Linking DNA replication checkpoint to MBF cell-cycle transcription reveals a distinct class of G1/S genes

Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA.
The EMBO Journal (Impact Factor: 10.43). 02/2012; 31(7):1798-810. DOI: 10.1038/emboj.2012.27
Source: PubMed


Reprogramming gene expression is crucial for DNA replication stress response. We used quantitative proteomics to establish how the transcriptional response results in changes in protein levels. We found that expression of G1/S cell-cycle targets are strongly up-regulated upon replication stress, and show that MBF, but not SBF genes, are up-regulated via Rad53-dependent inactivation of the MBF co-repressor Nrm1. A subset of G1/S genes was found to undergo an SBF-to-MBF switch at the G1/S transition, enabling replication stress-induced transcription of genes targeted by SBF during G1. This subset of G1/S genes is characterized by an overlapping Swi4/Mbp1-binding site and is enriched for genes that cause a cell cycle and/or growth defect when overexpressed. Analysis of the prototypical switch gene TOS4 (Target Of SBF 4) reveals its role as a checkpoint effector supporting the importance of this distinct class of G1/S genes for the DNA replication checkpoint response. Our results reveal that replication stress induces expression of G1/S genes via the Rad53-MBF pathway and that an SBF-to-MBF switch characterizes a new class of genes that can be induced by replication stress.

Download full-text


Available from: Robertus A M de Bruin
  • Source
    • "Studies in S. pombe indicate checkpoint control of MBF activity is mediated by Rad53-dependent regulation of the MBF activator Cdc10 [30] and the co-repressor Nrm1 [31]. Recent reports indicate Rad53 inactivates Nrm1 as part of the DNA replication checkpoint in S. cerevisiae [29,32]. Likewise, downregulation of SBF targets in both DNA replication checkpoint- and spindle assembly checkpoint-arrested cells, as in Cdc20-depleted cells, suggest that stabilization of Clb2 by inhibition of APCCdc20 may be responsible for control of this regulon (Figure 5a, Additional file 1: Figure S7 and S8). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background The coupling of cyclin dependent kinases (CDKs) to an intrinsically oscillating network of transcription factors has been proposed to control progression through the cell cycle in budding yeast, Saccharomyces cerevisiae. The transcription network regulates the temporal expression of many genes, including cyclins, and drives cell-cycle progression, in part, by generating successive waves of distinct CDK activities that trigger the ordered program of cell-cycle events. Network oscillations continue autonomously in mutant cells arrested by depletion of CDK activities, suggesting the oscillator can be uncoupled from cell-cycle progression. It is not clear what mechanisms, if any, ensure that the network oscillator is restrained when progression in normal cells is delayed or arrested. A recent proposal suggests CDK acts as a master regulator of cell-cycle processes that have the potential for autonomous oscillatory behavior.ResultsHere we find that mitotic CDK is not sufficient for fully inhibiting transcript oscillations in arrested cells. We do find that activation of the DNA replication and spindle assembly checkpoints can fully arrest the network oscillator via overlapping, but distinct mechanisms. Further, we demonstrate that the DNA replication checkpoint effector protein, Rad53, acts to arrest a portion of transcript oscillations in addition to its role in halting cell-cycle progression.Conclusions Our findings indicate that checkpoint mechanisms, likely via phosphorylation of network transcription factors, maintain coupling of the network oscillator to progression during cell-cycle arrest.
    Full-text · Article · Sep 2014 · Genome Biology
  • Source
    • "Because ribonucleotide reductase (Cdc22) is the target of HU and its expression is directly regulated by MBF (Lowndes et al., 1992), hyperactivation of the complex might help to overcome the block to DNA replication inflicted by the drug. Similar processes have been described in the distantly related budding yeast (de Bruin et al., 2008; Bastos de Oliveira et al., 2012; Travesa FIGURE 5: Cdc10 Ser-720 and Ser-732 are phosphorylated by Chk1, inactivating MBFdependent transcription. (A) Chk1 signals MBF through the C-terminal region of Cdc10. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We have previously shown that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1 resulting in the activation of the MBF-dependent transcription. This is essential to reinitiate DNA synthesis and for a correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA damaging agents. Thus, Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints on a single transcriptional complex.
    Full-text · Article · Sep 2013 · Molecular biology of the cell
  • Source
    • "Over 300 G1/S transcripts depend on SBF and/or MBF for their periodicity [7], [13], [14], [15]. The genes regulated by both can be further grouped into targets bound by both at the same time and switch genes, where an SBF-to-MBF switch takes place during the G1-to-S transition [16], [17]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: G1/S transcriptional regulation in the budding yeast Saccharomyces cerevisiae depends on three main transcriptional components, Swi4, Swi6 and Mbp1. These proteins constitute two transcription factor complexes that regulate over 300 G1/S transcripts, namely SBF (Swi4-Swi6) and MBF (Mbp1-Swi6). SBF and MBF are involved in regulating largely non-overlapping sets of G1/S genes via clearly distinct mechanisms. Here we establish and confirm protein-protein and protein-DNA interactions using specific polyclonal antisera to whole Swi6 and to the C-terminal domains of related proteins Swi4 and Mbp1. Our data confirm the protein-protein binding specificity of Swi4 and Mbp1 to Swi6 but not to each other, and support the binding specificity of the transcriptional inhibitor Whi5 to SBF and of the corepressor Nrm1 to MBF. We also show the DNA binding preference of Swi4 to the CLN2 promoter and Mbp1 to the RNR1 promoter, while Swi6 binds both promoters. Finally, we establish the binding dynamics of Swi4 and Whi5 to the CLN2 promoter during the cell cycle. These data confirm the binding specificity of the G1/S transcriptional regulators. Whereas previous observations were made using tagged Swi4, Swi6 and Mbp1, here we use specific polyclonal antisera to reestablish the protein-protein and protein-DNA interactions of these G1/S transcriptional components. Our data also reveal the dynamic changes in promoter binding of Swi4 during the cell cycle, which suggests a possible positive feedback loop involving Swi4.
    Full-text · Article · Apr 2013 · PLoS ONE
Show more