Dynamics and dispensability of variant-specific histone H1 Lys-26/Ser-27 and Thr-165 post-translational modifications

•Human histone H1.2 T165 is phosphorylated at S and G2/M phases of the cell cycle.•H1.2 T165 phosphorylation marks highly proliferative cancer cells.•H1.2 T165 is dispensable for cell proliferation and H1.2 binding to chromatin.•H1.4 S27 is phosphorylated at G2/M whether adjacent K26 is modified or not.•H1.4 K26 residue is required for cell proliferation and heterochromatin loading. In mammals, the linker histone H1, involved in DNA packaging into chromatin, is represented by a family of variants. H1 tails undergo post-translational modifications (PTMs) that can be detected by mass spectrometry. We developed antibodies to analyze several of these as yet unexplored PTMs including the combination of H1.4 K26 acetylation or trimethylation and S27 phosphorylation. H1.2-T165 phosphorylation was detected at S and G2/M phases of the cell cycle and was dispensable for chromatin binding and cell proliferation; while the H1.4-K26 residue was essential for proper cell cycle progression. We conclude that histone H1 PTMs are dynamic over the cell cycle and that the recognition of modified lysines may be affected by phosphorylation of adjacent residues.