Methods of epigenetic analysis
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-11208683-B2
Publication Date
2021-12-28
Expiration Date
2029-09-28
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Abstract
The present invention provides for methods of epigenetic analysis. In some cases, the methods may include obtaining a sample comprising a nucleic acid sequence. In some cases, the nucleic acid sequence may comprise one or more epigenetic marks. The methods may include performing a sequencing. The methods may include distinguishing a hydroxymethylated base from a methylated base.
Core Innovation
The invention provides novel methods for epigenetic analysis and regulation, primarily based on the discovery of a new enzymatic activity of the TET family of proteins, including TET1, TET2, TET3, and CXXC4. These enzymes catalyze the conversion of 5-methylcytosine to 5-hydroxymethylcytosine via hydroxylation, a modification undetectable by traditional methylation analyses.
The problem addressed relates to the need for improved understanding and manipulation of DNA methylation and demethylation processes, which are critical in mammalian development, cellular differentiation, pluripotency, aging, and oncogenesis. Existing methods could not distinguish between methylated and hydroxymethylated cytosine, hindering research and therapeutic development.
The invention further provides compositions and methods to promote cell reprogramming, improve cloning efficiency via nuclear transfer, generate stable human regulatory FOXP3+ T cells, detect and isolate 5-hydroxymethylcytosine in nucleic acids using specific antibodies and enzymatic tagging, and identify or screen for modulators of TET activity. The methods include use of TET enzymes or their derivatives to convert 5-methylcytosine to 5-hydroxymethylcytosine and subsequent detection through enzymatic glucosylation or chemical modification followed by antibody-based recognition.
Claims Coverage
The patent includes one independent claim defining a composition comprising three components: a methylcytosine dioxygenase, a DNA glucosyltransferase, and nucleic acid containing glucosylated 5-hydroxymethylcytosine.
Composition comprising specific enzymatic components
A mixture including a methylcytosine dioxygenase (notably TET enzymes), a DNA glucosyltransferase, and nucleic acid harboring glucosylated 5-hydroxymethylcytosine.
Use of TET enzymes as the methylcytosine dioxygenase
The methylcytosine dioxygenase component is specified as a TET family enzyme, including TET1, TET2, TET3, and CXXC4.
Incorporation of bacteriophage-encoded DNA glucosyltransferases
The DNA glucosyltransferase is encoded by bacteriophages of the T even family, including beta-glucosyltransferase and alpha-glucosyltransferase types.
Inclusion of DNA methyltransferase DNMT1
The composition may additionally include DNA methyltransferases such as DNMT1.
Provision of glucose donor substrates and buffers
The composition further contains glucose donor substrates like UDP-glucose and appropriate buffers for enzymatic activity.
These claims cover compositions combining enzymatically active TET proteins and bacteriophage-derived DNA glucosyltransferases with nucleic acids containing glucosylated 5-hydroxymethylcytosine, providing tools for detecting or manipulating epigenetic DNA modifications.
Stated Advantages
The invention allows modulation and regulation of DNA methylation and hydroxymethylation status, facilitating improved reprogramming of somatic cells into pluripotent stem cells.
It provides novel and specific detection methods for 5-hydroxymethylcytosine, overcoming the limitations of traditional methylation analysis techniques.
The invention enhances the efficiency of stem cell therapies and nuclear transfer cloning by targeting epigenetic regulation pathways.
It offers methods for diagnosis and treatment of cancers, particularly myeloid malignancies, by identifying and modulating TET activity and associated DNA modifications.
Documented Applications
Improving the generation of stable human regulatory FOXP3+ T cells using TET family enzymes or derivatives.
Enhancing the efficiency or rate of induced pluripotent stem cell (iPS) production from somatic cells by delivering catalytically active TET family enzymes in combination with pluripotency factors.
Increasing the efficiency of mammalian cloning by nuclear transfer via treatment with catalytically active TET family enzymes.
Detection and mapping of 5-hydroxymethylcytosine in genomes for research, disease diagnosis, and treatment monitoring, including cancer and myeloid malignancies.
Methods for screening small molecule or biological modulators of TET enzyme activity for therapeutic applications.
Use in stem cell therapies to increase pluripotency and direct differentiation.
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