Selective requirement for SAGA in Hog1-mediated gene expression depending on the severity of the external osmostress conditions.

Regulation of gene expression by the Hog1 stress-activated protein kinase is essential for proper cell adaptation to osmostress. Hog1 coordinates an extensive transcriptional program through the modulation of transcription. To identify systematically novel components of the transcriptional machinery required for osmostress-mediated gene expression, we performed an exhaustive genome-wide genetic screening, searching for mutations that render cells osmosensitive at high osmolarity and that are associated with reduced expression of osmoresponsive genes. The SAGA and Mediator complexes were identified as putative novel regulators of osmostress-mediated transcription. Interestingly, whereas Mediator is essential for osmostress gene expression, the requirement for SAGA is different depending on the strength of the extracellular osmotic conditions. At mild osmolarity, SAGA mutants show only very slight defects on RNA polymerase II (Pol II) recruitment and gene expression, whereas at severe osmotic conditions, SAGA mutants show completely impaired RNA Pol II recruitment and transcription of osmoresponsive genes. Thus, our results define an essential role for Mediator in osmostress gene expression and a selective role for SAGA under severe osmostress. Our results indicate that the requirement for a transcriptional complex to regulate a promoter might be determined by the strength of the stimuli perceived by the cell through the regulation of interactions between transcriptional complexes.

The mRNA export factor Sus1 is involved in Spt/Ada/Gcn5 acetyltransferase-mediated H2B deubiquitinylation through its interaction with Ubp8 and Sgf11.

Sus1 acts in nuclear mRNA export via its association with the nuclear pore-associated Sac3-Thp1-Cdc31 complex. In addition, Sus1 plays a role in transcription through its interaction with the Spt/Ada/Gcn5 acetyltransferase (SAGA) complex. Here, we have analyzed function and interaction of Sus1 within the SAGA complex. We demonstrate that Sus1 is involved in the SAGA-dependent histone H2B deubiquitinylation and maintenance of normal H3 methylation levels. By deletion analyses, we show that binding of Sus1 to SAGA depends on the deubiquitinylating enzyme Ubp8 and Sgf11. Moreover, a stable subcomplex between Sus1, Sgf11, and Ubp8 could be dissociated from SAGA under high salt conditions. In vivo recruitment of Sus1 to the activated GAL1 promoter depends on Ubp8 and vice versa. In addition, histones coenrich during SAGA purification in a Sus1-Sgf11-Ubp8-dependent way. Interestingly, sgf11 deletion enhances the mRNA export defect observed in sus1delta cells. Thus, the Sus1-Sgf11-Ubp8 module could work at the junction between SAGA-dependent transcription and nuclear mRNA export.

Control of cell cycle progression by the stress-activated Hog1 MAPK.

Activation of the stress-activated Hog1 kinase is essential for cell survival in response to high osmolarity. The Hog1 SAPK elicits the program for cell adaptation that includes modulation of several aspects of cell biology, such as, gene expression, translation and cell-cycle progression. In response to stress, the Hog1 SAPK mediates a transient cell cycle arrest required for proper cell adaptation. A recent report has described the mechanism underlying the effect of the SAPK at G1. Here, we propose that the mechanisms that involve cell cycle control in response to osmostress are of great complexity and that they could be conserved among eukaryotic cells.

Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1.

Activation of stress-activated protein kinases (SAPKs) is essential for proper cell adaptation to extracellular stimuli. The exposure of yeast cells to high osmolarity, or mutations that lead to activation of the Hog1 SAPK, result in cell-cycle arrest. The mechanisms by which Hog1 and SAPKs in general regulate cell-cycle progression are not completely understood. Here we show that Hog1 regulates cell cycle progression at the G1 phase by a dual mechanism that involves downregulation of cyclin expression and direct targeting of the CDK-inhibitor protein Sic1. Hog1 interacts physically with Sic1 in vivo and in vitro, and phosphorylates a single residue at the carboxyl terminus of Sic1, which, in combination with the downregulation of cyclin expression, results in Sic1 stabilization and inhibition of cell-cycle progression. Cells lacking Sic1 or containing a Sic1 allele mutated in the Hog1 phosphorylation site are unable to arrest at G1 phase after Hog1 activation, and become sensitive to osmostress. Together, our data indicate that the Sic1 CDK-inhibitor is the molecular target for the SAPK Hog1 that is required to modulate cell-cycle progression in response to stress.

