Expression of canonical MBF and SBF targets during replication stress. (A) Relative mRNA levels of the canonical SBF targets SVS1 and CLN2, and of the canonical MBF target CDC21 in cells synchronized by α-factor arrest and release, in the presence or absence of HU. Transcript levels are represented as a percentage of highest level (100%) observed in untreated samples. (B) Relative SVS1, CLN2 and CDC21 mRNA levels in wild-type, dun1Δ or sml1Δ/rad53Δ cells. The experiment was performed as described in Figure 1C.

SCB/MCB overlapping motifs at G1/S promoters provides the rationale for SBF and MBF competition. (A) Schematic representation of overlapping SBF (SCB) and MBF (MCB) DNA-binding motifs identified in the TOS4 promoter element (−232 to −214 from the ATG start codon). Grey shade represents SCB/MCB overlapping region. (B) ChIP analysis for Mbp1–myc and Swi4–myc at the TOS4 promoter in wild-type, swi4Δ and mbp1Δ cells. Analysis was performed in asynchronous cells and enrichment levels were accessed by qPCR and are shown as a percentage of WCE signal. Untagged cells were included as negative control. Average value and error bars are derived from experimental triplicates. (C) ChIP analysis for Mbp1–myc at TOS4 promoter in wild-type and swi4Δ cells. Experiment was performed as described in Figure 3A. (D) Relative mRNA levels of indicated genes during the cell cycle in wild-type, and cells expressing sic1Δp from the inducible GAL1 promoter. Cells were synchronized in G1 by α-factor, released from the arrest as detailed in Materials and methods and collected at the indicated times after release. Levels of mRNA were quantified by RT–qPCR and normalized to ACT1. Transcript levels are represented as a percentage of highest level (100%) observed in wild-type cells. Budding index (% budded cells) was measured as an indicator of synchrony. Average value and error bars are derived from biological triplicates.

Tos4 plays an important role in the replication stress response via interaction with HDACs. (A) FACS analysis of cells overexpressing TOS4 during S phase. Cells were synchronized in G1 by α-factor arrest and released from arrest in 2% galactose 1% raffinose for the indicated interval. (B) HU sensitivity of indicated strains. Four-fold serial dilutions were spotted on plates and grown for 2–3 days at 30°C. (C) Schematic representation of Tos4 protein displaying its FHA domain and the position of residues that were mutated to disrupt FHA domain function. (D) HU sensitivity of indicated strains. Four-fold serial dilutions were spotted on plates and grown for 2–3 days at 30°C. (E) Network of Tos4 FHA-interacting proteins during replication stress. According to available information in the Biogrid Database (http://thebiogrid.org/), the identified interacting proteins were grouped into two distinct complexes represented by the Rpd3 and Set3 HDACs. Grey lines represent previously known interactions between the different subunits that comprise both HDACs. Dotted line indicates the interactions identified in this work. (^*) Peptides for Hst1 were not detected in the MS experiment, but the interaction was validated by co-IP (see Supplementary Figure S6). (F) HU sensitivity of indicated strains. Four-fold serial dilutions were spotted on plates and grown for 2–3 days at 30°C.

(A) Parallel pathways for the replication checkpoint-dependent transcriptional regulation in budding yeast. Upon replication stress, Rad53 induces the expression of DDR genes via Dun1/Crt1 pathway and de-represses transcription of MBF-dependent targets required for replication stress response. (B) Regulation of TOS4 as a prototypical example of SBF-to-MBF subunit switch. TOS4 transcription is regulated via subunit switching and replication checkpoint signalling. During G1, SBF binds to TOS4 promoter and activates transcription. Upon entry into S phase, SBF dissociates from the TOS4 promoter in a Clb/CDK1-dependent manner leaving TOS4 promoter available for binding of MBF and its co-repressor Nrm1. In response to replication stress, Nrm1 dissociates from MBF in response to replication checkpoint activation leading to transcriptional de-repression of TOS4. (C) Proposed importance of SBF-to-MBF subunit switching. The SBF/MBF switch on G1/S promoters provides a fail-safe mechanism that prevents loss of periodicity and hyperaccumulation of dosage-sensitive genes during G1 in case of MBF loss of function. (^*) Indicate loss of periodicity in case of MBF ‘malfunction'.

