Nascent RNA signaling to yeast RNA Pol II during transcription elongation.

Transcription as the key step in gene expression is a highly regulated process. The speed of transcription elongation depends on the underlying gene sequence and varies on a gene by gene basis. The reason for this sequence dependence is not known in detail. Recently, our group studied the cross talk between the nascent RNA and the transcribing RNA polymerase by screening the Escherichia coli genome for RNA sequences with high affinity to RNA Pol by performing genomic SELEX. This approach led to the identification of RNA polymerase-binding APtamers termed "RAPs". RAPs can have positive and negative effects on gene expression. A subgroup is able to downregulate transcription via the activity of the termination factor Rho. In this study, we used a similar SELEX setup using yeast genomic DNA as source of RNA sequences and highly purified yeast RNA Pol II as bait and obtained almost 1300 yeast-derived RAPs. Yeast RAPs are found throughout the genome within genes and antisense to genes, they are overrepresented in the non-transcribed strand of yeast telomeres and underrepresented in intergenic regions. Genes harbouring a RAP are more likely to show lower mRNA levels. By determining the endogenous expression levels as well as using a reporter system, we show that RAPs located within coding regions can reduce the transcript level downstream of the RAP. Here we demonstrate that RAPs represent a novel type of regulatory RNA signal in Saccharomyces cerevisiae that act in cis and interfere with the elongating transcription machinery to reduce the transcriptional output.

INO80 represses osmostress induced gene expression by resetting promoter proximal nucleosomes.

The conserved INO80 chromatin remodeling complex is involved in regulation of DNA damage repair, replication and transcription. It is commonly recruited to the transcription start region and contributes to the establishment of promoter-proximal nucleosomes. We find a substantial influence of INO80 on nucleosome dynamics and gene expression during stress induced transcription. Transcription induced by osmotic stress leads to genome-wide remodeling of promoter proximal nucleosomes. INO80 function is required for timely return of evicted nucleosomes to the 5΄ end of induced genes. Reduced INO80 function in Arp8-deficient cells leads to correlated prolonged transcription and nucleosome eviction. INO80 and the related complex SWR1 regulate incorporation of the H2A.Z isoform at promoter proximal nucleosomes. However, H2A.Z seems not to influence osmotic stress induced gene regulation. Furthermore, we show that high rates of transcription promote INO80 recruitment to promoter regions, suggesting a connection between active transcription and promoter proximal nucleosome remodeling. In addition, we find that absence of INO80 enhances bidirectional promoter activity at highly induced genes and expression of a number of stress induced transcripts. We suggest that INO80 has a direct repressive role via promoter proximal nucleosome remodeling to limit high levels of transcription in yeast.

Ribosome quality control is a central protection mechanism for yeast exposed to deoxynivalenol and trichothecin.

BACKGROUND: The trichothecene mycotoxins deoxynivalenol (DON) and trichothecin (TTC) are inhibitors of eukaryotic protein synthesis. Their effect on cellular homeostasis is poorly understood. We report a systematic functional investigation of the effect of DON and TTC on the yeast Saccharomyces cerevisiae using genetic array, network and microarray analysis. To focus the genetic analysis on intracellular consequences of toxin action we eliminated the PDR5 gene coding for a potent pleiotropic drug efflux protein potentially confounding results. We therefore used a knockout library with a pdr5Δ strain background. RESULTS: DON or TTC treatment creates a fitness bottleneck connected to ribosome efficiency. Genes isolated by systematic genetic array analysis as contributing to toxin resistance function in ribosome quality control, translation fidelity, and in transcription. Mutants in the E3 ligase Hel2, involved in ribosome quality control, and several members of the Rpd3 histone deacetylase complex were highly sensitive to DON. DON and TTC have similar genetic profiles despite their different toxic potency. Network analysis shows a coherent and tight network of genetic interactions among the DON and TTC resistance conferring gene products. The networks exhibited topological properties commonly associated with efficient processing of information. Many sensitive mutants have a "slow growth" gene expression signature. DON-exposed yeast cells increase transcripts of ribosomal protein and histone genes indicating an internal signal for growth enhancement. CONCLUSIONS: The combination of gene expression profiling and analysis of mutants reveals cellular pathways which become bottlenecks under DON and TTC stress. These are generally directly or indirectly connected to ribosome biosynthesis such as the general secretory pathway, cytoskeleton, cell cycle delay, ribosome synthesis and translation quality control. Gene expression profiling points to an increased demand of ribosomal components and does not reveal activation of stress pathways. Our analysis highlights ribosome quality control and a contribution of a histone deacetylase complex as main sources of resistance against DON and TTC.

Sorbic acid stress activates the Candida glabrata high osmolarity glycerol MAP kinase pathway.

