Selective oxidation of 5-methylcytosine by TET-family proteins

• US 10,323,269 B2
• Filed: 02/23/2017
• Issued: 06/18/2019
• Est. Priority Date: 09/26/2008
• Status: Active Grant

• Current Assignee: Children's Medical Center Corporation, United States Department Of Health And Human Services
• Original Assignee: Children's Medical Center Corporation, United States Department Of Health And Human Services
• Inventors: Rao, Anjana, Tahiliani, Mamta, Koh, Kian Peng, Agarwal, Suneet, Iyer, Aravind
• Primary Examiner(s): Claytor, Renee
• Assistant Examiner(s): Clarke, Trent R
• Application Number:
  US15/440,815
• Publication Number:
  US 20180119113A1
• Time in Patent Office: 845 Days
• Field of Search: None
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/71 Oxidoreductases (EC 1.)
• CPC Class Codes: C12N 2501/724 Glycosyltransferases (EC 2.4.)
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2522/10 Nucleic acid binding proteins
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

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.

Claims

1. A method comprising contacting with, or delivering to, a human T

cell an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of an enzyme or a fragment thereof that oxidizes at least one methylated DNA base.

2. The method of claim 1, wherein the human T cell is isolated.

3. The method of claim 2, wherein the human T cell is a purified human CD4+ T cell.

4. The method of claim 1, further comprising contacting with, or delivering to, the human T cell a composition comprising at least one cytokine, growth factor, activating reagent, or combination thereof.

5. The method of claim 4, wherein the composition comprises the at least one cytokine, wherein the at least one cytokine comprises TGF-ß

.

6. The method of claim 1, wherein the enzyme or fragment thereof comprises a purified dioxygenase.

7. The method of claim 6, wherein the purified dioxygenase comprises a catalytically active TET family enzyme, a functional TET family derivative, a TET catalytically active fragment thereof, or any combination thereof.

8. The method of claim 1, wherein the method increases expression of FOXP3 in the human T cell.

9. The method of claim 1, wherein the method stabilizes expression of FOXP3 in the human T cell.

10. The method of claim 1, wherein the contacting with or the delivering to converts a 5-methylcytosine residue in the human T cell to a modified base.

11. The method of claim 10, wherein the modified base is formed by more than one oxidation reaction.

12. The method of claim 10, wherein the modified base comprises a 5-hydroxymethylcytosine residue.

13. The method of claim 1, wherein the method comprises contacting the human T cell with the enzyme or fragment thereof.

14. The method of claim 1, wherein the method comprises delivering the enzyme or fragment thereof to the human T cell.

15. The method of claim 1, wherein the enzyme or fragment thereof comprises TETI, TET2, TET3, CXXC4, or a catalytically active fragment of any of these.