Nuclear Localization of Hog1 MAPK is Not Necessary for Resistance to Hyperosmotic Stress
Ontology highlight
ABSTRACT: In budding yeast, this signaling pathway— the high-osmolarity glycerol (HOG) response —culminates in dual phosphorylation and nuclear translocation of the MAPK, Hog1 (ortholog of mammalian p38/SAPK). Induction of at least 50 genes requires nuclear Hog1, implying that transcriptional up-regulation is necessary to cope with hyperosmotic stress. Contrary to this expectation, we found that cells lacking the karyopherin (Nmd5) required for Hog1 nuclear import or in which Hog1 was permanently anchored at the plasma membrane(HOG1-CCAAX) (or both) withstood hyperosmotic challenge by three different solutes (1 M sorbitol, KCl or NaCl). In cells where activated Hog1 is excluded from the nucleus, there was little change in transcriptional program after exposure to hyperosmotic shock (comparable to hog1∆ cells), as judged by examining several diagnostic mRNAs and by global transcript measurements using microarrays. Systematic genetic analysis ruled out the need for any transcription factor known to be influenced by Hog1 (Hot1, Msn2, Msn4, Sko1 and Smp1). Keywords: Time course of stress response gene expression array
Project description:In budding yeast, this signaling pathway— the high-osmolarity glycerol (HOG) response —culminates in dual phosphorylation and nuclear translocation of the MAPK, Hog1 (ortholog of mammalian p38/SAPK). Induction of at least 50 genes requires nuclear Hog1, implying that transcriptional up-regulation is necessary to cope with hyperosmotic stress. Contrary to this expectation, we found that cells lacking the karyopherin (Nmd5) required for Hog1 nuclear import or in which Hog1 was permanently anchored at the plasma membrane(HOG1-CCAAX) (or both) withstood hyperosmotic challenge by three different solutes (1 M sorbitol, KCl or NaCl). In cells where activated Hog1 is excluded from the nucleus, there was little change in transcriptional program after exposure to hyperosmotic shock (comparable to hog1∆ cells), as judged by examining several diagnostic mRNAs and by global transcript measurements using microarrays. Systematic genetic analysis ruled out the need for any transcription factor known to be influenced by Hog1 (Hot1, Msn2, Msn4, Sko1 and Smp1). Keywords: Time course of stress response gene expression array The transcriptomes' of HOG1-GFP, hog1del, and HOG1-CCAAX strains before and after 60 min hyperosmotic shock with 1M sorbitol at 25C were compared. Three biological replicates were done, with the first biological replicate done in technical triplicate, and the final two biological replicates beind done in technical duplicate by in slide duplication of features. Several supplementary files attached to the Series are summarized below: GSE8703 B1, B2, B3 Tscombined_matrix files show the averages of the technical replicates for each biological replicate. GSE8703 B1, B2, B3 norm_to_0_matrix files show the fold change over the time course (log2 time 60 - log2 time 0) for each of the three strains in each biological replicate. GSE8703 Figure_S6 shows the 30 genes with the most significant difference between the HOG1-GFP strain and the hog1del strain as determined by SAM. Genes were identified as: >3 fold induction in HOG1-GFP over 60 minute hyperosmotic shock and <3 fold induction in hog1del over 60 minute hyperosmotic shock.
