Reconstituted histone methyltransferase complex and methods of identifyi ng modulators thereof

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.

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). 
     
     
         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). 
     
     
         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 8 , wherein the expressed reconstituted complex comprises a purification tag. 
     
     
         13 . 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. 
     
     
         14 . The method of  claim 8 , wherein the host cell is an insect cell. 
     
     
         15 . The method of  claim 8 , wherein the host cell is stably transformed with the heterologous nucleic acid sequences of (a) to (c). 
     
     
         16 . 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). 
     
     
         17 . The method of  claim 16 , wherein the one or more vectors are baculovirus vectors. 
     
     
         18 . The method of  claim 16 , 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. 
     
     
         19 . The method of  claim 16  further comprising transforming the host cell with the one or more vectors. 
     
     
         20 . 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.   
     
     
         21 . A kit for determining inhibition of histone methylation of lysine 27 of histone H3 (H3-K27), the kit comprising:
 (a) the reconstituted complex of  claim 1 ; 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.