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bioRxiv · 10.1101/2025.09.29.679131

A time-resolved atlas of histone modifications during mitotic entry

Kochanova, N. Y.·Borso, M.·Deb, M.·Wenn, S.·Ustun, I.·Samejima, K.·Forne, I.·Samejima, I.·Xie, L.·Paulson, J.·Earnshaw, W. C.·Imhof, A.
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Abstract

Mitotic chromosome formation is essential for faithful chromosome segregation in metazoans. While condensin complexes are critical for the formation of rod-shaped mitotic chromosomes, additional mechanisms--particularly those involving phosphorylation and deacetylation of specific histone residues--have been proposed to contribute a further 2- to 4-fold reduction in mitotic chromatin volume. In this study, we employ high-resolution mass spectrometry to determine the kinetics of histone modifications in cell cultures undergoing a highly synchronous mitotic entry at 2.5-minute resolution. Our analysis reveals three different programmes of histone H3 phosphorylation on T3, S10 and S28. These modifications are consistent with methyl-phos switches regulating the association of readers with chromatin other than at promoters. Mass spectrometry and quantitative ChIP-Seq reveal that H3 T3 phosphorylation is a general marker of heterochromatin and not specifically centromeres as previously suggested. Finally, we show that histone acetylation undergoes only modest changes as rod-shaped chromosomes form during unperturbed mitotic entry. Thus, previously reported reductions in acetylation associated with chromosome formation were apparently attributable to delays in mitotic exit used as part of mitotic synchronisation protocols. The mechanism of condensin-independent chromatin compaction in mitosis remains unexplained.

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Kochanova, N. Y., Borso, M., Deb, M., Wenn, S., Ustun, I., Samejima, K., Forne, I., Samejima, I., Xie, L., Paulson, J., Earnshaw, W. C., Imhof, A.. 2025-09-30. A time-resolved atlas of histone modifications during mitotic entry. https://doi.org/10.1101/2025.09.29.679131

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