Anja Rodenbrock, Marjolaine Roy, Liliana Pedro, Nancy Gauthier, Anne Labonté, Valérie Paquet, Michael Lässle, Lucille Beaudet, and Roberto Rodriguez-Suarez.
PerkinElmer Inc., 1744 William St., Montreal, Quebec, Canada H3J 1R4
In eukaryotes, the covalent modification of histones has a crucial role in chromatin architecture and plays an important part in a plethora of cellular processes, from chromatin remodeling and transcriptional regulation, to DNA repair and cell cycle control. While histone acetylation is generally associated to an open chromatin state and transcriptional activation, methylation of histones has been related to either activating or repressive functions. It is then clear that precise regulation of these events is critical for appropriate cellular development and function. In this regard, the abnormal level of several histone-modifying activities has been linked to pathological states, such as cancer or neurological disorders. Therefore, developing fast and robust assays for studying these enzymes could significantly accelerate research and the discovery process of clinically relevant compounds.
Here, we detail the development of enzymatic assays for histone H3K9 demethylases using two homogeneous nonradioactive technologies: time-resolved Förster resonance energy transfer, and a bead-based chemiluminescent proximity assay. To this end, a peptide substrate derived from the N-terminus of histone H3 (trimethylated on Lys 9) was used to analyze the catalytic activity of JMJD2A and JMJD2C demethylases. Optimized conditions were highly suitable for screening, with typical enzyme concentrations ranging from 1 to 10 nM, and methyl-peptide substrate between 100 and 300 nM. Required concentrations of alphaketoglutarate (2OG) ranged from 1 to 5 M, allowing sensitive detection of compounds which may compete with this cofactor. Complete assays could be carried out in less than 4 hours, and were further validated with the use of a known inhibitor, 2,4-PDCA. In brief, results shown herein will enable simple, robust, non-radioactive screenings of compound libraries for modulators of histone H3K9 demethylases.
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