Site-specific installation of methyl-lysine analogues into recombinant histones

• US 8,278,112 B2
• Filed: 12/20/2007
• Issued: 10/02/2012
• Est. Priority Date: 12/21/2006
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Shokat, Kevan, Simon, Matthew D.
• Primary Examiner(s): Gebreyesus, Kagnew H
• Application Number:
  US11/961,956
• Publication Number:
  US 20080199964A1
• Time in Patent Office: 1,748 Days
• Field of Search: None
• US Class Current: 436/86
• CPC Class Codes: C07K 1/1075 by covalent attachment of amino acids or peptide residues
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity

Abstract

The present invention provides reagents and methods for the introduction of analogues of methyl or acetyl lysine into histone proteins.

Claims

1. A method of introducing a site specific mono-, di, or tri-methylated lysine residue analogue into a histone H3 or H4 protein comprising the steps of:

(a) mutating the cysteine residues in the native sequence of the histone protein to a different amino acid residue;

(b) mutating at least one lysine residue in the amino acid sequence of the histone protein to a cysteine residue to generate a cysteine containing protein; and

(c) contacting the cysteine containing protein with a compound of the structure;

R—

CH2CH2-NH(Me)x, wherein R is a leaving group and x is 1, 2, or 3, thereby introducing a site specific mono-, di, or tri-methylated lysine residue analogue into a histone protein.

2. The method of claim 1, wherein the different amino acid residue is alanine.

3. The method of claim 1, wherein R is a halide.

4. The method of claim 3, wherein the halide is Cl, Br, or I.

5. The method of claim 1, wherein R is mesylate or tosylate.

6. The method of claim 1, wherein the specific lysine residue is K4, K9, K14, K27, K36, or K79 of histone H3.

7. The method of claim 1, wherein the specific lysine residue is K5, KS, K12, K16, or K20 of histone H4.

8. The method of claim 1, wherein the cysteine containing protein is treated with a reducing agent prior to step (c).

9. The method of claim 8, wherein the reducing agent is DTT.

10. An isolated histone H3 or H4 protein comprising a lysine to cysteine substitution, wherein the substituted cysteine comprises the side chain structure —

S—

CH2CH2-NH(Me)x, wherein x is 1, 2, or 3.

11. The isolated histone protein of claim 10, wherein the lysine to cysteine substitution is K4C, K9C, K14C, K27C, K36C, or K79C of histone H3.

12. The isolated histone protein of claim 10, wherein the lysine to cysteine substitution is K5C, K8C, K12C, K16C, or K20C of histone H4.

23. The isolated histone protein of claim 10, wherein the histone protein further comprises substitution of the cysteine residues in the native sequence of the histone protein to a different amino acid residue.

13. An isolated histone H3 or H4 protein comprising site specific mono-, di, or tri-methylated lysine residue analogues derived by a process comprising the steps of:

(a) mutating the cysteine residues in the native sequence of the H3 histone protein to alanine;

(b) mutating at least one lysine residue in the sequence of the H3 protein to a cysteine residue to generate a cysteine containing protein; and

(c) contacting the cysteine containing protein with a compound of the structure;

RCH2CH2-NH(Me)x, wherein R is a leaving group and x is 1, 2, or 3.

14. The isolated histone protein of claim 13, wherein R is a halide.

15. The isolated histone protein of claim 14, wherein the halide is Cl, Br, or I.

16. The method of claim 13, wherein R is mesylate or tosylate.

17. The isolated histone protein of claim 13, wherein the histone protein is histone H3 or H4.

18. The isolated histone protein of claim 13, wherein the specific lysine residue is K4, K9, K14, K27, K36, or K79 of histone H3.

19. The isolated histone protein of claim 13, wherein the specific lysine residue is K5, K8, K12, K16, or K20 of histone H4.

20. A method for assaying the effect of methylation of at least one lysine residue on a histone protein on the histone H3 or H4 protein'"'"'s activity comprising the steps of:

(a) mutating the cysteine residues in the native sequence of the histone H3 protein to alanine;

(b) mutating at least one lysine residue in the sequence of the histone protein to a cysteine residue to generate a cysteine containing histone protein;

(c) contacting the cysteine containing histone protein with a compound of the structure;

R—

CH2CH2-NH(Me)x, wherein R is a leaving group and x is 1, 2, or 3 to generate a methylated protein; and

(d) comparing the activity of the methylated histone protein with the histone protein without the mutation or with a protein derived after step (c) in which x is 0,

thereby assaying the effect of methylation of at least one lysine residue on the histone protein'"'"'s activity.

21. The method of claim 20, wherein the assaying comprises measuring octomer reconstitution.

22. The method of claim 20, wherein the assaying comprises measuring nucleosome remodeling.