Histone Chaperone Function in Nucleosome Assembly During DNA Replication
DOI:
https://doi.org/10.5281/zenodo.22756301Keywords:
histone chaperone, CAF-1, ASF1, MCM2, nucleosome assembly, DNA replication, iPOND, replication-coupled chromatin maturationAbstract
Faithful duplication of chromatin during S phase requires that parental histones be recycled and new histones be
deposited onto nascent DNA within seconds of fork passage, a process coordinated by a network of
replication-coupled histone chaperones whose individual contributions remain incompletely separated. Here we
combined isolation of proteins on nascent DNA coupled to mass spectrometry (iPOND-MS), nascent chromatin
capture sequencing, DNA fiber analysis and auxin-inducible degradation of the CAF-1 large subunit CHAF1A to
dissect how the CAF-1, ASF1 and MCM2 chaperone modules divide labour at the human replication fork.
iPOND-MS resolved a temporal chaperone handoff in which ASF1A/B peaked within 2 minutes of fork passage and
CAF-1 signal peaked between 2 and 10 minutes, coinciding with maturation of nucleosome spacing measured by
micrococcal nuclease digestion. Acute CHAF1A degradation for 30 minutes did not block fork progression but
reduced nascent nucleosome occupancy behind the fork by 46%, increased fork asymmetry and replication stress
marker gammaH2AX signal, and caused a marked redistribution of parental H3.1-K9me3 nucleosomes away from
their pre-replication genomic positions, indicating that CAF-1 is required both for new histone deposition and for
accurate positional inheritance of parental histones. Nascent chromatin capture sequencing after CHAF1A
depletion showed that heterochromatic domains were disproportionately affected, with a 2.3-fold greater loss of
nascent nucleosome density at constitutive heterochromatin than at euchromatin. Co-depletion of MCM2's
histone-binding domain further impaired parental H3-H4 transfer to the lagging strand without additively affecting
total nucleosome occupancy, consistent with MCM2 acting upstream of CAF-1 in a shared parental histone hand-off
pathway rather than as a redundant deposition route. These results define a chaperone division of labour in which
ASF1 supplies a shared new/old histone pool, MCM2 channels parental histones preferentially toward the lagging
strand, and CAF-1 performs the rate-limiting deposition step required for both new histone incorporation and faithful
epigenetic inheritance, with heterochromatin maintenance being particularly sensitive to its loss.
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