Genome-wide replication profiles indicate an expansive role for Rpd3L in regulating replication initiation timing or efficiency, and reveal genomic loci of Rpd3 function in Saccharomyces cerevisiae

Abstract

In higher eukaryotes, heritable gene silencing is associated with histone deacetylation and late replication timing. In Saccharomyces cerevisiae, the histone deacetylase Rpd3 regulates gene expression and also modulates replication timing; however, these mechanisms have been suggested to be independent, and no global association has been found between replication timing and gene expression levels. Using 5-Bromo-2'-deoxyuridine (BrdU) incorporation to generate genome-wide replication profiles, we identified >100 late-firing replication origins that are regulated by Rpd3L, which is specifically targeted to promoters to silence transcription. Rpd3S, which recompacts chromatin after transcription, plays a primary role at only a handful of origins, but subtly influences initiation timing globally. The ability of these functionally distinct Rpd3 complexes to affect replication initiation timing supports the idea that histone deacetylation directly influences initiation timing. Accordingly, loss of Rpd3 function results in higher levels of histone H3 and H4 acetylation surrounding Rpd3-regulated origins, and these origins show a significant association with Rpd3 chromatin binding and gene regulation, supporting a general link between histone acetylation, replication timing, and control of gene expression in budding yeast. Our results also reveal a novel and complementary genomic map of Rpd3L- and Rpd3S-regulated chromosomal loci.

Figure 1.

Early S-phase replication profiles identify Rpd3-regulated origins. Wild-type (WT) and rpd3Δ cells blocked in G1 phase with α-factor were synchronously released into fresh medium containing HU plus BrdU for 1 h and harvested for BrdU-IP-chip analysis. Data were processed as described in the Materials and Methods; chromosomes VI (A), V (B), and XIV (C) are shown. Individual peaks are denoted with gray dots, and peaks that are significantly different in height (P ≤ 0.001) between the strains are denoted with red dots; origins discussed in the text are indicated. (D) Horizontal lines in the plots indicate the lower quartile, median (red line), and upper quartile of wild-type BrdU peak heights assigned to the corresponding T_rep quartile (as indicated on the X-axis). Outliers are displayed with a red plus (+) sign. (E) BrdU peak heights determined for each origin in the rpd3Δ strain are plotted against the corresponding peak heights in the wild type; peaks that are significantly different in height in the rpd3Δ cells are indicated with red dots.

Figure 2.

Early S-phase replication profiles identify Rpd3S- and Rpd3L-regulated origins. eaf3Δ (A), dep1Δ (B), and dep1Δ eaf3Δ (C) cells were analyzed as described in the legend for Figure 1 and the resulting data for chromosome XIV are shown overlaid with the wild-type (WT) and rpd3Δ profiles. Peaks meeting significance criteria for initiation in rpd3Δ cells are indicated with green dots.

Figure 3.

Global comparison of BrdU peak heights in Rpd3S and Rpd3L mutants with corresponding wild-type peaks. BrdU peak heights for each origin in rco1Δ (A), eaf3Δ (B), set2Δ (C), dep1Δ (D), cti6Δ (E), dep1Δ eaf3Δ (F), ume6Δ (G), and ash1Δ (H) mutant strains are plotted against the corresponding BrdU peak heights in wild-type (WT) cells; peaks that are significantly different in height from the wild type are indicated with red dots.

Figure 4.

Pairwise correlation analysis comparing Rpd3S and Rpd3L replication profiles. Wild-type peak heights were subtracted from the corresponding peak heights of each mutant to yield a “difference vector” for every strain. The full pairwise Pearson's correlation matrix for the set of mutant strains was calculated using these difference vectors.

Figure 5.

Rpd3L-regulated origins are associated with Rpd3-regulated genes. (A) Venn diagram of overlap between (1) origins deregulated in rpd3Δ cells and (2) regions that show increased acetylation in rpd3Δ cells. (B) Venn diagram of overlap between intergenic regions containing origins that are (1) deregulated in dep1Δ cells, (2) flanked by genes deregulated in rpd3Δ or sin3Δ cells, and (3) flanked by genes that bind Rpd3 or Sin3 in their promoter regions.

Figure 6.

Different gene-origin configurations subject to regulation by Rpd3L and Rpd3S. See the Discussion. Red strips with points indicate ORF positions and direction of transcription, and gray strips indicate origin position. Vertical arrows indicate genes derepressed in rpd3Δ or sin3Δ cells, and spheres mark reported binding sites for the indicated proteins (P ≤ 0.05, unless indicated otherwise). Panels to the right show BrdU-incorporation tracings for the indicated strains.