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Development of High-Throughput Assays to Study Histone H3K4 Methyltransferases & H3K9 Methyl- and Acetyltransferases
Nathalie Rouleau, Liliana Pedro, Nancy Gauthier, Anne Labonté, Valérie Paquet, Anja Rodenbrock, Marjolaine Roy, Alexandre Marcil, Hendrick Plante, Lucille Beaudet, and Roberto Rodriguez-Suarez.
PerkinElmer, 1744 William St., Montreal, Quebec, Canada H3J 1R4
Sponsored by PerkinElmer
Post-translational modifications of histone proteins play an important part in a wide array of cellular processes including regulation of gene transcription, DNA repair, cell cycle, and metabolism control. For instance, transcriptional activation is associated with acetylation of Histone H3 on residues K9 and K14, and methylation on K4. On the other hand, gene repression has been linked to H3K9, H3K27, and H4K20 methylation. In this regard, the activity of several histone modifying enzymes (e.g., acetyl and methyltransferases, deacetylases, demethylases) have been linked to diseases such as cancer and neurological disorders. Thus, the development of simple and reliable assays for these enzymes could facilitate the identification of new modulatory compounds, eventually leading to the development of clinically relevant drugs.
In this work, we describe the development of enzymatic assays to perform screenings for modulators of H3K9 acetyltransferases, and H3K4 and H3K9 methyltransferases, using homogeneous proximity assays based on two non-radioactive technologies: LANCE® Ultra timeresolved fluorescence energy transfer and AlphaLISA® bead-based chemiluminescent assays. To this end, we used as substrate a biotinylated peptide derived from the N-terminus of Histone H3 (residues 1-21) to assay acetyltransferase p300 and methyltransferases G9a and SET7/9. In all cases, we found conditions highly suitable for screening: enzyme concentrations between 0.05 to 5 nM and substrate concentrations between 50 and 500 nM. Acetyl-CoA or Sadenosylmethionine (SAM) concentrations were in the 0.1-20 M range, enabling sensitive screenings for compounds competing with these cofactors. Entire assays could be carried out in less than 4 hours, with order-of-potency for known inhibitors (anacardic acid, sinefungin, SAH) in good correlation with published literature.
The results presented herein demonstrate how to optimally assess p300, G9a, and SET7/9 activities in vitro using a biotinylated H3-derived peptide in non-radioactive, homogeneous assay formats. This could enable simple and fast screenings of compound libraries, facilitating the discovery of novel modulators of histone modifying enzymes.
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