Abstract
BackgroundThe 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.ConclusionsOur findings indicate that checkpoint mechanisms, likely via phosphorylation of network transcription factors, maintain coupling of the network oscillator to progression during cell-cycle arrest.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0446-7) contains supplementary material, which is available to authorized users.
Highlights
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
Mitotic CDKs are known to both inhibit and activate specific transcription factors within the network oscillator [14] (Figure 1a), and we have shown that CDKs play a role in controlling oscillation amplitude and period of the network oscillator [6]
Persistent Clb2/Cdk1 affects the function of specific network transcription factors To ask whether persistent levels of mitotic CDK (Clb2/ Cdk1) could freeze the network oscillator, we used a strain in which the anaphase promoting complex (APC) activator, Cdc20, is conditionally expressed from a modified GAL1 promoter (PGALL-CDC20) in a cdc20Δ background [15]
Summary
Persistent Clb2/Cdk affects the function of specific network transcription factors To ask whether persistent levels of mitotic CDK (Clb2/ Cdk1) could freeze the network oscillator, we used a strain in which the anaphase promoting complex (APC) activator, Cdc, is conditionally expressed from a modified GAL1 promoter (PGALL-CDC20) in a cdc20Δ background [15]. The positive feedback between Clb2/Cdk and SFF results in persistent target expression, but not at elevated levels compared to normally cycling cells or Cdc20depleted cells (Figure 5c, Additional file 1: Figure S9) This finding indicates some unknown mechanism may modulate the transcript level of SFF-regulated genes. Swi5- and Ace2-regulated clusters exhibit similar transcript dynamics in cells arrested by the two checkpoints, as well as Cdc20-depleted cells (Figure 5d, Additional file 1: Figure S10) Taken together, these observations suggest that persistent Clb2/Cdk acts in concert with other checkpoint effectors to fully arrest the network oscillator. A number of SBF-regulated genes, such as Yox, become periodic in the absence of Rad activity, suggesting that the DNA replication checkpoint kinases are responsible for arresting SBF-mediated transcription in addition to MBF/Nrm1-regulated genes (Figure 6d, Additional file 1: Figure S12) Taken together, these findings indicate that Rad and its downstream effectors are responsible for regulating a large fraction of checkpoint-regulated genes. Because the transcriptional program does not appear fully WT after the loss of Rad, it is likely that there are effectors in addition to Rad and APCCdc acting on the network during the replication checkpoint
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