The MAPK Hog1 recruits Rpd3 histone deacetylase to activate osmoresponsive genes.

Regulation of gene expression by mitogen-activated protein kinases (MAPKs) is essential for proper cell adaptation to extracellular stimuli. Exposure of yeast cells to high osmolarity results in rapid activation of the MAPK Hog1, which coordinates the transcriptional programme required for cell survival on osmostress. The mechanisms by which Hog1 and MAPKs in general regulate gene expression are not completely understood, although Hog1 can modify some transcription factors. Here we propose that Hog1 induces gene expression by a mechanism that involves recruiting a specific histone deacetylase complex to the promoters of genes regulated by osmostress. Cells lacking the Rpd3-Sin3 histone deacetylase complex are sensitive to high osmolarity and show compromised expression of osmostress genes. Hog1 interacts physically with Rpd3 in vivo and in vitro and, on stress, targets the deacetylase to specific osmostress-responsive genes. Binding of the Rpd3-Sin3 complex to specific promoters leads to histone deacetylation, entry of RNA polymerase II and induction of gene expression. Together, our data indicate that targeting of the Rpd3 histone deacetylase to osmoresponsive promoters by the MAPK Hog1 is required to induce gene expression on stress.

Sources and concentrations of indoor nitrogen dioxide in Barcelona, Spain.

Sources and concentrations of indoor nitrogen dioxide (NO2) were examined in Barcelona, Spain, during 1996-1999. A total of 340 dwellings of infants participating in a hospital-based cohort study were selected from different areas of the city. Passive filter badges were used for indoor NO2 measurement over 7-30 days. Dwelling inhabitants completed a questionnaire on housing characteristics and smoking habits. Data on outdoor NO2 concentrations were available for the entire period of the study in the areas of the city where indoor concentrations were determined. Bivariate analysis was performed to investigate relationships between indoor NO2 concentrations on one hand and outdoor NO2 concentrations, housing, and occupant characteristics on the other. Stepwise multiple linear regression was performed with variables that were found to have a significant bivariate relationship. Indoor NO2 mean values ranged between 23.57 ppb in 1996 and 27.02 ppb in 1999, with the highest yearly value of 27.82 ppb in 1997. In the same time period, mean outdoor NO2 concentration ranged between 25.26 and 25.78 ppb with a peak of 30.5 ppb in 1998. Multiple regression analysis showed that principal sources of indoor NO2 concentrations were the use of a gas cooker, the absence of an extractor fan when cooking, and cigarette smoking. The absence of central heating was also associated with higher NO2 concentrations. Finally, each ppb increase in outdoor NO2 was associated with a 1% increase in indoor concentrations.

Osmostress-induced transcription by Hot1 depends on a Hog1-mediated recruitment of the RNA Pol II.

In budding yeast, the mitogen-activated protein kinase (MAPK) Hog1 coordinates the transcriptional program required for cell survival upon osmostress. The Hot1 transcription factor acts downstream of the MAPK and regulates a subset of Hog1-responsive genes. In response to high osmolarity, Hot1 targets Hog1 to specific osmostress-responsive promoters. Here, we show that assembly of the general transcription machinery at Hot1-dependent promoters depends on the presence of Hot1 and active Hog1 MAPK. Unexpectedly, recruitment of RNA polymerase (Pol) II complex to target promoters does not depend on the phosphorylation of the Hot1 activator by the MAPK. Hog1 interacts with the RNA Pol II and with general components of the transcription machinery. More over, when tethered to a promoter as a LexA fusion protein, Hog1 activates transcription in a stress- regulated manner. Thus, anchoring of active Hog1 to promoters by the Hot1 activator is essential for recruitment and activation of RNA Pol II. The mammalian p38 also interacts with the RNA Pol II, which might suggest a conserved mechanism for regulation of gene expression by SAPKs among eukaryotic cells.