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Full Description
This volume provides methods used to investigate histone methyltransferase function. Chapters guide readers through a comprehensive set of approaches that detail phylogenetic diversity, histone demethylase activities in vitro, generating chromatin substrates, auto-methylation, quantification of metabolites, protein purification, crystallization, X-ray structure, cryogenic electron microscopy, assessing genome-wide patterns, CUT&Tag in mouse embryonic tissues, chemical biology approaches, peptide SPOT arrays, nascent chromatin capture, ectopic protein tethering, computational models, and development of methyltransferase inhibitors. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.
Authoritative and cutting-edge, Histone Methyltransferases: Methods and Protocols aims to be a useful and practical guide to new researchers and experts looking to expand their knowledge.
Contents
Not all is SET for methylation: Evolution of eukaryotic protein methyltransferases.- Detection and quantification of histone methyltransferase activity in vitro.- In Vitro Histone Demethylase Assays.- Preparation and characterization of chromatin templates for histone methylation assays.- Techniques to study automethylation of histone methyltransferases and its functional impact.- Rodriguez Profiling the regulation of histone methylation and demethylation by metabolites and metals.- Determination of histone methyltransferase structure by crystallography.- Determination of histone methyltransferase structures in complex with the nucleosome by cryogenic electron microscopy.- Development and validation of antibodies targeting site-specific histone methylation.- Genetic, genomic and imaging approaches to dissect the role of Polycomb group epigenetic regulators in mice.- Profiling histone methylation in low numbers of cells.- Automated CUT&RUN using the KingFisher Duo Prime.- Bioinformatics methods for ChIP-seq histone analysis.- Characterization of SET-domain histone lysine methyltransferase substrates using a cofactor S-adenosyl-L-methionine surrogate.- Specificity analysis of protein methyltransferases and discovery of novel substrates using SPOT peptide arrays.- Identifying specific protein interactors of nucleosomes carrying methylated histones using mass spectrometr.- Investigating mitotic inheritance of histone post-translational modifications by triple pSILAC coupled to Nascent Chromatin Capture.- Investigating mitotic inheritance of histone modifications using tethering.- Investigating histone modification dynamics by mechanistic computational modelling.- Screening for small molecule inhibitors of histone methyltransferases.
