Volume 54, Issue 4, Pages 691-697 (May 2014) The Histone Deacetylases Sir2 and Rpd3 Act on Ribosomal DNA to Control the Replication Program in Budding Yeast  Kazumasa Yoshida, Julien Bacal, Damien Desmarais, Ismaël Padioleau, Olga Tsaponina, Andrei Chabes, Véronique Pantesco, Emeric Dubois, Hugues Parrinello, Magdalena Skrzypczak, Krzysztof Ginalski, Armelle Lengronne, Philippe Pasero  Molecular Cell  Volume 54, Issue 4, Pages 691-697 (May 2014) DOI: 10.1016/j.molcel.2014.04.032 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 54, 691-697DOI: (10.1016/j.molcel.2014.04.032) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 BrdU-IP-Chip Analysis of DNA Replication Profiles in HDAC Mutants (A) Wild-type (PP872), sir2Δ (PP367), hos2Δ (PP1369), hst1Δ (PP1364), hda1Δ (PP1368), sum1Δ (PP1365), hos1Δ (PP1553), hos3Δ (PP1554), hst2Δ (PP1555), hst3Δ hst4Δ (PP1370), rpd3Δ (PP978), and rad53-11 (PP37) cells were arrested in G1 phase with α factor and released for 60 min into YPD medium containing 200 mM HU and 400 μg/ml BrdU. BrdU-labeled DNA was immunoprecipitated and hybridized on Affymetrix tiling arrays, as described previously (Crabbé et al., 2010). Signal log ratio of BrdU enrichment relative to input DNA is shown for a representative region of chromosome XIV. Active early and late origins are indicated with open and filled arrowheads, respectively. Significant BrdU peaks are marked in blue. (B) Number of active origins in the indicated strains. Early and late origins are shown in gray and blue, respectively. Differences relative to wild-type cells are indicated for each strain. (C) BrdU-IP-chip analysis of origin firing in wild-type, sir2Δ and rpd3Δ cells collected in early S phase in the absence of HU. (D) Scatter plot of BrdU incorporation at early origins in rpd3Δ (orange) and sir2Δ (blue) mutants, relative to wild-type cells. BrdU incorporation (arbitrary units) was determined for a 2 kb region centered on replication origins. Molecular Cell 2014 54, 691-697DOI: (10.1016/j.molcel.2014.04.032) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Origin Usage in sir2Δ Cells Is Inversely Correlated with Initiation at the rDNA Array (A) Wild-type (PP872), fob1Δ (PP1730), fob1Δ cells with a 20-copy rDNA array (PP1758), sir2Δ (PP367), sir2Δ fob1Δ (PP1734), and sir2Δ fob1Δ cells with a 20-copy rDNA array (PP1762) were BrdU-labeled as indicated in Figure 1. In (A), BrdU incorporation at rDNA origins was monitored by BrdU-IP followed by qPCR. BrdU levels were expressed relative to wild-type cells. Error bars represent SD for three experiments. (B) Relative activity of rDNA origins (relative BrdU incorporation) and single-copy origins (relative number of active origins) in HU-arrested cells, as determined by BrdU-IP-qPCR and BrdU-IP-chip, respectively. (C and D) Quantitation of ERC levels in the indicated strains. A shorter exposure of the autoradiography is shown for the rDNA array (rDNA). Error bars indicate SD for six experiments. (E) ChIP-qPCR analysis of acetylated histone H4 (H4ac) at E-pro, rARS and C-pro (35S gene). Error bars indicate SD for two experiments. Molecular Cell 2014 54, 691-697DOI: (10.1016/j.molcel.2014.04.032) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Rpd3 and Sir2 Have an Opposite Effect on rDNA Replication (A and B) BrdU-IP-seq analysis of DNA replication at the rDNA (A) and single-copy sequences (B). Wild-type (PP872), sir2Δ (PP367), rpd3Δ (PP978), and rpd3Δ sir2Δ (PP1449) cells were synchronized in G1 and were released for 60 min in YPD medium containing 200 mM HU and 400 μg/ml BrdU. Newly replicated DNA was immunoprecipitated and sequenced by NGS. BrdU incorporation at ribosomal DNA (A) and at a representative region of chromosome XI (B) is shown. Asterisks indicate origins that are differentially activated in sir2Δ and rpd3Δ cells. The map of the 9.1 kb rDNA unit shows the position of the 35S and 5S rRNA gene, the replication origin (ARS), and the replication fork barrier (RFB). (C) Number of active origins in single-copy regions of the yeast genome, as determined by BrdU-IP-seq (two biological replicates). (D) Scatter plot of BrdU signal at 2 kb fragments centered on replication origins in rpd3Δ (blue) and rpd3Δ sir2Δ (orange) cells, relative to BrdU signal in wild-type cells (arbitrary units). Molecular Cell 2014 54, 691-697DOI: (10.1016/j.molcel.2014.04.032) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Overexpression of Limiting Initiation Factors Restores the Activation of Early Origins in sir2Δ Cells (A) Wild-type and sir2Δ cells containing one or multiple copies of a GAL-SLD3/SLD7/CDC45 construct inserted at the TRP1 locus (Tanaka et al., 2011) were synchronized in G1 and were released for 60 min in YPGal medium containing 200 mM HU and 400 μg/ml BrdU, as indicated (Tanaka et al., 2011). Newly replicated DNA was immunoprecipitated and was analyzed on tiling arrays. Low overexpression correspond to wt (PP2022) and sir2Δ (PP2020) cells containing one insertion. High overexpression correspond to wt (PP2023) and sir2Δ (PP2021) cells containing multiple insertions of the construct. (B and C) Scatter plot of BrdU signal at 2 kb fragments centered on replication origins in wild-type cells and sir2Δ cells overexpressing (orange) or not (blue) initiation factors to different levels. (D) Model of the regulation of replication timing by Sir2 and Rpd3. Early, mid, and late origins are activated sequentially during S phase through the recycling of limiting initiation factors (orange arrows). A fraction of these factors also activate rDNA origins in the nucleolus. In the absence of Sir2, silencing is decreased at the rDNA array and more rDNA origins are accessible to initiation factors. As a consequence, the activation of early- to mid-S phase origins is delayed. In the absence of Rpd3, silencing increases at the rDNA in a Sir2-dependent manner. The pool of available initiation factors increases, leading to the premature activation of chromosomal origins. Deletion of the SIR2 gene in rpd3Δ mutants restores normal rDNA replication and suppresses the premature activation of origins in the rest of the genome. Molecular Cell 2014 54, 691-697DOI: (10.1016/j.molcel.2014.04.032) Copyright © 2014 Elsevier Inc. Terms and Conditions