Hog1 activation delays mitotic exit via phosphorylation of Net1.

Adaptation to environmental changes is crucial for cell fitness. In Saccharomyces cerevisiae, variations in external osmolarity trigger the activation of the stress-activated protein kinase Hog1 (high-osmolarity glycerol 1), which regulates gene expression, metabolism, and cell-cycle progression. The activation of this kinase leads to the regulation of G1, S, and G2 phases of the cell cycle to prevent genome instability and promote cell survival. Here we show that Hog1 delays mitotic exit when cells are stressed during metaphase. Hog1 phosphorylates the nucleolar protein Net1, altering its affinity for the phosphatase Cdc14, whose activity is essential for mitotic exit and completion of the cell cycle. The untimely release of Cdc14 from the nucleolus upon activation of Hog1 is linked to a defect in ribosomal DNA (rDNA) and telomere segregation, and it ultimately delays cell division. A mutant of Net1 that cannot be phosphorylated by Hog1 displays reduced viability upon osmostress. Thus, Hog1 contributes to maximizing cell survival upon stress by regulating mitotic exit.

A novel mechanism for the prevention of transcription replication conflicts.

Transcription and replication complexes can coincide in space and time. Such coincidences may result in collisions that trigger genomic instability. The phosphorylation of Mrc1 by different signaling kinases is part of a general mechanism that serves to delay replication in response to different stresses that trigger a massive transcriptional response in S phase. This mechanism prevents Transcription-Replication Conflicts and maintains genomic integrity in response to unscheduled massive transcription during S phase.

Multiple signaling kinases target Mrc1 to prevent genomic instability triggered by transcription-replication conflicts.

Conflicts between replication and transcription machineries represent a major source of genomic instability and cells have evolved strategies to prevent such conflicts. However, little is known regarding how cells cope with sudden increases of transcription while replicating. Here, we report the existence of a general mechanism for the protection of genomic integrity upon transcriptional outbursts in S phase that is mediated by Mrc1. The N-terminal phosphorylation of Mrc1 blocked replication and prevented transcription-associated recombination (TAR) and genomic instability during stress-induced gene expression in S phase. An unbiased kinome screening identified several kinases that phosphorylate Mrc1 at the N terminus upon different environmental stresses. Mrc1 function was not restricted to environmental cues but was also required when unscheduled transcription was triggered by low fitness states such as genomic instability or slow growth. Our data indicate that Mrc1 integrates multiple signals, thereby defining a general safeguard mechanism to protect genomic integrity upon transcriptional outbursts.

Dealing with transcriptional outbursts during S phase to protect genomic integrity.

Transcription during S phase needs to be spatially and temporally regulated to prevent collisions between the transcription and replication machineries. Cells have evolved a number of mechanisms to make both processes compatible under normal growth conditions. When conflict management fails, the head-on encounter between RNA and DNA polymerases results in genomic instability unless conflict resolution mechanisms are activated. Nevertheless, there are specific situations in which cells need to dramatically change their transcriptional landscape to adapt to environmental challenges. Signal transduction pathways, such as stress-activated protein kinases (SAPKs), serve to regulate gene expression in response to environmental insults. Prototypical members of SAPKs are the yeast Hog1 and mammalian p38. In response to stress, p38/Hog1 SAPKs control transcription and also regulate cell cycle progression. When yeast cells are stressed during S phase, Hog1 promotes gene induction and, remarkably, also delays replication by directly affecting early origin firing and fork progression. Therefore, by delaying replication, Hog1 plays a key role in preventing conflicts between RNA and DNA polymerases. In this review, we focus on the genomic determinants and mechanisms that make compatible transcription with replication during S phase to prevent genomic instability, especially in response to environmental changes.

Coordinated control of replication and transcription by a SAPK protects genomic integrity.

Upon environmental changes or extracellular signals, cells are subjected to marked changes in gene expression. Dealing with high levels of transcription during replication is critical to prevent collisions between the transcription and replication pathways and avoid recombination events. In response to osmostress, hundreds of stress-responsive genes are rapidly induced by the stress-activated protein kinase (SAPK) Hog1 (ref. 6), even during S phase. Here we show in Saccharomyces cerevisae that a single signalling molecule, Hog1, coordinates both replication and transcription upon osmostress. Hog1 interacts with and phosphorylates Mrc1, a component of the replication complex. Phosphorylation occurs at different sites to those targeted by Mec1 upon DNA damage. Mrc1 phosphorylation by Hog1 delays early and late origin firing by preventing Cdc45 loading, as well as slowing down replication-complex progression. Regulation of Mrc1 by Hog1 is completely independent of Mec1 and Rad53. Cells carrying a non-phosphorylatable allele of MRC1 (mrc1(3A)) do not delay replication upon stress and show a marked increase in transcription-associated recombination, genomic instability and Rad52 foci. In contrast, mrc1(3A) induces Rad53 and survival in the presence of hydroxyurea or methyl methanesulphonate. Therefore, Hog1 and Mrc1 define a novel S-phase checkpoint independent of the DNA-damage checkpoint that permits eukaryotic cells to prevent conflicts between DNA replication and transcription, which would otherwise lead to genomic instability when both phenomena are temporally coincident.

