A time-resolved atlas of histone modifications during mitotic entry

Mitotic chromosome formation is essential for faithful chromosome segregation in metazoans. Although condensin complexes are critical for the formation of rod-shaped mitotic chromosomes, histone phosphorylation and deacetylation have been proposed to contribute to a further 2- to 4-fold reduction in mitotic chromatin volume. Here, we employ high-resolution mass spectrometry to determine the kinetics of histone modifications in cell cultures undergoing highly synchronous mitotic entry. Our analysis reveals three temporally distinct programs of histone H3 phosphorylation on T3, S10, and S28 that could differentially regulate the association of readers with chromatin via methyl-phos switching. Mass spectrometry, quantitative chromatin immunoprecipitation sequencing (ChIP-seq), ChIP-qPCR, and immunofluorescence analyses reveal that H3 T3 phosphorylation is a mitosis-specific marker of heterochromatin, whose deposition requires H3K9me3. Finally, we show that histone acetylation undergoes only modest changes as rod-shaped chromosomes form during unperturbed mitotic entry. Thus, deacetylation does not drive mitotic chromosome formation. The mechanism of condensin-independent chromatin compaction in mitosis remains unexplained.