Checkpoints couple transcription network oscillator dynamics to cell-cycle progression

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. Results: Here 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.

The CDK-APC/C Oscillator Predominantly Entrains Periodic Cell-Cycle Transcription

“…Some (but not all) SBF-regulated genes were reported to fire in cdc20Δ -blocked cells in [Bristow et al, 2014]. We observe strong, initial repression of the SBF-regulated CLN2 promoter, consistent with the well-established ability of Clb-Cdk (which accumulates to high levels in cdc20Δ -blocked cells) to repress SBF [Amon et al, 1993] (Fig.…”

“…S4 P) and SIC1 pulses (Fig. 4 J) were seen in our experiments in arrested cells but not in [Bristow et al, 2014]. These events occurred with great inter-cell timing variability, thus are nearly undetectable in a population experiment.…”

“…In our hands, the MET-CDC20 block is rather leaky; single-cell analysis was crucial for identifying and removing escaping cells from analysis. We have no information to suggest leakiness of the cdc20Δ block in [Bristow et al, 2014]. However, any leakage that does occur will contribute to an average increase in SBF gene expression and in our experience, leakage in cdc20Δ -blocked cells is very hard to catch in bulk culture, since the leaked cells promptly re-arrest in the next cell cycle.…”

“…It is important to compare our results with experimental results supporting the GTO model [Orlando et al, 2008, Simmons Kovacs et al, 2012, Bristow et al, 2014]. …”

“…2 D right) could drive the global cell cycle transcription program without CDK-APC/C regulation. Evidence for such a CDK-APC/C-independent global transcriptional oscillator (GTO) has been reported in yeast cells; hundreds of genes oscillate in mutant strains with crippled CDK-APC/C oscillations [Orlando et al, 2008, Simmons Kovacs et al, 2012, Bristow et al, 2014]. …”