Distinct regions of ATF/CREB proteins Atf1 and Pcr1 control recombination hotspot ade6–M26 and the osmotic stress response

The Atf1 protein of Schizosaccharomyces pombe contains a bZIP (DNA-binding/protein dimerization) domain characteristic of ATF/CREB proteins, but no other functional domains or clear homologs have been reported. Atf1-containing, bZIP protein dimers bind to CRE-like DNA sites, regulate numerous stress responses, and activate meiotic recombination at hotspots like ade6–M26. We defined systematically the organization of Atf1 and its heterodimer partner Pcr1, which is required for a subset of Atf1-dependent functions. Surprisingly, only the bZIP domain of Pcr1 is required for hotspot activity and tethering of Atf1 to ade6 promotes recombination in the absence of its bZIP domain and the Pcr1 protein. Therefore the recombination–activation domain of Atf1-Pcr1 heterodimer resides exclusively in Atf1, and Pcr1 confers DNA-binding site specificity in vivo. Atf1 has a modular organization in which distinct regions affect differentially the osmotic stress response (OSA) and meiotic recombination (HRA, HRR). The HRA and HRR regions are necessary and sufficient to activate and repress recombination, respectively. Moreover, Atf1 defines a family of conserved proteins with discrete sequence motifs in the functional domains (OSA, HRA, HRR, bZIP). These findings reveal the functional organization of Atf1 and Pcr1, and illustrate several mechanisms by which bZIP proteins can regulate multiple, seemingly disparate activities

Deciphering the role of Yap4 phosphorylation under stress conditions

The existence of molecular mechanisms of response, repair and adaptation, many of which are greatly conserved across nature, gives to the cell with the plasticity it requires to adjust to its ever-changing environment, a homeostatic event that is termed the stress response. In the budding yeast Saccharomyces cerevisiae there is a particular family of transcription factors, the Yap family, which has been shown to have a relevant role in yeast adaptation to several stress conditions. In particular, Yap1 is the major regulator of the transcriptional response to oxidative stress and Yap2 and Yap8 play important roles upon cadmium and arsenic exposure, respectively.(...

PKA-chromatin association at stress responsive target genes from Saccharomyces cerevisiae

Gene expression regulation by intracellular stimulus-activated protein kinases is essential for cell adaptation to environmental changes. There are three PKA catalytic subunits in Saccharomyces cerevisiae: Tpk1, Tpk2, and Tpk3 and one regulatory subunit: Bcy1. Previously, it has been demonstrated that Tpk1 and Tpk2 are associated with coding regions and promoters of target genes in a carbon source and oxidative stress dependent manner. Here we studied five genes, ALD6, SED1, HSP42, RPS29B, and RPL1B whose expression is regulated by saline stress. We found that PKA catalytic and regulatory subunits are associated with both coding regions and promoters of the analyzed genes in a stress dependent manner. Tpk1 and Tpk2 recruitment was completely abolished in catalytic inactive mutants. BCY1 deletion changed the binding kinetic to chromatin of each Tpk isoform and this strain displayed a deregulated gene expression in response to osmotic stress. In addition, yeast mutants with high PKA activity exhibit sustained association to target genes of chromatin-remodeling complexes such as Snf2-catalytic subunit of the SWI/SNF complex and Arp8-component of INO80 complex, leading to upregulation of gene expression during osmotic stress. Tpk1 accumulation in the nucleus was stimulated upon osmotic stress, while the nuclear localization of Tpk2 and Bcy1 showed no change. We found that each PKA subunit is transported into the nucleus by a different β-karyopherin pathway. Moreover, β-karyopherin mutant strains abolished the chromatin association of Tpk1 or Tpk2, suggesting that nuclear localization of PKA catalytic subunits is required for its association to target genes and properly gene expression

The PKC, HOG and Ca2+ signalling pathways co-ordinately regulate chitin synthesis in Candida albicans

Open Access via PMC2649417Peer reviewedPublisher PD

cAMP/PKA signaling balances respiratory activity with mitochondria dependent apoptosis via transcriptional regulation