Weak organic acids such as sorbic acid are important food preservatives and powerful fungistatic agents. These compounds accumulate in the cytosol and disturb the cellular pH and energy homeostasis. Candida glabrata is in many aspects similar to Saccharomyces cerevisiae. However, with regard to confrontation to sorbic acid, two of the principal response pathways behave differently in C. glabrata. In yeast, sorbic acid stress causes activation of many genes via the transcription factors Msn2 and Msn4. The C. glabrata homologs CgMsn2 and CgMsn4 are apparently not activated by sorbic acid. In contrast, in C. glabrata the high osmolarity glycerol (HOG) pathway is activated by sorbic acid. Here we show that the MAP kinase of the HOG pathway, CgHog1, becomes phosphorylated and has a function for weak acid stress resistance. Transcript profiling of weak acid treated C. glabrata cells suggests a broad and very similar response pattern of cells lacking CgHog1 compared to wild type which is over lapping with but distinct from S. cerevisiae. The PDR12 gene was the highest induced gene in both species and it required CgHog1 for full expression. Our results support flexibility of the response cues for general stress signaling pathways, even between closely related yeasts, and functional extension of a specific response pathway.

Yeast protein phosphatase 2A-Cdc55 regulates the transcriptional response to hyperosmolarity stress by regulating Msn2 and Msn4 chromatin recruitment.

We have identified Cdc55, a regulatory B subunit of protein phosphatase 2A (PP2A), as an essential activating factor for stress gene transcription in Saccharomyces cerevisiae. The presence of PP2A-Cdc55 is required for full activation of the environmental stress response mediated by the transcription factors Msn2 and Msn4. We show that PP2A-Cdc55 contributes to sustained nuclear accumulation of Msn2 and Msn4 during hyperosmolarity stress. PP2A-Cdc55 also enhances Msn2-dependent transactivation, required for extended chromatin recruitment of the transcription factor. We analyzed a possible direct regulatory role for PP2A-Cdc55 on the phosphorylation status of Msn2. Detailed mass spectrometric and genetic analysis of Msn2 showed that stress exposure causes immediate transient dephosphorylation of Msn2 which is not dependent on PP2A-Cdc55 activity. Furthermore, the Hog1 mitogen-activated protein kinase pathway activity is not influenced by PP2A-Cdc55. We therefore propose that the PP2A-Cdc55 phosphatase is not involved in cytosolic stress signal perception but is involved in a specific intranuclear mechanism to regulate Msn2 and Msn4 nuclear accumulation and chromatin association under stress conditions.

Interplay of dynamic transcription and chromatin remodeling: lessons from yeast.

Regulation of transcription involves dynamic rearrangements of chromatin structure. The budding yeast Saccharomyces cerevisiae has a variety of highly conserved factors necessary for these reconstructions. Chromatin remodelers, histone modifiers and histone chaperones directly associate to promoters and open reading frames of exposed genes and facilitate activation and repression of transcription. We compare two distinct patterns of induced transcription: Sustained transcribed genes switch to an activated state where they remain as long as the induction signal is present. In contrast, single pulsed transcribed genes show a quick and strong induction pulse resulting in high transcript levels followed by adaptation and repression to basal levels. We discuss intensively studied promoters and coding regions from both groups for their co-factor requirements during transcription. Interplay between chromatin restructuring factors and dynamic transcription is highly variable and locus dependent.

Regulation of Candida glabrata oxidative stress resistance is adapted to host environment.

The human fungal pathogen Candida glabrata is related to Saccharomyces cerevisiae but has developed high resistance against reactive oxygen species. We find that induction of conserved genes encoding antioxidant functions is dependent on the transcription factors CgYap1 and CgSkn7 which cooperate for promoter recognition. Superoxide stress resistance of C. glabrata is provided by superoxide dismutase CgSod1, which is not dependent on CgYap1/Skn7. Only double mutants lacking both CgSod1 and CgYap1 were efficiently killed by primary mouse macrophages. Our results suggest that in C. glabrata the regulation of key genes providing stress protection is adopted to meet a host-pathogen situation.

Cooperation between the INO80 complex and histone chaperones determines adaptation of stress gene transcription in the yeast Saccharomyces cerevisiae.

In yeast, environmental stresses provoke sudden and dramatic increases in gene expression at stress-inducible loci. Stress gene transcription is accompanied by the transient eviction of histones from the promoter and the transcribed regions of these genes. We found that mutants defective in subunits of the INO80 complex, as well as in several histone chaperone systems, exhibit extended expression windows that can be correlated with a distinct delay in histone redeposition during adaptation. Surprisingly, Ino80 became associated with the ORFs of stress genes in a stress-specific way, suggesting a direct function in the repression during adaptation. This recruitment required elongation by RNA polymerase (Pol) II but none of the histone modifications that are usually associated with active transcription, such as H3 K4/K36 methylation. A mutant lacking the Asf1-associated H3K56 acetyltransferase Rtt109 or Asf1 itself also showed enhanced stress-induced transcript levels. Genetic data, however, suggest that Asf1 and Rtt109 function in parallel with INO80 to restore histone homeostasis, whereas Spt6 seems to have a function that overlaps that of the chromatin remodeler. Thus, chromatin remodeling by INO80 in cooperation with Spt6 determines the shape of the expression profile under acute stress conditions, possibly by an elongation-dependent mechanism.