Project description:Snf1 and TORC1 are two global regulators that sense the nutrient availability and regulate the cell growth in yeast Saccharomyces cerevisiae. Here we undertook a systems biology approach to study the effect of deletion of these genes and investigate the interaction between Snf1 and TORC1 in regulation of gene expression and cell metabolism. 3 mutant strains (snf1?, tor1?, snf1?tor1?) together with 1 reference strain grown under both glucose-limited or amonia-limited defined media with three biological replicates for each strain
Project description:The existence of two chromatin structures in the rDNA locus was previously demonstrated for a large variety of organisms, ranging from yeast to human. In yeast there are about 150-200 rRNA genes organized in tandem repeats. Almost half of them are transcribed and largely depleted of nucleosomes (active/open), the other half is not transcribed and is assembled in regular arrays of nucleosomes (inactive/closed). It is proposed that RNA polymerase-I (RNAPI) transcription-elongation removes nucleosomes from closed rRNA genes (opening), and that soon after DNA replication there is deposition of nucleosomes on the open rRNA genes (closing). In G1 arrested cells, nearly all rRNA genes are depleted of nucleosomes, but most of them are not transcribed (inactive/open). In relation to the research article by Charton et al. (Mutat. Res.), the data presented here are on the hydroxyurea concentration-dependent inhibition of yeast culture growth, on cell cycle arrest before completion of genome replication, and on the opening of rRNA gene chromatin. As comparison, data are presented for yeast arrested in the G1-phase of the cell cycle by the pheromone ?-factor.
Project description:Fpr1 (FK506-sensitive proline rotamase 1), a protein of the FKBP12 (FK506-binding protein 12 kDa) family in Saccharomyces cerevisiae, is a primary target for the immunosuppressive agents FK506 and rapamycin. Fpr1 inhibits calcineurin and TORC1 (target of rapamycin complex 1) when bound to FK506 and rapamycin, respectively. Although Fpr1 is recognised to play a crucial role in the efficacy of these drugs, its physiological functions remain unclear. In a hmo1? (high mobility group family 1-deleted) yeast strain, deletion of FPR1 induced severe growth defects, which could be alleviated by increasing the copy number of RPL25 (ribosome protein of the large subunit 25), suggesting that RPL25 expression was affected in hmo1?fpr1? cells. In the current study, extensive chromatin immunoprecipitation (ChIP) and ChIP-sequencing analyses revealed that Fpr1 associates specifically with the upstream activating sequences of nearly all RPG (ribosomal protein gene) promoters, presumably in a manner dependent on Rap1 (repressor/activator site binding protein 1). Intriguingly, Fpr1 promotes the binding of Fhl1/Ifh1 (forkhead-like 1/interacts with forkhead 1), two key regulators of RPG transcription, to certain RPG promoters independently of and/or cooperatively with Hmo1. Furthermore, mutation analyses of Fpr1 indicated that for transcriptional function on RPG promoters, Fpr1 requires its N-terminal domain and the binding surface for rapamycin, but not peptidyl-prolyl isomerase activity. Notably, Fpr1 orthologues from other species also inhibit TORC1 when bound to rapamycin, but do not regulate transcription in yeast, which suggests that these two functions of Fpr1 are independent of each other.
Project description:Effect of FLO8 or MSS11 deletion and -overexpression on yeast transcript profiles compared to wild type in laboratory yeast strains ?1278b and S288c. We used two laboratory yeast strains that behave different with regard to adhesion phenotypes. By comparing yeast deleted in either FLO8 or MSS11 to wild type, or yeast overexpressing these genes, in both genetic backgrounds, we investigate the role of Flo8p and Mss11p on yeast transcription. By using similar growth conditions to what we use for adhesion phenotype determination we aim to correlate transcription profile changes to yeast behaviour (phenotypes).