Background Appropriate control of mitochondrial function, morphology and biogenesis are crucial determinants of the general health of eukaryotic cells. It is therefore imperative that we understand the mechanisms that co-ordinate mitochondrial function with environmental signaling systems. The regulation of yeast mitochondrial function in response to nutritional change can be modulated by PKA activity. Unregulated PKA activity can lead to the production of mitochondria that are prone to the production of ROS, and an apoptotic form of cell death. Results We present evidence that mitochondria are sensitive to the level of cAMP/PKA signaling and can respond by modulating levels of respiratory activity or committing to self execution. The inappropriate activation of one of the yeast PKA catalytic subunits, Tpk3p, is sufficient to commit cells to an apoptotic death through transcriptional changes that promote the production of dysfunctional, ROS producing mitochondria. Our data implies that cAMP/PKA regulation of mitochondrial function that promotes apoptosis engages the function of multiple transcription factors, including HAP4, SOK2 and SCO1. Conclusions We propose that in yeast, as is the case in mammalian cells, mitochondrial function and biogenesis are controlled in response to environmental change by the concerted regulation of multiple transcription factors. The visualization of cAMP/TPK3 induced cell death within yeast colonies supports a model that PKA regulation plays a physiological role in coordinating respiratory function and cell death with nutritional status in budding yeast

Deciphering dynamic dose responses of natural promoters and single cis elements upon osmotic and oxidative stress in yeast

[EN] Fine-tuned activation of gene expression in response to stress is the result of dynamic interactions of transcription factors with specific promoter binding sites. In the study described here we used a time-resolved luciferase reporter assay in living Saccharomyces cerevisiae yeast cells to gain insights into how osmotic and oxidative stress signals modulate gene expression in a dose-sensitive manner. Specifically, the dose-response behavior of four different natural promoters (GRE2, CTT1, SOD2, and CCP1) reveals differences in their sensitivity and dynamics in response to different salt and oxidative stimuli. Characteristic dose-response profiles were also obtained for artificial promoters driven by only one type of stress-regulated consensus element, such as the cyclic AMP-responsive element, stress response element, or AP-1 site. Oxidative and osmotic stress signals activate these elements separately and with different sensitivities through different signaling molecules. Combination of stress-activated cis elements does not, in general, enhance the absolute expression levels; however, specific combinations can increase the inducibility of the promoter in response to different stress doses. Finally, we show that the stress tolerance of the cell critically modulates the dynamics of its transcriptional response in the case of oxidative stress.This work was supported by the Ministerio de Economa y Competitividad (grant BFU2011-23326 to M.P.) and the Ministerio de Ciencia e Innovacion (predoctoral FPI grant to A.R.).Dolz Edo, L.; Rienzo, A.; Poveda Huertes, D.; Pascual-Ahuir Giner, MD.; Proft, MH. (2013). Deciphering dynamic dose responses of natural promoters and single cis elements upon osmotic and oxidative stress in yeast. Molecular and Cellular Biology. 33(11):2228-2240. https://doi.org/10.1128/MCB.00240-13222822403311Gasch, A. P., Spellman, P. T., Kao, C. M., Carmel-Harel, O., Eisen, M. B., Storz, G., … Brown, P. O. (2000). 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Mechanisms of Cell Cycle Control Revealed by a Systematic and Quantitative Overexpression Screen in S. cerevisiae

Regulation of cell cycle progression is fundamental to cell health and reproduction, and failures in this process are associated with many human diseases. Much of our knowledge of cell cycle regulators derives from loss-of-function studies. To reveal new cell cycle regulatory genes that are difficult to identify in loss-of-function studies, we performed a near-genome-wide flow cytometry assay of yeast gene overexpression-induced cell cycle delay phenotypes. We identified 108 genes whose overexpression significantly delayed the progression of the yeast cell cycle at a specific stage. Many of the genes are newly implicated in cell cycle progression, for example SKO1, RFA1, and YPR015C. The overexpression of RFA1 or YPR015C delayed the cell cycle at G2/M phases by disrupting spindle attachment to chromosomes and activating the DNA damage checkpoint, respectively. In contrast, overexpression of the transcription factor SKO1 arrests cells at G1 phase by activating the pheromone response pathway, revealing new cross-talk between osmotic sensing and mating. More generally, 92%–94% of the genes exhibit distinct phenotypes when overexpressed as compared to their corresponding deletion mutants, supporting the notion that many genes may gain functions upon overexpression. This work thus implicates new genes in cell cycle progression, complements previous screens, and lays the foundation for future experiments to define more precisely roles for these genes in cell cycle progression