Project description:When challenged with osmotic shock, S. cerevisiae induces hundreds of genes, despite a global reduction in transcriptional capacity. The mechanisms that regulate this rapid reallocation of transcriptional resources are not known. Here we show that redistribution of RNA Pol II upon stress requires the stress-responsive MAP kinase Hog1. We find that Hog1 and RNA Pol II co-localize to open reading frames that bypass global transcriptional repression, and that these targets are specified by two osmotic stress-responsive transcription factors. The combination of reduced global transcription with a gene-specific override mechanism allows cells to rapidly switch their transcriptional program in response to stress. ChIP-sequencing of S. cervisiae RNA Pol II, Hog1, Sko1 and Hot1 Processed data file descriptions: ORFcounts.txt: table of summed ChIP-seq reads that align to each ORF (normalized by reads per kilobase per million) promoter_counts.txt: table of summed ChIP-seq reads that align to each promoter (1kb upstream, normalized by reads per kilobase per million) downstream_counts.txt: table of summed ChIP-seq reads that align 3' regions (50-500 bp downstream, normalized by reads per kilobase per million) Sko1_peak_list.txt: table of peaks found by PeakSeq Hot1_peak_list.txt: table of peaks found by PeakSeq
Project description:Yeast Hog1-3HA ChIP-Chip in triplicate Keywords: ChIP-Chip Yeast Hog1 ChIP-Chip was performed in MAP51 (3HA-Hog1) in triplicate using anti-HA (12CA5 monoclonal antibody)
Project description:Based on studies in S. pombe (Chen et al., 2003), we predicted that conditions which activate C. albicans Hog1 would result in the induction of a common set of genes that are regulated by this SAPK (Smith et al., 2004). Hence in this study we compared the global transcriptional responses of wild-type and hog1 C. albicans cells to environmental stresses that are known to activate the Hog1 SAPK. We compared the homozygous hog1/hog1 null mutant (JC50) with the isogenic HOG1 reintegrant (hog1/hog1/HOG1: JC52) because this controlled for any secondary mutations that might have been introduced during the construction of the null mutant. We have shown that this reintegrant is indistinguishable from its parental wild-type strain RM1000 (HOG1/HOG1) with respect to their stress phenotypes (Smith et al., 2004) and their expression of stress genes (Supplementary Data). Three conditions were chosen for transcript profiling: osmotic stress imposed by 0.3 M NaCl, oxidative stress imposed by 5 mM H2O2, and heavy metal stress imposed by 0.5 mM CdSO4. Each of these treatments stimulates the phosphorylation and nuclear accumulation of this Hog1 SAPK within a 10-min time frame (Smith et al., 2004). Furthermore, significant differences in stress regulated gene expression are observed within this time scale (Enjalbert et al., 2003; Smith et al., 2004). Hence, we analyzed the C. albicans transcriptome after a 10-min exposure to each stress condition. Although some stress genes might be missed by analyzing a single time point, most C. albicans stress genes are induced within 10 min (Enjalbert et al., 2003). At least four independent biological replicates were analyzed for each condition
Project description:The stress-activated protein kinase Hog1 is best known for its role in osmotic stress but is also activated by a variety of mechanistically distinct environmental stressors including heat shock, ER stress, and arsenic. In the osmotic stress response, the signal is sensed upstream and relayed to Hog1 via a kinase cascade. Here we identify a novel mode of Hog1 function whereby Hog1 senses arsenic though direct physical interaction requiring three conserved cysteine residues located adjacent to the catalytic loop. These residues are essential for Hog1's role in protecting against arsenic, but completely dispensable for osmotic stress, and mediate Hog1's cellular localization upon arsenic exposure. Hog1 in turn regulates arsenic detoxification by phosphorylating the transcription factor Yap8, promoting its nuclear localization, and stimulating transcription of its only known targets, Arr2 and Arr3, which promote arsenic efflux. Arsenic binding was also observed with the related human kinases Erk1 and Erk2, suggesting that this may be a conserved aspect of this kinase family. These data provide a mechanistic basis for understanding how stress-activated kinases can sense individual threats and carry out highly specific adaptive responses.
Project description:To monitor changes in the transcriptome following activation of Hog1 per se, we used a previously established inducible expression system of intrinsically active Hog1 proteins
Project description:The high osmolarity glycerol response (HOG) pathway plays a pivotal role in the stress response, virulence regulation, and differentiation of fungi, including Cryptococcus neoformans that causes fatal meningoencephalitis. Core signaling components of in the HOG pathway, including the Tco-Ypd1-Ssk1 phosphorelay system and the Ssk2-Pbs2-Hog1 MAPK module, have been elucidated but its downstream transcription factors remain unclear. Here we demonstrated that Atf1 with a basic leucine zipper domain is the transcription factor downstream of Hog1 in C. neoformans. We found that ATF1 expression was differentially regulated by oxidative damaging agents, mainly in a Hog1-dependent, but Mpk1-independent, manner. Interestingly, Atf1 not only promoted oxidative stress response and adaptation, but also played an opposing role to Hog1 in the process. Atf1 primarily localized to the nucleus under both unstressed and oxidative stress conditions in a Hog1-independent manner. Our data demonstrated that Atf1 promoted pheromone production and sexual differentiation under negative control by Hog1. Finally, a DNA microarray-based transcriptome analysis of the atf1∆ mutant under unstressed and oxidative stress conditions revealed that Atf1 regulated oxidative stress response genes, including a sulfiredoxin gene (SRX1). Intriguing, the array data further demonstrated that Atf1 modulated basal expression of genes involved in DNA repair and genotoxic stress response. Supporting this, we found that the atf1∆ mutant was highly sensitive to genotoxic agents. In conclusion, this study provided a further insight into the Hog1-dependent oxidative and genotoxic stress response and differentiation mechanism of C. neoformans.
Project description:We did transcription profiling on the effect of hog1 deletion, gene involved in cell osmotic stress and cell wall stress response Keywords: cell wall stress response