Topoisomerases depletion intensifies the prereplication H3K9ac. (A) Experimental scheme. Cells were grown in the permissive temperature (24°C), synchronized using α-factor, and 30 min before release transferred to the restrictive temperature (34°C). Cells were released from G1 arrest and sampled every 3 min for DNA staining, RNA-seq, and ChIP-seq of H3K9ac. Binding of RNA polymerase II was measured using ChIP-seq in the synchronized time-point. Gene expression changes of cell-cycle-regulated genes are shown in Supplemental Figure S11A. (B) S-phase progression. Flow cytometry analysis of DNA-stained cells collected at the indicated time-points. (C) Topoisomerase depletion arrests DNA replication. Same as Figure 1F (top) for the indicated strains on chromosome VII. Dashed lines denote confirmed replication origins (based on OriDB) that intersect with regions that were replicated in the double mutant (Nieduszynski et al. 2007). A high-resolution view of a 30-kb region is shown in the inset, together with the corresponding H3K9ac profile. Top panel represents genes. (Blue) Watson; (green) Crick. Dashed lines in inset indicate the replicated region and the replication origin. For all chromosomes, see Supplemental Figure S11B. (D) Topoisomerase depletion represses gene expression in replicated regions. Log2 changes in gene expression, relative to synchronized cells, averaged over all genes (∼400) positioned in regions that were replicated in the topoisomerase-depleted strain. (E) H3K9ac on chromosome VII. H3K9ac normalized by the synchronized time-point. The inset is a blow-up of the indicated region (also marked in C). (F) H3K9ac in replicated regions. All replicated regions in top2-tsΔtop1 were ordered according to their length. DNA abundance (left) and the H3K9ac levels (right) at these regions, 45 min after release, normalized by the synchronized time-point, are plotted. Dashed lines depict the edges of the replicated region. (G) H3K9ac precedes replication. Same as Figure 4B for one of the two clusters replicated in top2-tsΔtop1. For the second cluster, see Supplemental Figure S11C.