Antagonistická regulace pomocí globálních transkripčních faktorů Tup1p a Cyc8p u Flo11 a Flo11-dependentních fenotypů u divokých kvasinek

Biofilmy jsou běžným způsobem růstu kvasinek, při kterém buňky adherují jak k sobě navzájem tak i k abiotickým povrchům za vzniku složitých mnohobuněčných struktur. Společné soužití v biofilmech poskytuje buňkám několik výhod ve srovnání s planktonními kulturami. Mezi ně patří nepochybně ochrana a odolnost vůči antimikrobiálním látkám, stresovým faktorům prostředí nebo imunitnímu napadení hostitele. Biofilmy se nacházejí v mnoha prostředích a hrají důležité role v komerčních průmyslových odvětvích. Mohou však být také extrémně nebezpečné v klinickém prostředí. Existuje tedy velký zájem o studium biofilmů a o to, jak je eliminovat. V této studii jsme použili biofilm divokého kmene kvasinky Saccharomyces cerevisiae jako ideální systém pro zkoumání potenciálních funkcí komplexu transkripčních korepresorů Cyc8-Tup1 při regulaci buněčné adheze a tvorby biofilmu na agarovém médiu a na rozhraní pevné látky a kapaliny. Neočekávaně jsme zjistili, že Cyc8p a Tup1p antagonisticky řídí tvorbu strukturovaných biofilmových kolonií na pevných médiích prostřednictvím modulace expresí genu FLO11. Samotný Cyc8p působí jako klíčový represor FLO11, zatímco Tup1p podporuje tvorbu biofilmových kolonií a indukuje expresi FLO11 inhibicí represivní funkce Cyc8p a zároveň zabraňuje degradaci Flo11p možnou inhibicí...Biofilms are a common mode of yeast growth in which cells adhere to each other and adhere to biotic and abiotic surfaces to form complex multicellular structures. Living together in biofilms provides cells with several benefits, compared to planktonic cells such as protection and resistance to antimicrobials, environmental stresses and host immune attacks. Biofilms may play many important roles in commercial industries. But they are considered to be extremely dangerous in clinical settings. There is thus great interest in studying biofilms and how to eliminate them. In this study, we used wild yeast Saccharomyces cerevisiae colony biofilm as an ideal system to investigate potential functions of the yeast Cyc8p-Tup1p transcriptional corepressor complex in the regulation of yeast adhesion and biofilm formation on agar and at solid-liquid interfaces. Unexpectedly, we found that Cyc8p and Tup1p antagonistically control FLO11 expression and the formation of structured biofilm colonies on agar. Cyc8p itself acts as a key repressor of FLO11 and biofilm colony formation, whereas Tup1p promotes the formation of biofilm colonies and induces FLO11 expression by inhibiting the repressive function of Cyc8p and preventing Flo11p degradation possibly by inhibiting an extracellular protease. Other typical features...Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologieFaculty of SciencePřírodovědecká fakult

CRISPRi screens reveal genes modulating yeast growth in lignocellulose hydrolysate.

BACKGROUND: Baker's yeast is a widely used eukaryotic cell factory, producing a diverse range of compounds including biofuels and fine chemicals. The use of lignocellulose as feedstock offers the opportunity to run these processes in an environmentally sustainable way. However, the required hydrolysis pretreatment of lignocellulosic material releases toxic compounds that hamper yeast growth and consequently productivity. RESULTS: Here, we employ CRISPR interference in S. cerevisiae to identify genes modulating fermentative growth in plant hydrolysate and in presence of lignocellulosic toxins. We find that at least one-third of hydrolysate-associated gene functions are explained by effects of known toxic compounds, such as the decreased growth of YAP1 or HAA1, or increased growth of DOT6 knock-down strains in hydrolysate. CONCLUSION: Our study confirms previously known genetic elements and uncovers new targets towards designing more robust yeast strains for the utilization of lignocellulose hydrolysate as sustainable feedstock, and, more broadly, paves the way for applying CRISPRi screens to improve industrial fermentation processes