Background: Gene expression in eukaryotic cells can be governed by histone variants, which replace replication-coupled histones, conferring unique chromatin properties. MacroH2A1 is a histone H2A variant containing a domain highly similar to H2A and a large non-histone (macro) domain. MacroH2A1, in turn, is present in two alternatively exon-spliced isoforms: macroH2A1.1 and macroH2A1.2, which regulate cell plasticity and proliferation in a remarkably distinct manner. The N-terminal and the C-terminal tails of H2A histones stem from the nucleosome core structure and can be target sites for several post-translational modifications (PTMs). MacroH2A1.1 and macroH2A1.2 isoforms differ only in a few amino acids and their ability to bind NAD-derived metabolites, a property allegedly conferring their different functions in vivo. Some of the modifications on the macroH2A1 variant have been identified, such as phosphorylation (T129, S138) and methylation (K18, K123, K239). However, no study to our knowledge has analyzed extensively, and in parallel, the PTM pattern of macroH2A1.1 and macroH2A1.2 in the same experimental setting, which could facilitate the understanding of their distinct biological functions in health and disease. Methods: We used a mass spectrometry-based approach to identify the sites for phosphorylation, acetylation, and methylation in green fluorescent protein (GFP)-tagged macroH2A1.1 and macroH2A1.2 expressed in human hepatoma cells. The impact of selected PTMs on macroH2A1.1 and macroH2A1.2 structure and function are demonstrated using computational analyses. Results: We identified K7 as a new acetylation site in both macroH2A1 isoforms. Quantitative comparison of histone marks between the two isoforms revealed significant differences in the levels of phosphorylated T129 and S170. Our computational analysis provided evidence that the phosphorylation status in the intrinsically disordered linker region in macroH2A1 isoforms might represent a key regulatory element contributing to their distinct biological responses. Conclusions: Taken together, our results report different PTMs on the two macroH2A1 splicing isoforms as responsible for their distinct features and distribution in the cell.

Department of Biology Faculty of Medicine Masaryk University Kamenice 753 5 62500 Brno Czech Republic

Department of Molecular Medicine Sapienza University of Rome Viale Regina Elena 291 00161 Rome Italy

Department of Translational Stem Cell Biology Medical University of Varna 9002 Varna Bulgaria

International Clinical Research Center St'Anne University Hospital Pekařská 53 65691 Brno Czech Republic

IRCCS Casa Sollievo della Sofferenza Bioinformatics Unit Viale Cappuccini 1 71013 San Giovanni Rotondo Italy

Laboratory of Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 753 5 62500 Brno Czech Republic

Mendel Centre for Plant Genomics and Proteomics Central European Institute of Technology Masaryk University Kamenice 753 5 62500 Brno Czech Republic

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