Histone Methyltransferase Assay in vitro

Materials and Reagents

1. ¹⁴C labeled-methyl-adenosyl-L-methionine (SAMS) (Sigma-Aldrich)
   
2. Purified recombinant histones (from E. coli)
   
3. Histone H3 (Roche Diagnostics, catalog number: 1034758 )
   
4. Core histones (Sigma-Aldrich, catalog number: H9250 )
   
5. Mononuclosomes and Oligonucleosomes (see procedure)
   
6. Coomassie brilliant blue R-250
   
7. MiliQ water
   
8. SDS-PAGE
   
9. KCl
   
10. MgCl₂
    
11. Sucrose
    
12. ZnCl₂
    
13. β-mercaptoethanol
    
14. MAB buffer (see Recipes)
    

Equipment

1. Water bath

Procedure

1. Substrate
   Purified recombinant Histones (Ding et al., 2007), histone H3, core histones, mononuclosome and oligonucleosome (from Hela cells or recombinant ones from E.coli).
   Note: The mononucleosomes and oligonucleosomes prepared from Hela cells were gifts from Yi Zhang (UNC Chapel Hill, USA). The recombinant oligonucleosomes from E. coli were gifts from Bing Zhu (NIBS, Beijing, China).
   
   
2. Reaction
   A mixture containing 1-5 μl enzyme (such as SDG725), 5 μl 4x MAB buffer, 250 nCi ¹⁴C labeled SAM and 1 μg substrates to 20 μl volume, is kept at 37 °C for 1-2 h. The reaction is stopped by adding 5 μl 5x SDS-PAGE loading buffer and boiling at 100 °C for 5 min, and 5 μl samples will be analyzed by 15% SDS-PAGE gel electrophoresis and visualized by Coomassie brilliant blue R-250. The SDS-PAGE gel is dried and exposed to X-ray films or scanned by Typhoon (GE) (15% refers to the concentration of Acr/Bis. Different histones can be separated more clearly by 15% SDS-PAGE).
   

Recipes

1. MAB buffer
   1x MAB buffer
   50 mM Tris-Cl (pH 8.5)
   20 mM KCl
   10 mM MgCl₂
   250 mM Sucrose
   100 μM ZnCl₂
   10 mM β-mercaptoethanol
   

Acknowledgments

The protocol was adapted from previously published papers (Rea et al., 2000; Ding et al., 2007).

References

1. Ding, B., Zhu, Y., Gao J., Yu, Y., Cao, K. M., Shen W. H., Dong, A. (2007) Molecular characterization of three rice SET-domain proteins. Plant Sci 172:1072-1078.
   
2. Rea, S., Eisenhaber, F., O'Carroll, D., Strahl, B. D., Sun, Z. W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C. P., Allis, C. D. and Jenuwein, T. (2000). Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406(6796): 593-599.
   
3. Sui, P., Jin, J., Ye, S., Mu, C., Gao, J., Feng, H., Shen, W. H., Yu, Y. and Dong, A. (2012). H3K36 methylation is critical for brassinosteroid-regulated plant growth and development in rice. Plant J 70(2): 340-347.