Reconstituted histone methyltransferase complex and methods of identifying modulators thereof

• US 20050266473A1
• Filed: 05/27/2005
• Published: 12/01/2005
• Est. Priority Date: 06/01/2004
• Status: Active Grant

• Current Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: Zhang, Yi, Cao, Ru
• Granted Patent: US 7,563,589 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C12N 2799/026 where the vector is derived from a baculovirus
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12Y 201/01043 Histone-lysine N-methyltransferase (2.1.1.43)
• CPC Class Codes: G01N 2333/91011 Methyltransferases (general) (2.1.1.)

Abstract

The present invention provides a reconstituted complex including EED, EZH2 and SUZ12 wherein the reconstituted complex has histone methyltransferase (HMTase) activity for lysine 27 of histone H3 (H3-K27). The reconstituted complex may further include RbAp48, AEBP2 or both. Also disclosed are methods of producing the reconstituted complex, methods of identifying compounds that inhibit the HTMase activity of the reconstituted complex and methods of identifying candidate compounds for treating cancer. Reagents and kits including the reconstituted complex are further provided.

Claims

1. A reconstituted complex comprising EED, EZH2 and SUZ12 wherein the reconstituted complex has histone methyltransferase (HMTase) activity for lysine 27 of histone H3 (H3-K27).

2. The reconstituted complex of claim 1, wherein EED, EZH2 and SUZ12 are mammalian proteins, drosophila proteins, nematode proteins, plant proteins or combinations thereof.

3. The reconstituted complex of claim 1 further comprising RbAp48, AEBP2 or both, wherein RbAp48 and/or AEBP2 are mammalian proteins, drosophila proteins, nematode proteins, plant proteins or combinations thereof.

4. The reconstituted complex of claim 1, wherein the reconstituted complex has enzyme activity comparable to the enzyme activity of a native complex.

5. The reconstituted complex of claim 1, wherein the reconstituted complex has substrate specificity comparable to the substrate specificity of a native complex.

6. The reconstituted complex of claim 1, wherein the reconstituted complex has a purity level of at least 60% (w/w).

8. A method of producing the reconstituted complex of claim 1, comprising:

providing a host cell comprising;

(a) a heterologous nucleic acid sequence encoding an EED protein;

(b) a heterologous nucleic acid sequence encoding an EZH2 protein; and

(c) a heterologous nucleic acid sequence encoding a SUZ12 protein; and

culturing the host cell under conditions sufficient for expression of the proteins and production of the reconstituted complex.

9. The method of claim 8 further comprising isolating the expressed reconstituted complex from the cultured host cell or a culture medium from the cultured host cell.

10. The method of claim 9, wherein isolating the expressed reconstituted complex comprises binding the expressed reconstituted complex to a solid support.

11. The method of claim 10, wherein the solid support comprises nickel, glutathione or an antibody directed to the expressed reconstituted complex.

12. The method of claim 10 further comprising eluting the expressed reconstituted complex from the solid support.

13. The method of claim 8, wherein the expressed reconstituted complex comprises a purification tag.

14. The method of claim 8, wherein the host cell further comprises (a) a nucleic acid sequence encoding a RbAp48 protein, (b) a nucleic acid sequence encoding an AEBP2 protein, or (c) both.

15. The method of claim 8, wherein the host cell is an insect cell.

16. The method of claim 15, wherein the insect cell is an Sf9 cell.

17. The method of claim 8, wherein the host cell is stably transformed with the heterologous nucleic acid sequences of (a) to (c).

18. The method of claim 8, wherein the host cell comprises one or more recombinant vectors comprising the heterologous nucleic acid sequences of (a) to (c).

19. The method of claim 18, wherein the vector is a baculovirus vector.

20. The method of claim 18, wherein the one or more vectors comprise a vector comprising a heterologous nucleic acid sequence encoding the EED protein, a separate vector comprising a heterologous nucleic acid sequence encoding the EZH2 protein, and a further separate vector comprising a heterologous nucleic acid sequence encoding the SUZ12 protein.

21. The method of claim 18 further comprising transforming the host cell with the one or more vectors.

7. A reconstituted complex consisting essentially of EED, EZH2 and SUZ12, wherein the reconstituted complex has histone methyltransferase (HMTase) activity specific for lysine 27 of histone H3 (H3-K27).

22. A host cell comprising:

(a) a heterologous nucleic acid sequence encoding an EED protein;

(b) a heterologous nucleic acid sequence encoding an EZH2 protein; and

(c) a heterologous nucleic acid sequence encoding a SUZ12 protein.

23. The host cell of claim 22, wherein the host cell is an insect cell.

24. The host cell of claim 23, wherein the insect cell is an Sf9 cell.

25. The host cell of claim 22, wherein the host cell is stably transformed with the heterologous nucleic acid sequences of (a) to (c).

26. The host cell of claim 22, wherein the host cell comprises one or more recombinant vectors comprising the heterologous nucleic acid sequences of (a) to (c).