Replication stress-induced reprogramming of gene expression includes transcriptional up-regulation of G1/S genes. (A) Proteomic analysis of changes in protein abundance following replication stress. Cells were arrested in G1 and released for 20 or 120 min in media containing 100 mM HU. Rad53 is activated at ∼25 min after release from G1 arrest. Shown results are the average ratio (120/20 min) for each of the 2862 proteins identified and quantified in the experiment. (B) Relative mRNA levels of indicated genes in cells synchronized by α-factor arrest and release, in the presence or absence of HU. Transcript levels are represented as a fold over 0 min for the Crt1 targets and a fold over max. unt. 100% for the G1/S targets. (C) Relative mRNA levels of indicated genes in wild-type, dun1Δ or sml1Δ/rad53Δ cells. Expression levels, at indicated time points (min), are presented as fold induction of levels detected in time 0. For samples treated with HU, 100 mM HU was added 20 min after release from α-factor arrest. Levels of mRNA were quantified by RT–qPCR and normalized to ACT1.

An SBF-to-MBF switch at the promoter of the G1/S gene TOS4. (A) ChIP analysis for Swi4–myc or Mbp1–myc at the TOS4, SVS1, CDC21 and CLN1 promoters during the G1 and S phase of the cell cycle. Analysis was performed in G1 synchronized cells released from α-factor arrest and samples were taken at the indicated intervals. (A) Enrichment levels were accessed by qPCR and are presented as a percentage of highest obtained level in the separate Swi4 ChIPs and Mbp1 ChIPs after normalization to whole cell extract (WCE) levels. Average value and error bars are derived from biological replicates. (B) Relative mRNA levels of indicated genes during the cell cycle in wild-type, swi4Δ and mbp1Δ cells. Cells were synchronized in G1 by α-factor, released from the arrest and collected at the indicated times after release. Levels of mRNA were quantified by RT–qPCR and normalized to ACT1. Transcript levels are represented as a percentage of highest level (100%) observed in wild-type cells.

Nrm1 represses expression of MBF targets during unperturbed S phase but not during replication stress. Relative mRNA levels of indicated genes in wild-type and nrm1Δ cells in the presence or absence of HU. For HU treatment, 100 mM HU was added 20 min after release from α-factor arrest. Levels of mRNA were quantified by RT–qPCR, normalized to ACT1, and presented as fold induction of levels detected in time 0 in wild-type cells. Budding index (% budded cells) was measured as an indicator of synchrony.

SBF-to-MBF switch at the promoter of other G1/S genes. (A) Schematic representation of overlapping SCB and MCB motifs identified in the MCD1 promoter element (–336 to –329 from the ATG start codon). Grey shade represents SCB/MCB overlapping region. (B) ChIP analysis for Swi4–myc or Mbp1–myc at the MCD1 promoter during the G1 and S phase of the cell cycle. Experiment was performed as described in Figure 3A. (C, D) Relative mRNA levels of MCD1 during the cell cycle in (C) wild-type, swi4Δ and mbp1Δ cells and (D) wild-type and cells expressing sic1Δp from the inducible GAL1 promoter. Experiments were performed as described in Figures 3B and 4D, respectively. (E) ChIP analysis for Swi4–myc or Mbp1–myc at the promoter of the indicated genes. Analysis was performed in G1 synchronized cells released from α-factor arrest and samples were taken at the indicated intervals. G1: 0 min, G1/S: 30 min and S phase: 60 min after release. Enrichment levels were accessed by qPCR and are presented as the ratio between Swi4–myc/Mbp1–myc percentages after normalization of each ChIP for its corresponding maximum value. (F) Percentage (%) of dosage-sensitive genes as compared with the number of genes tested in the combined genome-wide overexpression studies of Sopko et al (2006) and Yoshikawa et al (2011) for the indicated gene categories. The number of genes tested for overexpression in each of the categories is indicated in parenthesis.