SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS

• US 20170219589A1
• Filed: 04/10/2017
• Published: 08/03/2017
• Est. Priority Date: 09/26/2008
• Status: Abandoned Application

• 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, The United States of America As Represented By The Secretary of Agriculture
• Inventors: Rao, Anjana, Tahiliani, Mamta, Koh, Kian Peng, Agarwal, Suneet, Iyer, Aravind
• Application Number:
  US15/483,297
• Publication Number:
  US 20170219589A1
• Time in Patent Office: Days
• 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:

measuring a presence of a methylated cytosine in a nucleotide sequence from a subject, wherein the subject is a subject having a cancer or at risk of developing the cancer; and

comparing the presence of the methylated cytosine in the nucleotide sequence from the subject to a presence of a methylated cytosine in a reference.

2. The method of claim 1, wherein the methylated cytosine is a 5-hydroxymethylcytosine.

3. The method of claim 2, wherein the measuring comprises contacting an agent to the 5-hydroxymethylcytosine in the nucleotide sequence, wherein the agent comprises an antibody, an antigen-binding portion thereof, an intrabody, or a protein.

4. The method of claim 3, wherein the agent selectively binds to the 5-hydroxymethylcytosine in the nucleotide sequence.

5. The method of claim 3, wherein the agent is engineered to increase its binding affinity or selectivity for the 5-hydroxymethylcytosine.

6. The method of claim 3, wherein the agent comprises a label or a tag.

7. The method of claim 1, wherein the measuring comprises imaging analysis or mass spectrometry.

8. The method of claim 1, wherein the measuring comprises a thin-layer chromatography, a blotting assay or a linked enzyme mediated substrate conversion.

9. The method of claim 1, wherein the nucleotide sequence is obtained from a tissue sample of the subject.

10. The method of claim 9, wherein the tissue sample comprises bone marrow.

11. The method of claim 9, wherein the tissue sample comprises a diseased tissue sample.

12. The method of claim 1, further comprising diagnosing the subject as having cancer based on the presence of the methylated cytosine in the nucleotide sequence from the subject.

13. The method of claim 12, wherein if the subject is diagnosed as having the cancer, selecting a treatment for the subject.

14. The method of claim 13, wherein the treatment comprises administering a composition that modulates catalytically active TET family enzymes, functional TET family derivatives, TET catalytic fragments thereof, or any combination thereof.

15. The method of claim 1, further comprising stratifying a risk of the subject as having cancer based on the presence of methylated cytosine in the nucleotide sequence from the subject.

16. The method of claim 1, wherein the reference is a normal reference, and wherein a difference between the presence of methylated cytosine in the nucleotide sequence and the presence of methylated cytosine in the normal reference is indicative of the subject having the cancer.

17. The method of claim 1, wherein the reference comprises a diseased reference, and wherein a presence of methylated cytosine in the nucleotide sequence that is similar to the presence of methylated cytosine in the reference is indicative of the subject having the cancer.

18. The method of claim 1, wherein the presence of the methylated cytosine in the nucleotide sequence comprises a level of methylated cytosine, and wherein the level of methylated cytosine from the subject is higher than a level of the methylated cytosine in the reference.

19. The method of claim 1, wherein the presence of the methylated cytosine in the nucleotide sequence comprises a level of methylated cytosine, and wherein the level of methylated cytosine from the subject is lower than a level of the methylated cytosine in the reference.

20. The method of claim 16, wherein the normal reference comprises a normal tissue sample, a normal cell sample, or a combination thereof obtained from a subject.

21. The method of claim 17, wherein the diseased reference comprises a diseased tissue sample, a diseased cell sample, or a combination thereof obtained from a subject.

22. The method of claim 1, further comprising treating the subject.

23. The method of claim 12, further comprising treating the subject.

24. The method of claim 13, further comprising treating the subject.