Selective oxidation of 5-methylcytosine by tet-family proteins
AssigneesChildren's Medical Center Corporation, The United States of America, as represented by the Secretary, Department of Health and Human Services
InventorsAnjana Rao, Mamta Tahiliani, Kian Peng Koh, Suneet Agarwal, Aravind Iyer
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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.
Claims30
A method for improving the generation of stable human Foxp3+ T cells, the method comprising contacting with, or delivering to, a human T cell an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET family catalytically active fragment thereof, or combination thereof.
A method for improving efficiency or rate with which an induced pluripotent stem (iPS) cell is produced from an adult somatic cell, the method comprising contacting with, or delivering to a somatic cell, an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active thereof, or combination thereof.
A method for improving efficiency of cloning a mammal by nuclear transfer or nuclear transplantation, the method comprising contacting a nucleus extracted from a cell to be cloned with an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, during a nuclear transfer protocol.
A kit for the detection and purification of methylcytosine and 5-hydroxymethylcytosine, the kit comprising:
- (a) one or more catalytically active TET family enzymes, functional TET family derivatives, or TET catalytically active fragments thereof for the conversion of methylcytosine to 5-hydroxymethylcytosine;
- (b) one or more enzymes encoded by bacteriophages of the “T even” family;
- (c) one or more glucose or glucose-derivative donor substrates;
- (d) one or more proteins to detect glucose or glucose-derivative modified nucleotides;
- (e) standard DNA purification columns, buffers, and substrate solutions;
- and
- (f) packaging materials and instructions therein to use said kits.
Dependencies are inferred from the claim text.