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Protein degradation during the cell cycle is controlled by the opposing activities of ubiquitin ligases and deubiquitinating enzymes (DUBs). Although the functions of ubiquitin ligases in the cell cycle have been studied extensively, the roles of DUBs in this process are less well understood. Here, we used an overexpression screen to examine the specificities of each of the 21 DUBs in budding yeast for 37 cell cycle–regulated proteins. We find that DUBs up-regulate specific subsets of proteins, with five DUBs regulating the greatest number of targets. Overexpression of Ubp10 had the largest effect, stabilizing 15 targets and delaying cells in mitosis. Importantly, UBP10 deletion decreased the stability of the cell cycle regulator Dbf4, delayed the G1/S transition, and slowed proliferation. Remarkably, deletion of UBP10 together with deletion of four additional DUBs restored proliferation to near–wild-type levels. Among this group, deletion of the proteasome-associated DUB Ubp6 alone reversed the G1/S delay and restored the stability of Ubp10 targets in ubp10Δ cells. Similarly, deletion of UBP14, another DUB that promotes proteasomal activity, rescued the proliferation defect in ubp10Δ cells. Our results suggest that DUBs function through a complex genetic network in which their activities are coordinated to facilitate accurate cell cycle progression.

Upon exposure to environmental stressors, cells transiently arrest the cell cycle while they adapt and restore homeostasis. A challenge for all cells is to distinguish between stress signals and coordinate the appropriate adaptive response with cell cycle arrest. Here we investigate the role of the phosphatase calcineurin (CN) in the stress response and demonstrate that CN activates the Hog1/p38 pathway in both yeast and human cells. In yeast, the MAPK Hog1 is transiently activated in response to several well-studied osmostressors. We show that when a stressor simultaneously activates CN and Hog1, CN disrupts Hog1-stimulated negative feedback to prolong Hog1 activation and the period of cell cycle arrest. Regulation of Hog1 by CN also contributes to inactivation of multiple cell cycle-regulatory transcription factors (TFs) and the decreased expression of cell cycle-regulated genes. CN-dependent downregulation of G1/S genes is dependent upon Hog1 activation, whereas CN inactivates G2/M TFs through a combination of Hog1-dependent and -independent mechanisms. These findings demonstrate that CN and Hog1 act in a coordinated manner to inhibit multiple nodes of the cell cycle-regulatory network. Our results suggest that crosstalk between CN and stress-activated MAPKs helps cells tailor their adaptive responses to specific stressors., Author summary In order to survive exposure to environmental stress, cells transiently arrest the cell division cycle while they adapt to the stress. Several kinases and phosphatases are known to control stress adaptation programs, but the extent to which these signaling pathways work together to tune the stress response is not well understood. This study investigates the role of the phosphatase calcineurin in the stress response and shows that calcineurin inhibits the cell cycle in part by stimulating the activity of the Hog1/p-38 stress-activated MAPK in both yeast and human cells. Crosstalk between stress response pathways may help cells mount specific responses to diverse stressors and to survive changes in their environment.

SummaryUpon exposure to environmental stressors, cells transiently arrest the cell cycle while they adapt and restore homeostasis. A challenge for all cells is to distinguish between diverse stress signals and coordinate the appropriate adaptive response with cell cycle arrest. Here we investigate the role of the stress-activated phosphatase calcineurin (CN) in this process and show that CN utilizes multiple pathways to control the cell cycle. Upon activation, CN inhibits transcription factors (TFs) that regulate the G1/S transition through activation of the stress-activated MAPK Hog1. In contrast, CN inactivates G2/M TFs through a combination of Hog1-dependent and -independent mechanisms. These findings demonstrate that CN and Hog1 act in a coordinated manner at multiple nodes of the cell cycle-regulatory network to rewire gene expression and arrest cells in response to stress. Our results suggest that crosstalk between CN and stress-activated MAPKs helps cells tailor their adaptive responses to specific stressors.

The transcription factor Hcm1 is a key regulator of chromosome segregation and genome stability. The phosphatase calcineurin directly inactivates Hcm1 in response to environmental stress, which inhibits proliferation. Hcm1 functions as a rheostat, whose phosphorylation state affects the rate of proliferation., Cyclin-dependent kinase (Cdk1) orchestrates progression through the cell cycle by coordinating the activities of cell-cycle regulators. Although phosphatases that oppose Cdk1 are likely to be necessary to establish dynamic phosphorylation, specific phosphatases that target most Cdk1 substrates have not been identified. In budding yeast, the transcription factor Hcm1 activates expression of genes that regulate chromosome segregation and is critical for maintaining genome stability. Previously we found that Hcm1 activity and degradation are stimulated by Cdk1 phosphorylation of distinct clusters of sites. Here we show that, upon exposure to environmental stress, the phosphatase calcineurin inhibits Hcm1 by specifically removing activating phosphorylations and that this regulation is important for cells to delay proliferation when they encounter stress. Our work identifies a mechanism by which proliferative signals from Cdk1 are removed in response to stress and suggests that Hcm1 functions as a rheostat that integrates stimulatory and inhibitory signals to control cell proliferation.

During mitosis chromosomes are condensed to facilitate their segregation, through a process mediated by the condensin complex. Although several factors that promote maximal condensin activity during mitosis have been identified, the mechanisms that downregulate condensin activity during interphase are largely unknown. Here, we demonstrate that Ycg1, the Cap-G subunit of budding yeast condensin, is cell cycle-regulated with levels peaking in mitosis and decreasing as cells enter G1 phase. This cyclical expression pattern is established by a combination of cell cycle-regulated transcription and constitutive degradation. Interestingly, overexpression of YCG1 and mutations that stabilize Ycg1 each result in delayed cell-cycle entry and an overall proliferation defect. Overexpression of no other condensin subunit impacts the cell cycle, suggesting that Ycg1 is limiting for condensin complex formation. Consistent with this possibility, we find that levels of intact condensin complex are reduced in G1 phase compared to mitosis, and that increased Ycg1 expression leads to increases in both levels of condensin complex and binding to chromatin in G1. Together, these results demonstrate that Ycg1 levels limit condensin function in interphase cells, and suggest that the association of condensin with chromosomes must be reduced following mitosis to enable efficient progression through the cell cycle., Author Summary Chromosome conformation is cell cycle-regulated so that chromosomes are highly compacted to facilitate their segregation during mitosis, and decondensed during interphase to facilitate DNA-dependent processes such as replication and transcription. Understanding how chromosomes transition between these different states is important for understanding how cells maintain a stable genome. The condensin complex is an essential five-subunit complex that controls chromosome condensation in all eukaryotes. In this study, we show that expression of the Cap-G/Ycg1 subunit of condensin in budding yeast is cell cycle-regulated, and that its reduced expression during interphase limits condensin function. When this regulation is disrupted, and Ycg1 is constitutively expressed, progression through interphase is delayed. Emerging evidence indicates that individual condensin subunits are also expressed at limiting levels in metazoan cells, which suggests that cell-cycle regulation of an individual condensin subunit is a conserved mechanism that coordinates condensin function with the cell cycle.