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Identifying Specific Protein Interactors of Nucleosomes Carrying Methylated Histones Using Quantitative Mass Spectrometry

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Histone Methyltransferases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2529))

Abstract

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.

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Acknowledgements

We would like to thank Ann-Christine König from the Helmholtz Zentrum München Research Unit Protein Science for help with the quantification of the label-free mass spectrometry data for the H3K4me3 and H3K9me3 dinucleosome pull-down experiments. The development of these protocols was supported by funding from the Human Frontiers Science Program Organization, the Medical Research Council (Grant No. MC_UP_1102/2), the European Research Council (ERC StG No. 309952), the Deutsche Forschungsgemeinschaft (DFG Project No. 431163844 and 213249687/SFB 1064), and the Helmholtz Gesellschaft to T.B.

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Author notes

  1. Andrey Tvardovskiy and Nhuong Nguyen contributed equally to this work.

Authors and Affiliations

  1. Institute of Functional Epigenetics, Helmholtz Zentrum München, Neuherberg, Germany

    Andrey Tvardovskiy & Till Bartke

  2. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

    Nhuong Nguyen

Authors
  1. Andrey Tvardovskiy
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  2. Nhuong Nguyen
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  3. Till Bartke
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Corresponding author

Correspondence to Till Bartke .

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Editors and Affiliations

  1. Institute Curie, INSERM U934/CNRS UMR 3215, Paris, France

    Raphaël Margueron

  2. Institute Curie, INSERM U934/CNRS UMR 3215, Paris, France

    Daniel Holoch

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Tvardovskiy, A., Nguyen, N., Bartke, T. (2022). Identifying Specific Protein Interactors of Nucleosomes Carrying Methylated Histones Using Quantitative Mass Spectrometry. In: Margueron, R., Holoch, D. (eds) Histone Methyltransferases. Methods in Molecular Biology, vol 2529. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2481-4_16

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  • DOI: https://doi.org/10.1007/978-1-0716-2481-4_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2480-7

  • Online ISBN: 978-1-0716-2481-4

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