Selective oxidation of 5-methylcytosine by TET-family proteins

• US 10,612,076 B2
• Filed: 04/10/2017
• Issued: 04/07/2020
• Est. Priority Date: 09/26/2008
• Status: Active Grant

• Current Assignee: Children's Medical Center Corporation, United States Department Of Health And Human Services (Government of the United States of America)
• Original Assignee: Children's Medical Center Corporation
• 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:
  US15/483,282
• Publication Number:
  US 20170218338A1
• Time in Patent Office: 1,093 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:

regulating a methylation status of one or more cytosine bases in an adult cell that is isolated and purified, wherein the regulating comprises contacting the adult cell with an effective amount of;

(i) an enzyme or fragment thereof that converts;

(a) 5-methylcytosine to 5-hydroxymethylcytosine,

(b) 5-methylcytosine to an acid beyond hydroxymethlyation, or

(c) 5-methylcytosine to an aldehyde beyond hydroxymethlyation;

(ii) an inhibitor that decreases or prevents hydroxymethylation, wherein the inhibitor comprises siRNA or shRNA;

or

(iii) a combination thereof, and

wherein the methylation status of the one or more cytosine bases in the adult cell is regulated.

2. The method of claim 1, wherein the adult cell is a naive T cell.

3. The method of claim 1, wherein the adult cell is a peripheral T cell.

4. The method of claim 1, wherein the adult cell is a purified human CD4+ T cell.

5. The method of claim 2, wherein the regulating differentiates the naive T cell to a regulatory T cell, and wherein the regulatory T cell is a Foxp3+ T cell.

6. The method of claim 1, wherein the one or more cytosine bases are positioned in a CpG site, a CpG island, or a combination thereof in the adult cell.

7. The method of claim 1, further comprising contacting the adult cell with a cytokine, a growth factor, or an activating reagent.

8. The method of claim 7, wherein the adult cell is contacted with the cytokine, and wherein the cytokine comprises TGF-beta.

9. The method of claim 7, wherein the adult cell is contacted with the activating agent, and wherein the activating reagent comprises retinoic acid.

10. The method of claim 2, wherein the naive T cell comprises a plurality of naive T cells, and wherein a percentage of regulatory T cells generated from the regulating of (a) is at least 5% higher in the plurality of naive T cells than a control population of naive T cells generated without the effective amount of (a).

11. The method of claim 2, wherein the regulating differentiates the naive T cell to a regulatory T cell, and wherein the regulatory T cell maintains a characteristic function in the absence of an exogenous differentiation factor.

12. The method of claim 11, wherein the characteristic function comprises a suppression of proliferation of naive T cells.

13. The method of claim 1, wherein the method comprises contacting the adult cell with an effective amount of the enzyme or fragment thereof in (i) that is a recombinant enzyme or a fragment of a recombinant enzyme.

14. The method of claim 1, wherein the method comprises contacting the adult cell with an effective amount of the enzyme or fragment thereof in (i) that comprises a mutation in a sequence corresponding to the enzyme or fragment thereof.

15. The method of claim 1, wherein the method comprises contacting the adult cell with an effective amount of the enzyme or fragment thereof in (i) that comprises at least a portion of a TET family enzyme or a fragment thereof.

16. The method of claim 1, wherein the method comprises contacting the adult cell with an effective amount of the enzyme or fragment thereof in (i) that is TETI, TET2, TET3, CXXC4, a fragment of any of these, or any combination thereof.

17. The method of claim 1, wherein the method comprises contacting the adult cell with an effective amount of the enzyme or fragment thereof in (i) that comprises a nucleic acid sequence having at least about 90% sequence homology to any one of:

SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, or SEQ ID NO;

5.