The expression of the thousands of genes present in the human cell is regulated by dynamic changes in chromatin compaction throughout the different regions of the genome. The activity of chromatin-remodeling machineries, as well as changes in DNA-methylation patterns and histone post-translational modifications by specialized enzymes, are all biochemical strategies that cells employ to modulate gene expression.
For instance, DNA methylation on CpG islands as well as methylation of histone H3 on specific lysine residues are events associated with chromatin condensation leading to transcriptional repression. Conversely, lysine acetylation of histones is generally related to open chromatin structures with higher rates of transcription.
In this regard, there is a growing body of evidence that associates the activity of both histone methyltransferases (HMTs) and acetyltransferases (HATs) with different human pathologies, from malignancies to neuropathies. Thus, the development of molecular probes for these enzyme families could enable biologists to study in more depth the complex role of histone modifications in gene regulation, and provide a starting point to new drug discovery.
A variety of radioactive-based assays have been reported in the literature for identifying compounds that modulate the activity of histone-modifying enzymes. Scintillation proximity assays (SPA) and FlashPlate® have been successfully developed to monitor the activity of HMTs by measuring the transfer of the methyl group from the radiolabeled co-factor, S-adenosyl-L-methionine (SAM), to the ε-amino residue of lysines on histone-derived peptidic substrates. However, as HMTs can perform either mono-, di-, or tri-methylation of lysines, measuring the incorporation of radioactive methyl groups does not provide information on the nature of the final reaction products.
Mass spectroscopy (MS) is a non-radioactive alternative that has also been employed for studying the activity of HMTs and HATs. This technology allows the simultaneous characterization of the different methyl-lysine forms produced by certain HMTs, i.e., mono-, di-, or tri-methylated lysines. However, while recent technical advancements continue to improve MS throughput, plate-based detection assays remain the standard for speed and sensitivity in HTS applications.