Identifying Specific Protein Interactors of Nucleosomes Carrying Methylated Histones Using Quantitative Mass Spectrometry
Andrey Tvardovskiy, Nhuong Nguyen, Till Bartke (2022) Identifying Specific Protein Interactors of Nucleosomes Carrying Methylated Histones Using Quantitative Mass Spectrometry. Methods in Molecular Biology (MIMB) vol 2529: 327-403. DOI: 10.1007/978-1-0716-2481-4_16
Chemical modification of histone proteins by methylation plays a central role in chromatin regulation by recruiting epigenetic “readers” via specialized binding domains. Depending on the degree of methylation, the exact modified amino acid, and the associated reader proteins histone methylations are involved in the regulation of all DNA-based processes, such as transcription, DNA replication, and DNA repair. Here we present methods to identify histone methylation readers using a mass spectrometry–linked nucleosome affinity purification approach. We provide detailed protocols for the generation of semisynthetic methylated histones, their assembly into biotinylated nucleosomes, and the identification of methylation-specific nucleosome-interacting proteins from nuclear extracts via nucleosome pull-downs and label-free quantitative proteomics. Due to their versatility, these protocols allow the identification of readers of various histone methylations, and can also be adapted to different cell types and tissues, and other types of modifications.