The Hog1 SAPK controls the Rtg1/Rtg3 transcriptional complex activity by multiple regulatory mechanisms.

Cells modulate expression of nuclear genes in response to alterations in mitochondrial function, a response termed retrograde (RTG) regulation. In budding yeast, the RTG pathway relies on Rtg1 and Rtg3 basic helix-loop-helix leucine Zipper transcription factors. Exposure of yeast to external hyperosmolarity activates the Hog1 stress-activated protein kinase (SAPK), which is a key player in the regulation of gene expression upon stress. Several transcription factors, including Sko1, Hot1, the redundant Msn2 and Msn4, and Smp1, have been shown to be directly controlled by the Hog1 SAPK. The mechanisms by which Hog1 regulates their activity differ from one to another. In this paper, we show that Rtg1 and Rtg3 transcription factors are new targets of the Hog1 SAPK. In response to osmostress, RTG-dependent genes are induced in a Hog1-dependent manner, and Hog1 is required for Rtg1/3 complex nuclear accumulation. In addition, Hog1 activity regulates Rtg1/3 binding to chromatin and transcriptional activity. Therefore Hog1 modulates Rtg1/3 complex activity by multiple mechanisms in response to stress. Overall our data suggest that Hog1, through activation of the RTG pathway, contributes to ensure mitochondrial function as part of the Hog1-mediated osmoadaptive response.

The p38 and Hog1 SAPKs control cell cycle progression in response to environmental stresses.

In response to environmental stresses, cells need to activate an adaptive program to maximize cell progression and survival. Stress-activated protein kinases (SAPK) are key signal transduction kinases required to respond to stress. Prototypical members of SAPKs are the yeast Hog1 and mammalian p38. Upon stress, those enzymes play a critical role in mounting the adaptive responses to stress such as the regulation of metabolism and the control of gene expression. In addition, a major function of SAPKs in response to stress is to modulate cell cycle progression. In this review, we focus on the role of Hog1 and p38 in the control of cell cycle progression in response to environmental stresses.

Time-dependent quantitative multicomponent control of the G1-S network by the stress-activated protein kinase Hog1 upon osmostress.

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to hyperosmotic stress activates the SAPK Hog1, which delays cell cycle progression through G1 by direct phosphorylation of the cyclin-dependent kinase (CDK) inhibitor Sic1 and by inhibition of the transcription of the genes encoding the G1 cyclins Cln1 and 2. Additional targets of Hog1 may also play a role in this response. We used mathematical modeling and quantitative in vivo experiments to define the contributions of individual components of the G1-S network downstream of Hog1 to this stress-induced delay in the cell cycle. The length of the arrest depended on the degree of stress and the temporal proximity of the onset of the stress to the commitment to cell division, called "Start." Hog1-induced inhibition of the transcription of the gene encoding cyclin Clb5, rather than that of the gene encoding Cln2, prevented entry into S phase upon osmostress. By controlling the accumulation of specific cyclins, Hog1 delayed bud morphogenesis (through Clns) and delayed DNA replication (through Clb5). Hog1-mediated phosphorylation and degradation of Sic1 at Start prevented residual activity of the cyclin/CDK complex Clb5/Cdc28 from initiating DNA replication before adaptation to the stress. Thus, our work defines distinct temporal roles for the actions of Hog1 on Sic1 and cyclins in mediating G1 arrest upon hyperosmotic stress.

A Dbf4 mutant contributes to bypassing the Rad53-mediated block of origins of replication in response to genotoxic stress.

An intra-S phase checkpoint slows the rate of DNA replication in response to DNA damage and replication fork blocks in eukaryotic cells. In the budding yeast Saccharomyces cerevisiae, such down-regulation is achieved through the Rad53 kinase-dependent block of origins of replication. We have identified the Rad53 phosphorylation sites on Dbf4, the activator subunit of the essential S phase Dbf4-dependent kinase, and generated a non-phosphorylatable Dbf4 mutant (dbf4(7A)). We show here that dbf4(7A) is a bona fide intra-S phase checkpoint bypass allele that contributes to abrogating the Rad53 block of origin firing in response to genotoxic stress.

Cyclin regulation by the s phase checkpoint.