27. The host cell of claim 26, wherein the vector is a baculovirus vector.

28. The host cell of claim 26, wherein the one or more vectors comprise a vector comprising a heterologous nucleic acid sequence encoding the EED protein, a separate vector comprising a heterologous nucleic acid sequence encoding the EZH2 protein, and a further separate vector comprising a heterologous nucleic acid sequence encoding the SUZ12 protein.

29. A method of identifying a compound that modulates the histone methyltransferase (HMTase) activity for lysine 27 of histone H3 (H3-K27) of a reconstituted complex comprising EED, EZH2, and SUZ12, the method comprising:

contacting the reconstituted complex with a histone substrate in the presence of a test compound; and

detecting the level of H3-K27 methylation under conditions sufficient to provide H3-K27 methylation, wherein a change in H3-K27 methylation as compared with the level of H3-K27 methylation in the absence of the test compound indicates that the test compound is a modulator of the H3-K27 HTMase activity of the reconstituted complex.

30. The method of claim 29, wherein EED, EZH2 and SUZ12 are mammalian proteins, drosophila proteins, nematode proteins, plant proteins or combinations thereof.

31. The method of claim 29, wherein the reconstituted complex further comprises RbAp48, AEBP2 or both, wherein RbAp48 and/or AEBP2 are mammalian proteins, drosophila proteins, nematode proteins, plant proteins or combinations thereof.

32. The method of claim 29, wherein a reduction in H3-K27 methylation as compared with the level of H3-K27 methylation in the absence of the test compound indicates that the test compound is an inhibitor of the H3-K27 HMTase activity of the reconstituted complex.

33. The method of claim 29, wherein an increase in H3-K27 methylation as compared with the level of H3-K27 methylation in the absence of the test compound indicates that the test compound is an activator of the H3-K27 HMTase activity of the reconstituted complex.

34. The method of claim 29, wherein the histone substrate is a core histone, a histone octamer, a mononucleosome, a dinucleosome or an oligonucleosome.

35. The method of claim 29, wherein the histone substrate is a dinucleosome.

36. The method of claim 29, wherein the method is a cell-based method.

37. The method of claim 29, wherein the method is a cell-free method.

38. A method of identifying a candidate compound for treating cancer, comprising:

contacting a reconstituted complex comprising EED, EZH2 and SUZ12 with a histone substrate in the presence of a test compound; and

detecting the level of histone methylation of lysine 27 of histone H3 (H3-K27) under conditions sufficient to provide H3-K27 methylation, wherein a reduction in H3-K27 methylation as compared with the level of H3-K27 methylation in the absence of the test compound indicates that the test compound is a candidate compound for the treatment of cancer.

39. The method of claim 38, wherein EED, EZH2 and SUZ12 are mammalian proteins, drosophila proteins, nematode proteins, plant proteins or combinations thereof.

40. The method of claim 38, wherein the reconstituted complex further comprises RbAp48, AEBP2 or both, wherein RbAp48 and/or AEBP2 are mammalian proteins, drosophila proteins, nematode proteins, plant proteins or combinations thereof.

41. A method of inhibiting methylation of lysine 27 of histone H3 (H3-K27), comprising contacting a cell with an inhibitor of the H3-K27 histone methyltransferase (HMTase) activity of a reconstituted complex comprising EED, EZH2 and SUZ12.

42. The method of claim 41, wherein the cell is a cultured cell.

43. The method of claim 41, wherein the cell is a cell in vivo in a subject.

44. A reagent for increasing sensitivity of detection of methylation of lysine 27 of histone H3 (H3-K27), comprising:

a reconstituted complex comprising EED, EZH2 and SUZ12; and

optionally, a suspension medium.

45. The reagent of claim 44, wherein EED, EZH2 and SUZ12 are mammalian proteins, drosophila proteins, nematode proteins, plant proteins or combinations thereof.

46. The reagent of claim 44, wherein the reconstituted complex further comprises RbAp48, AEBP2 or both, wherein RbAp48 and/or AEBP2 are mammalian proteins, drosophila proteins, nematode proteins, plant proteins or combinations thereof.

47. A kit for determining inhibition of histone methylation of lysine 27 of histone H3 (H3-K27), the kit comprising:

(a) a reconstituted complex comprising EED, EZH2 and SUZ12; and

(b) written instructions for methods for determining inhibition of H3-K27 methylation comprising determining the inhibition of H3-K27 methyltransferase (HTMase) activity of the reconstituted complex, and optionally additional reagents or apparatus for carrying out methods for determining inhibition of HTMase activity.

48. The kit of claim 47, wherein EED, EZH2 and SUZ12 are mammalian proteins, drosophila proteins, nematode proteins, plant proteins or combinations thereof.

49. The kit of claim 47, wherein the reconstituted complex further comprises RbAp48, AEBP2 or both, wherein RbAp48 and/or AEBP2 are mammalian proteins, drosophila proteins, nematode proteins, plant proteins or combinations thereof.