The Use Of A Fluorescent Polarization Assay Using SET7/9 Histone Lysine MethyltransferaseSource: BioTek Instruments, Inc.
By Peter J. Brescia, Applications Scientist, BioTek Instruments, Inc.
There continues to be increasing interest in epigenetic mechanisms of control that alter chromatin structure and influence gene regulation. Posttranslational modifications (PTMs) of histones have been shown to provide a complex mechanism of influencing chromatin structure that play a primary role in many DNA regulatory processes. Structural studies of nucleosomes have shown that highly basic histone amino (N)-terminal tails remain unstructured, allowing contacts to be made with neighboring nucleosomes. It has subsequently been shown that modifications to the N-terminal tails indeed influence global chromatin structure due to these inter-nucleosomal contacts. While structural changes are a result of histone modifications, recruitment of proteins and protein complexes have also been shown to correlate with specific histone modifications. Once recruited, these proteins perform a variety of enzymatic activities and regulate a range of DNA processes including remodeling, repair, replication and recombination, as well as transcriptional regulation.
Histone modifications include acetylation, phosphorylation, methylation, deamination, and ubiquitylation, to name a few. Histone methylation has been shown to primarily occur on the sidechains of lysine and arginine residues and has been widely studied as a potential druggable target due to the association of some histone methylation states with disease. Thus, histone methyltransferases are divided into two groups, lysine or arginine, based on the target residue. Histone lysine methyltransferases, or HKMTs, that target N-terminal lysine residues generally contain a SET domain responsible for the methyltransferase activity. While relatively specific for a particular N-terminal residue within the histone tail, some HKMTs can target additional non-histone proteins. Methyl groups from the cofactor S-adenosylmethionine (SAM) are transferred by HKMTs to the ε-amino group of target lysine residues. The methylation state can vary from one to three groups per residue depending on the HKMT.
Furthermore, it has been shown that the methylation state is influenced by adjacent residues of the N-terminal tail1. SET7/9 (KMT7) is a mono-methyltransferase targeting H3K4, as well as several non-histone targets such as TAF10, p53, and viral Tat, and can be used as a model to study SET domain histone methylation. The Set7/9 SAM-Screener Assay is a fluorescence polarization assay designed around a small molecule fluorescent probe that binds to the SET7/9 SAM binding site. When bound to SET7/9, an increase in fluorescence polarization is seen when compared to free probe. The probe is displaced by the endogenous cofactor SAM as well as known SAM-binding site inhibitors (e.g. sinefungin). Probe binding is not altered by SET7/9 substrate peptides, suggesting a binding modality that is purely dependent upon the SAM-binding site. Here we demonstrate the use of automated methods to improve assay performance in a 384-well microplate format suitable for highthroughput screening (HTS) applications.