Rtt109 promotes nucleosome replacement ahead of the replication fork

Abstract

DNA replication perturbs chromatin by triggering the eviction, replacement, and incorporation of nucleosomes. How this dynamic is orchestrated in time and space is poorly understood. Here, we apply a genetically encoded sensor for histone exchange to follow the time-resolved histone H3 exchange profile in budding yeast cells undergoing slow synchronous replication in nucleotide-limiting conditions. We find that new histones are incorporated not only behind, but also ahead of the replication fork. We provide evidence that Rtt109, the S-phase-induced acetyltransferase, stabilizes nucleosomes behind the fork but promotes H3 replacement ahead of the fork. Increased replacement ahead of the fork is independent of the primary Rtt109 acetylation target H3K56 and rather results from Vps75-dependent Rtt109 activity toward the H3 N terminus. Our results suggest that, at least under nucleotide-limiting conditions, selective incorporation of differentially modified H3s behind and ahead of the replication fork results in opposing effects on histone exchange, likely reflecting the distinct challenges for genome stability at these different regions.

Figure 1.

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.

Figure 2.

H3 exchange follows replication fork progression. (A,B) Histone incorporation precedes the replication fork and correlates with the replication rate. (A) As in Figure 1E, adding the myc and HA epitope of the exchange sensor marking new and all histones, respectively. Note the different color scales for the epitopes. For additional repeats, see Supplemental Figure S1. n = 4 time courses. (B) myc and HA correlation to replication fraction and rate, analyzed as in Figure 1D. (C,D) Scaling of histone exchange with replicated fraction and replication rate. The genome was clustered based on the annotated RT, and the indicated profile was averaged within each of the 96 clusters (median). (C) The average H3 exchange level (myc/HA) as a function of the replicated fraction (top) or replication rate (bottom). Clusters are colored by their respective RT; time points 70–150 of the time course are shown (for all time points, see Supplemental Fig. S2). The scaling of exchange with the replication rate is summarized in D, displaying the slopes of the linear fits in C (blue lines) (Supplemental Fig. S2). Shading indicates SE (n = 4).

Figure 3.

Scaling of H3 exchange with the replication rate depends on Rtt109. (A,B) Exchange in RTT109-deleted cells is dominated by the replicated fraction, rather than the replication rate: (A,B) As in Figure 2C and 2B, respectively. For all time points, see Supplemental Figure S3. (C) Exchange at the replication fork is decreased in RTT109-deleted cells. The average fork exchange rate in RTT109-deleted cells is plotted against the corresponding rate in wild-type cells. Error bars indicate the SE (n = 3 for rtt109, n = 4 for WT). Color intensity indicates the replicated fraction at loci with the earliest RT, and dot size indicates time. (D) Replication-independent histone dynamics are invariant to RTT109 deletion. Mean myc level of nucleosomes that have not yet been replicated at the indicated time points in wild-type versus RTT109-deleted cells (n = 4 and 3, respectively). Highlighted are the rapidly exchanging nucleosomes (as determined in G₁ arrest) used for the linear fit (pink dotted line and equation). The one-to-one line is shown in blue.

Figure 4.

N-terminal H3 acetylation mediates histone exchange ahead of the fork. (A–C) VPS75-deleted cells lose replication-associated K9ac and display reduced H3 exchange ahead of the fork but maintain stable nucleosomes behind it. (A,B) As in Figure 2C and 2A, respectively, for VPS75-deleted cells (n = 2) (see also Supplemental Figure S4). HA data for times 50 and 90 were interpolated (see Methods). Of note is the delayed H3K9 acetylation (B; cf. to wild-type cells Fig. 2A; Supplemental Fig. S1A). Note the different color scales for the epitopes. (C) As in Figure 3C with the addition of the vps75 mutant. (D) Rtt109's dual role in regulating histone exchange during DNA replication. A schematic model describing the main findings: Rtt109-dependent acetylation of K56 stabilizes nucleosomes behind the replication fork, whereas acetylation of K9 promotes nucleosome replacement ahead of the fork. See Discussion.