Chromatin replication dynamics during slowed S phase. (A,B) Profiling histone H3 modifications and exchange dynamics during slowed replication. (A) Time-resolved chromatin immunoprecipitation (ChIP-seq) and DNA-seq experiments were performed to follow replication in cells synchronously released into 200 mM hydroxyurea (HU) to slow replication. DNA levels, H3 abundance (HA), H3 incorporation (myc) (see Yaakov et al. 2021), and H3K56 and H3K9 acetylations were measured. (B) Replication dynamics are captured by the change in read coverage, shown here on a segment of Chromosome X. The annotated replication time (RT) in unperturbed S phase is included (Yabuki et al. 2002). Annotated origins of replication initiation (ORIs) (Siow et al. 2012) with RT < 21 min are shown as green dots. H3K9ac coverage is additionally shown after normalizing to that of nonreplicating, G1-arrested cells (log2). (C,D) H3K56ac correlates with DNA abundance, whereas H3K9ac accumulates ahead of the replication fork. ORIs were clustered by their RT, and profiles around each ORI were aligned and averaged. (C) Shown are G1-normalized profiles within each of the four ORI clusters (note the scales for the various antibodies) and the fork position as calculated from the DNA profile (red dots). (D) Data for H3K9ac or H3K56ac (n = 2 time courses) are summarized by comparing the profiles of these modifications with the DNA profile (replicated fraction) or its change along the genome (replication rate; see Methods). Color intensity indicates time, and the dot size indicates the divergence (1 − correlation) between the replicated fraction and rate. (E) H3K9ac accumulates ahead of the replication fork. Cross-correlation between the temporal changes of the indicated acetylation with DNA dynamics is shown as a function of the time delay; black dots indicate the delay with the highest correlation (for repeat, see Supplemental Fig. S1). H3K56ac coincides with DNA content, whereas H3K9ac precedes it. Note the different color scales for the epitopes.
