Abstract
How histone post-translational modifications (PTMs) are inherited through the cell cycle remains poorly understood. Canonical histones are made in the S phase of the cell cycle. Combining mass spectrometry-based technologies and stable isotope labeling by amino acids in cell culture, we question the distribution of multiple histone PTMs on old versus new histones in synchronized human cells. We show that histone PTMs can be grouped into three categories according to their distributions. Most lysine mono-methylation and acetylation PTMs are either symmetrically distributed on old and new histones or are 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 the following three human cell types: HeLa, 293T, and human foreskin fibroblast 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 Lys-9 di- or tri-methylations, they are not required for the asymmetric distribution of Ser-10 phosphorylation 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 cross-talk between H3S10ph and H3K9me2.
Highlights
In eukaryotes, histone proteins facilitate the packaging of DNA molecules
We show that histone post-translational modifications (PTMs) can be grouped into three categories according to their distributions
Because canonical histone proteins are only synthesized in S phase [1, 2], any heavy Arg-labeled histones were newly synthesized histones and could be detected by the subsequent mass spectrometry analysis
Summary
Systematic Analysis of the Distribution of Histone PTMs in Mitosis—To investigate the distribution of histone PTMs on old versus new histones during mitosis, we used pulse-SILAC (stable isotope labeling by amino acids in cell culture) [28] followed by mass spectrometry techniques. Most lysine di- and tri-methylations were enriched on the old histones, including H4K20me2/3, H3K79me2/3, H3K9me, H3.1/ 2K27me2/3, H3.1/2K36me2/3, H3.3K27me, and H3.3K36me2/ 3 These results extended previous findings from us and others demonstrating that new H3K9me and H3K27me are synthesized rather slowly and do not complete until G1 (9 –11). Whereas H3K4me was enriched on old H3 (Table 1) This result demonstrates for the first time that me2/3 marks could be restored before mitosis and suggests that the mechanisms underlying the inheritance of H3K4me and other Kme marks are distinct. Another example is that H3K79me1/2/3s were all asymmetrically distributed on the old H3 in mitosis (Table 1), suggesting that the sole methyltransferase for H3K79, Dot1l [31], was preferentially recruited to old histone H3. As H3S10ph was the most abundant form of histone Ser/Thr phosphorylation (Table 1 and Fig. 2B), we first confirmed the Relative Abundance (%) Normalized distribution at 11h (Log2)
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