Documented Applications
Reprogramming somatic cells into induced pluripotent stem cells with increased efficiency and speed.
Improving cloning efficiency in mammals by nuclear transfer or nuclear transplantation protocols.
Generating stable human regulatory Foxp3+ T cells for immunological tolerance and therapeutic applications.
Diagnosing and treating myeloid cancers, including myeloproliferative disorders, myelodysplastic syndromes, acute myeloid leukemia, systemic mastocytosis, and chronic myelomonocytic leukemia.
Mapping and detecting 5-hydroxymethylcytosine in genome-wide methylation studies and epigenetic analyses.
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