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-29-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.[Keywords: DNA replication timing; replication origin; chromatin; histone deacetylase; 5-Bromo-29-deoxyuridine (BrdU); S-phase checkpoint; microarrays] Supplemental material is available at http://www.genesdev.org.

Defining replication origin efficiency using DNA fiber assays

“…Data generated by microarray-based assays support the view that initiation of DNA replication follows a temporal program that is imposed by epigenetic mechanisms and is stably transmitted to daughter cells (Knott et al 2009;Raghuraman et al 2001). However, this model has been recently challenged by Shao and colleagues, who showed that all yeast chromosomes VI have different replication profile when analyzed at the singlemolecule level (Czajkowsky et al 2008).…”

“…Hence, early origins replicate late when they are moved to late-replicating regions of the genome, whereas initiation at late origins is advanced when they are moved to early regions (Lucas and Feng 2003). Multiple factors affect replication timing in eukaryotic cells, including histone modifications, subnuclear localization, and gene activity (Hiratani et al 2009;Knott et al 2009;Lucas and Feng 2003). Replication timing is also controlled by checkpoint kinases, which detect stalled replication forks and prevent the activation of late origins until the replication stress is relieved (Tourriere and Pasero 2007).…”

Conservation of replication timing reveals global and local regulation of replication origin activity

“…The replication time of telomere proximal regions is regulated by telomere length with shorter telomeres replicated earlier (Bianchi and Shore 2007;Lian et al 2011). Chromatin modifications have also been implicated in the regulation of replication origin activation time (Vogelauer et al 2002;Knott et al 2009); however, no clear correlations between specific chromatin modifications and replication time have been identified. In summary, the molecular mechanisms responsible for differences in replication time remain elusive (Sclafani and Holzen 2007).…”

Nucleosome occupancy as a novel chromatin parameter for replication origin functions

“…Because of these reports, many studies have tried to establish the parameters of chromatin that influence origin firing. We and others have shown that histone modifications, especially acetylation, can affect origin timing and efficiency (Vogelauer et al 2002;Iizuka et al 2006;Knott et al 2009;Unnikrishnan et al 2010). Moreover, genome-wide nucleosome mapping showed that nucleosome positions dictate the association of Mcm2-7 with origins (Belsky et al 2015).…”