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-11072818-B2
Publication Date
2021-07-27
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. It is based upon the discovery of a novel catalytic activity in TET family proteins, specifically TET1, TET2, TET3, and CXXC4, which can convert 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation.
This hydroxylase activity of the TET family enzymes is surprising and enables modulation of DNA methylation status, influencing pluripotency and differentiation in cells, including improving the efficiency of generating induced pluripotent stem cells (iPS). The presence of 5-hydroxymethylcytosine correlates with pluripotency, while its loss is associated with differentiation.
The problem addressed is the unknown molecular basis of active DNA demethylation, a process important in embryogenesis, cell differentiation, aging, and cancer. Existing methods to detect cytosine methylation cannot distinguish 5-hydroxymethylcytosine, limiting the ability to study and manipulate DNA epigenetic states. The invention also addresses the lack of suitable methods and tools to identify and modulate TET protein activity in physiological contexts and diseases such as cancer.
Claims Coverage
The claims cover methods using enzymes that oxidize 5-methylcytosine to 5-hydroxymethylcytosine, specifically TET family proteins, including their functional derivatives and catalytic fragments, applied to nucleic acid sequences from somatic cells. The claims encompass both contacting nucleic acids with these enzymes and delivering the enzymes to nucleic acids.
Method of conversion of 5-methylcytosine to 5-hydroxymethylcytosine using TET family proteins
Contacting a nucleic acid sequence with an enzyme or fragment thereof that oxidizes at least one methylated DNA base to convert 5-methylcytosine to 5-hydroxymethylcytosine or a modified base, wherein the enzyme is a catalytically active TET family protein, functional derivative, or catalytic fragment.
Use of purified and isolated dioxygenase enzymes
Employing purified and isolated dioxygenases capable of hydroxylating 5-methylcytosine, specifically catalytically active TET family enzymes and their derivatives or fragments, to mediate the conversion.
Application to somatic cells and delivery methods
Applying the method to nucleic acid sequences derived from somatic cells and performing the conversion either by directly contacting or delivering the enzymes or fragments thereto.
The claims focus on the inventive catalytic activity of TET family enzymes and their use to hydroxylate 5-methylcytosine in nucleic acids, enabling modulation of methylation states. This includes purified enzymes or fragments, their functional derivatives, and focuses on methods involving contacting or delivering these enzymes to nucleic acids, especially from somatic cells, broadening applications in epigenetic regulation and stem cell biology.
Stated Advantages
Provides novel methods and reagents to modulate DNA methylation by converting 5-methylcytosine to 5-hydroxymethylcytosine.
Enables improvement in reprogramming efficiency and rate of induced pluripotent stem cells production.
Allows generation of stable regulatory Foxp3+ human T cells by modulating DNA methylation status.
Introduces novel diagnostic and therapeutic approaches for cancers, especially myeloid cancers, by detecting defects in TET function and 5-hydroxymethylcytosine levels.
Facilitates detection, isolation, and mapping of 5-hydroxymethylcytosine in DNA with new enzymatic and antibody-based tools, enhancing epigenetic research and diagnostic potential.
Documented Applications
Regulating and detecting DNA cytosine methylation status, including conversion of 5-methylcytosine to 5-hydroxymethylcytosine.
Improving reprogramming of somatic cells into induced pluripotent stem (iPS) cells, increasing efficiency and rate of generation.
Improving cloning efficiency in mammals by nuclear transfer or transplantation using TET family enzymes.
Production of stable human regulatory Foxp3+ T cells for immunological applications.
Diagnosis, treatment, and monitoring of myeloid cancers such as myeloproliferative disorders, myelodysplastic syndromes, acute myeloid leukemia, systemic mastocytosis, and chronic myelomonocytic leukemia.
Screening for modulators of TET family enzymatic activity to identify anti-cancer agents or therapeutic inhibitors/activators.
Methods for detecting and isolating 5-hydroxymethylcytosine in nucleic acids using enzymatic tagging, antibody detection, and bisulfite conversion techniques.
Interested in licensing this patent?