Preferential phosphorylation on old histones during early mitosis in human cells

Abstract How histone posttranslational modifications (PTMs) are inherited through cell cycle remains poorly understood. Canonical histones are made in the S phase of cell cycle. Combining mass spectrometry-based technologies and stable isotope labeling by amino acids in cell culture (SILAC), we interrogate the distribution of multiple histone PTMs on old versus new histones in synchronized human cells. We show that histone PTMs can be grouped to three categories according to their distributions. Most lysine mono-methylation and acetylation PTMs are either symmetrically distributed on old and new histones or enriched on new histones. In contrast, most di- and tri-methylation PTMs are enriched on old histones, suggesting that the inheritance of different PTMs is regulated distinctly. Intriguingly, old and new histones are distinct in their phosphorylation status during early mitosis in three human cell types: Hela, 293T and human foreskin fibroblast (HFF) cells. The mitotic hallmark H3S10ph is predominantly associated with old H3 at early mitosis and becomes symmetric with the progression of mitosis. This same distribution was observed with other mitotic phosphorylation marks, including H3T3/T6ph, H3.1/2S28ph and H1.4S26ph, but not S28/S31ph on the H3 variant H3.3. Although H3S10ph often associates with the neighboring K9 di- or tri-methylation, they are not required for the asymmetric distribution of S10ph on the same H3 tail. Inhibition of the kinase Aurora B does not change the distribution despite significant reduction of H3S10ph levels. However, K9me2 abundance on the new H3 is significantly reduced after Aurora B inhibition, suggesting a crosstalk between H3S10ph and H3K9me2.