Histone Phosphorylation Dynamics During Mitotic Chromosome Condensation
DOI:
https://doi.org/10.5281/zenodo.22761427Keywords:
H3S10ph, H3T3ph, Aurora B, condensin, mitosis, chromosome condensation, live imaging, phospho-proteomicsAbstract
Histone H3 phosphorylation at serine 10 (H3S10ph) and threonine 3 (H3T3ph), deposited by Aurora B and Haspin
kinases respectively, accumulates on mitotic chromosomes coincident with condensation, but whether these events
are mechanistically required for condensation itself, as opposed to a parallel consequence of mitotic entry, and how
their dynamics relate to condensin loading, has remained incompletely resolved. We combined live-cell imaging of a
phospho-specific H3S10ph biosensor with synchronized-release ChIP-seq for H3S10ph, H3T3ph and the
condensin subunit NCAPH across mitotic entry in HeLa cells, alongside Aurora B and Haspin kinase inhibition and
quantitative phospho-proteomics. Live imaging showed that H3S10ph began in a punctate, pericentromeric pattern
approximately 8 minutes before nuclear envelope breakdown and spread to fill the chromosome arms within 4
minutes, preceding visible condensation by 2-3 minutes in every cell examined. ChIP-seq showed that NCAPH
loading closely tracked H3S10ph spread rather than preceding it, with genome-wide NCAPH occupancy at
prometaphase correlating strongly with H3S10ph signal at the immediately preceding time point. Acute Aurora B
inhibition with ZM447439 blocked H3S10ph spread, prevented normal NCAPH loading, and produced grossly
under-condensed chromosomes, whereas Haspin inhibition with 5-ITu, which selectively blocked H3T3ph,
permitted normal arm condensation but produced defective centromeric cohesion and segregation errors.
Phospho-proteomics identified condensin subunit NCAPD2 as a direct Aurora B substrate, and a
phosphorylation-deficient NCAPD2 mutant reduced condensin arm loading and produced under-condensed
chromosomes despite normal H3S10ph deposition, indicating that Aurora B promotes condensation through direct
condensin phosphorylation separable from its histone substrate. These results define spatially distinct roles for
Aurora B-dependent H3S10ph and condensin phosphorylation, and for Haspin-dependent H3T3ph, in arm
condensation versus centromeric cohesion during mitotic entry.
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