In eukaryotic cells a surveillance mechanism, the S phase checkpoint, detects and responds to DNA damage and replication stress, protecting DNA replication and arresting cell cycle progression. We show here that the S phase cyclins Clb5 and Clb6 are regulated in response to genotoxic stress in the budding yeast Saccharomyces cerevisiae. Clb5 and Clb6 are responsible for the activation of the specific Cdc28 cyclin-dependent kinase activity that drives the onset and progression of the S phase. Intriguingly, Clb5 and Clb6 are regulated by different mechanisms. Thus, the presence of Clb6, which is eliminated early in an unperturbed S phase, is stabilized when replication is compromised by replication stress or DNA damage. Such stabilization depends on the checkpoint kinases Mec1 and Rad53. The stabilization of Clb6 levels is a dynamic process that requires continued de novo protein synthesis, because the cyclin remains subject to degradation. It also requires the activity of the G(1) transcription factor Mlu1 cell cycle box-binding factor (MBF) in the S phase, whereas Dun1, the checkpoint kinase characteristically responsible for the transcriptional response to genotoxic stress, is dispensable in this case. On the other hand, two subpopulations of endogenous Clb5 can be distinguished according to turnover in an unperturbed S phase. In the presence of replication stress, the unstable Clb5 pool is stabilized, and such stabilization requires neither MBF transcriptional activity nor de novo protein synthesis.

The stress-activated protein kinase Hog1 mediates S phase delay in response to osmostress.

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress activates the Hog1 SAPK, which modulates cell cycle progression at G1 and G2 by the phosphorylation of elements of the cell cycle machinery, such as Sic1 and Hsl1, and by down-regulation of G1 and G2 cyclins. Here, we show that upon stress, Hog1 also modulates S phase progression. The control of S phase is independent of the S phase DNA damage checkpoint and of the previously characterized Hog1 cell cycle targets Sic1 and Hsl1. Hog1 uses at least two distinct mechanisms in its control over S phase progression. At early S phase, the SAPK prevents firing of replication origins by delaying the accumulation of the S phase cyclins Clb5 and Clb6. In addition, Hog1 prevents S phase progression when activated later in S phase or cells containing a genetic bypass for cyclin-dependent kinase activity. Hog1 interacts with components of the replication complex and delays phosphorylation of the Dpb2 subunit of the DNA polymerase. The two mechanisms of Hog1 action lead to delayed firing of origins and prolonged replication, respectively. The Hog1-dependent delay of replication could be important to allow Hog1 to induce gene expression before replication.

Keratin 7 promoter selectively targets transgene expression to normal and neoplastic pancreatic ductal cells in vitro and in vivo.

Keratin 7 is expressed in simple epithelia but is expressed at low or undetectable levels in gastrointestinal epithelial cells. In the pancreas, it is present in ductal but not in acinar cells. K7 mRNA is overexpressed in pancreatic cancers. Here we use luciferase reporter assays to analyze the tissue-specific regulatory elements of murine keratin 7 (Krt7) promoter in vitro and in vivo. All elements required for appropriate cell and tissue specificity in reporter assays are present within the Krt7 -234 bp sequence. This fragment appears more selective to pancreatic ductal cells than the Krt19 promoter. GC-rich sequences corresponding to putative Sp1, AP-2 binding sites are essential for in vitro activity. Krt7-LacZ transgenic mice were generated to analyze in vivo activity. Sequences located 1.5 or 0.25 kb upstream of the transcription initiation site drive reporter expression to ductal, but not acinar, cells in transgenic mice. LacZ mRNA was detected in the pancreas as well as in additional epithelial tissues--such as the intestine and the lung--using both promoter constructs. An AdK7Luc adenovirus was generated to assess targeting selectivity in vivo by intravenous injection to immunocompetent mice and in a xenograft model of pancreatic cancer. The -0.25 kb region showed pancreatic selectivity, high activity in pancreatic cancers, and sustained transgene expression in xenografts. In conclusion, the krt7 promoter is useful to target pancreatic ductal adenocarcinoma cells in vitro and in vivo.

Distinct phosphatases mediate the deactivation of the DNA damage checkpoint kinase Rad53.

The DNA damage checkpoint regulates DNA replication and arrests cell cycle progression in response to genotoxic stress. In Saccharomyces cerevisiae, the protein kinase Rad53 plays a central role in preventing genomic instability and maintaining viability in the presence of replication stress and DNA damage. Activation of Rad53 depends on phosphorylation by the upstream kinase Mec1, followed by autophosphorylation on multiple residues. Also critical for cell viability, the molecular mechanism of Rad53 deactivation remains incompletely understood. Rad53 dephosphorylation after repair of a persistent double strand break in G(2)/M has been shown to depend on the presence of the PP2C-type phosphatases Ptc2 and Ptc3. More recently, the PP2A-like protein phosphatase Pph3 has been shown to be required to dephosphorylate Rad53 after DNA methylation damage in S phase. However, we show here that Ptc2/3 are dispensable for Rad53 deactivation after replication stress or DNA methylation damage. Pph3 is also dispensable for the deactivation of Rad53 after replication stress. In addition, Rad53 kinase activity is still deactivated in pph3 null cells after DNA methylation damage, despite persistent Rad53 hyperphosphorylation. Finally, a strain in which the three phosphatases are deleted shows a severe defect in Rad53 kinase deactivation after DNA methylation damage but not after replication stress. In all, our results suggest that distinct phosphatases operate to return Rad53 to its basal state after different genotoxic stresses and that a yet unidentified phosphatase may be responsible for the deactivation of Rad53 after replication stress.