Quantitative analysis of post-translational modifications of histone H3 variants during the cell cycle

Histones participate in epigenetic regulation via dynamic post-translational modifications (PTMs) of histone variants. Comprehensive characterization of histone markers, especially those for the histone variants, could help to decipher the mechanism of epigenetic regulation. However, correctly profiling histone PTMs and its variants using mass spectrometry remains a challenge. Here, we developed an improved, specific and sensitive LC separation in conjunction with a high throughput multiple reaction monitoring combined with stable isotope-labeled internal standards (MRM-SIS) based quantitative method for histone H3 variants in the study of epigenetic regulation in the cell cycle. PTM patterns and the overall abundance of the three main histone H3 variants from Karpas 422 cells were analyzed and quantified simultaneously during different cell stages. The methylation pattern varied between different sites and modification states during the cell cycle. The canonical H3.1 presented regular patterns on K27 and K36, similar to H3.2, albeit differing from variant H3.3. H3.3 K36me2 increased from G1, S to G2 phase, whereas the same marker decreased in both H3.1 and H3.2. This novel discovery inspires more focus and research on the histone variants behavior and function during cell cycle. Moreover, this improved method could be applied to unveil PTMs dynamics of histone variants in several biological processes.