A chemical biology toolbox to study protein methyltransferases and epigenetic signaling

© 2019, The Author(s). Protein methyltransferases (PMTs) comprise a major class of epigenetic regulatory enzymes with therapeutic relevance. Here we present a collection of chemical probes and associated reagents and data to elucidate the function of human and murine PMTs in cellular studies. Our collection provides inhibitors and antagonists that together modulate most of the key regulatory methylation marks on histones H3 and H4, providing an important resource for modulating cellular epigenomes. We describe a comprehensive and comparative characterization of the probe collection with respect to their potency, selectivity, and mode of inhibition. We demonstrate the utility of this collection in CD4 + T cell differentiation assays revealing the potential of individual probes to alter multiple T cell subpopulations which may have implications for T cell-mediated processes such as inflammation and immuno-oncology. In particular, we demonstrate a role for DOT1L in limiting Th1 cell differentiation and maintaining lineage integrity. This chemical probe collection and associated data form a resource for the study of methylation-mediated signaling in epigenetics, inflammation and beyond

A scintillation proximity assay for histone demethylases.

Covalent modifications, such as methylation and demethylation of lysine residues in histones, play important roles in chromatin dynamics and the regulation of gene expression. The lysine demethylases (KDMs) catalyze the demethylation of lysine residues on histone tails and are associated with diverse human diseases, including cancer, and are therefore proposed as targets for the therapeutic modulation of gene transcription. High-throughput assays have been developed to find inhibitors of KDMs, most of which are fluorescence-based assays. Here we report the development of a coupled scintillation proximity assay (SPA) for 3 KDMs: KDM1A (LSD1), KDM3A (JMJD1A), and KDM4A (JMJD2A). In this assay methylated peptides are first demethylated by a KDM, and a protein methyltransferase (PMT) is added to methylate the resulting peptide with tritiated S-(5'-adenosyl)-l-methionine. The enzyme activities were optimized and kinetic parameters were determined. These robust coupled assays are suitable for screening KDMs in 384-well format (Z' factors of 0.70-0.80), facilitating discovery of inhibitors in the quest for cancer therapeutics

A scintillation proximity assay for histone demethylases.

Covalent modifications, such as methylation and demethylation of lysine residues in histones, play important roles in chromatin dynamics and the regulation of gene expression. The lysine demethylases (KDMs) catalyze the demethylation of lysine residues on histone tails and are associated with diverse human diseases, including cancer, and are therefore proposed as targets for the therapeutic modulation of gene transcription. High-throughput assays have been developed to find inhibitors of KDMs, most of which are fluorescence-based assays. Here we report the development of a coupled scintillation proximity assay (SPA) for 3 KDMs: KDM1A (LSD1), KDM3A (JMJD1A), and KDM4A (JMJD2A). In this assay methylated peptides are first demethylated by a KDM, and a protein methyltransferase (PMT) is added to methylate the resulting peptide with tritiated S-(5'-adenosyl)-l-methionine. The enzyme activities were optimized and kinetic parameters were determined. These robust coupled assays are suitable for screening KDMs in 384-well format (Z' factors of 0.70-0.80), facilitating discovery of inhibitors in the quest for cancer therapeutics