Abstract
Histone dynamics constitute an important layer of gene regulations associated with development and growth in multicellular eukaryotes. They also stand as key determinants of plant responses to environmental changes. Histone dynamics include the exchange of histone variants as well as post-translational modifications of their amino acid residues (such as acetylation and mono/di/trimethylation), commonly referred to as histone marks. Investigating histone dynamics with a focus on combinatorial changes occurring at their residues will greatly help unravel how plants achieve phenotypic plasticity.
Mass spectrometry (MS) analysis offers unequaled resolution of the abundance of histone variants and of their marks. Indeed, relative to other techniques such as western blot or genome-wide profiling, this powerful technique allows quantifying the relative abundances of histone forms, as well as revealing coexisting marks on the same histone molecule. Yet, while MS-based histone analysis has proven efficient in several animals and other model organisms, this method stands out as more challenging in plants. One major challenge is the isolation of sufficient amounts of pure, high-quality histones, likely rendered difficult by the presence of the cell wall, for sufficiently deep and resolutive identification of histone species.
In this chapter, we describe a straightforward MS-based proteomic method, implemented to characterize histone marks from Arabidopsis thaliana seedling tissues and cell culture suspensions. After acid extraction of histones, in vitro propionylation of free lysine residues, and digestion with trypsin, a treatment at highly basic pH allows obtaining sharp spectral signals of biologically relevant histone peptide forms.
The method workflow described here shall be used to measure changes in histone marks between Arabidopsis thaliana genotypes, along developmental time-courses, or upon various stresses and treatments.
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Acknowledgments
We thank Anne-Marie Boisson for help with cell suspension preparation, Laura Turchi for help with Fig. 1 preparation and assembly, and Steve Jacobsen, Fredy Barneche, Lars Henning, and Andrzej Jerzmanowski, whose protocols inspired the one described in this chapter. This project has been partially supported by the Idex funding for Université Grenoble Alpes (ANR-15-IDEX-02) and by the Grenoble Alliance for Integrated Structural and Cell Biology (GRAL), a program of the Chemistry Biology Health Graduate School of Université Grenoble Alpes (ANR-17-EURE-0003), as well as the proteomics platform via ProFI [ANR-10-INBS-08].
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Geshkovski, V. et al. (2025). Quantitative Profiling of Histone Variants and Posttranslational Modifications by Tandem Mass Spectrometry in Arabidopsis. In: Baroux, C., Tatout, C. (eds) Methods for Plant Nucleus and Chromatin Studies. Methods in Molecular Biology, vol 2873. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-4228-3_2
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DOI: https://doi.org/10.1007/978-1-0716-4228-3_2
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