Chemical Biology Approaches for Characterization of Epigenetic Regulators D. Barsyte-Lovejoy*, M.M. Szewczyk*, P. Prinos*, E. Lima-Fernandes*, S. Ackloo*, C.H. Arrowsmith* ,†,1 * Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada † Princess Margaret Cancer Centre, University of Toronto, Toronto, ON, Canada 1 Corresponding author: e-mail address: carrow@uhnresearch.ca Contents 1. Introduction 2 1.1 Chemical Probes and Their Use in Biology 2 1.2 Epigenetics and Protein Methyltransferases 3 2. Validation of Chemical Probes for Use in Cell-Based Experiments 3 2.1 Requirements for Chemical Probes 3 2.2 Biomarker Assays: How to Ensure Your Chemical Probe Is Active in Cells 4 2.3 Assay Readout Choice 5 2.4 Biomarker Assays for PMTs 6 2.5 Timing and Other Considerations for Cell-Based Overexpression Assays 13 3. Inhibitor Enabled Discovery 14 3.1 Inhibitor Handling and Inhibitor Libraries 14 3.2 Phenotypic Assays Using Chemical Probes 15 3.3 Demonstration of On-Target Phenotypic Effects 16 4. Conclusions 21 Acknowledgments 22 References 22 Abstract Chemical biology approaches are a powerful means to functionally characterize epige- netic regulators such as histone modifying enzymes. We outline experimental protocols and best practices for the cellular characterization and use of “chemical probes” that selectively inhibit protein methyltransferases, many of which methylate histones to reg- ulate heritable gene expression patterns. We describe biomarker assays to validate the probes in specific cellular systems, and provide guidelines for their use in functional characterization of methyltransferases including detailed protocols, examples, and con- trols. Together these techniques enable precision manipulation of cellular epigenomes and the exploration of the therapeutic potential of epigenetic targets in human disease. Methods in Enzymology # 2016 Elsevier Inc. ISSN 0076-6879 All rights reserved. http://dx.doi.org/10.1016/bs.mie.2016.01.011 1 ARTICLE IN PRESS