Project description:Tip growth in fungi involves highly polarized secretion and modification of the cell wall at the growing tip. The genetic requirements for initiating polarized growth are perhaps best understood for the model budding yeast Saccharomyces cerevisiae. Once the cell is committed to enter the cell cycle by activation of G1 cyclin/cyclin-dependent kinase (CDK) complexes, the polarity regulator Cdc42 becomes concentrated at the presumptive bud site, actin cables are oriented toward that site, and septin filaments assemble into a ring around the polarity site. Several minutes later, the bud emerges. Here, we investigated the mechanisms that regulate the timing of these events at the single-cell level. Septin recruitment was delayed relative to polarity establishment, and our findings suggest that a CDK-dependent septin "priming" facilitates septin recruitment by Cdc42. Bud emergence was delayed relative to the initiation of polarized secretion, and our findings suggest that the delay reflects the time needed to weaken the cell wall sufficiently for the cell to bud. Rho1 activation by Rom2 occurred at around the time of bud emergence, perhaps in response to local cell-wall weakening. This report reveals regulatory mechanisms underlying the morphogenetic events in the budding yeast.
Project description:Spore germination in the yeast Saccharomyces cerevisiae is a process in which non-dividing haploid spores re-enter the mitotic cell cycle and resume vegetative growth. To study the signals and pathways underlying spore germination we examined the global changes in gene expression and followed cell-cycle and germination markers during this process.We find that the germination process can be divided into two distinct stages. During the first stage, the induced spores respond only to glucose. The transcription program during this stage recapitulates the general transcription response of yeast cells to glucose. Only during the second phase are the cells able to sense and respond to other nutritional components in the environment. Components of the mitotic machinery are involved in spore germination but in a distinct pattern. In contrast to the mitotic cell cycle, growth-related events during germination are not coordinated with nuclear events and are separately regulated. Thus, genes that are co-induced during G1/S of the mitotic cell cycle, the dynamics of the septin Cdc10 and the kinetics of accumulation of the cyclin Clb2 all exhibit distinct patterns of regulation during spore germination, which allow the separation of cell growth from nuclear events.Taken together, genome-wide expression profiling enables us to follow the progression of spore germination, thus dividing this process into two major stages, and to identify germination-specific regulation of components of the mitotic cell cycle machinery.
Project description:14-3-3 proteins form a family of highly conserved eukaryotic proteins involved in a wide variety of cellular processes, including signalling, apoptosis, cell-cycle control and transcriptional regulation. More than 150 binding partners have been found for these proteins. The yeast Saccharomyces cerevisiae has two genes encoding 14-3-3 proteins, BMH1 and BMH2. A bmh1 bmh2 double mutant is unviable in most laboratory strains. Previously, we constructed a temperature-sensitive bmh2 mutant and showed that mutations in RTG3 and SIN4, both encoding transcriptional regulators, can suppress the temperature-sensitive phenotype of this mutant, suggesting an inhibitory role of the 14-3-3 proteins in Rtg3-dependent transcription [van Heusden and Steensma (2001) Yeast 18, 1479-1491]. In the present paper, we report a genome-wide transcription analysis of a temperature-sensitive bmh2 mutant. Steady-state mRNA levels of 60 open reading frames were increased more than 2.0-fold in the bmh2 mutant, whereas those of 78 open reading frames were decreased more than 2.0-fold. In agreement with our genetic experiments, six genes known to be regulated by Rtg3 showed elevated mRNA levels in the mutant. In addition, several genes with other cellular functions, including those involved in gluconeogenesis, ergosterol biosynthesis and stress response, had altered mRNA levels in the mutant. Our data show that the yeast 14-3-3 proteins negatively regulate Rtg3-dependent transcription, stimulate the transcription of genes involved in ergosterol metabolism and in stress response and are involved in transcription regulation of multiple other genes.
Project description:Downregulation of specific transcripts is one of the mechanisms utilized by eukaryotic checkpoint systems to prevent cell cycle progression. Here we identified and explored such a mechanism in the yeast Saccharomyces cerevisiae. It involves the Mec1-Rad53 kinase cascade, which attenuates G(2)/M-specific gene transcription upon genotoxic stress. This inhibition is achieved via multiple Rad53-dependent inhibitory phosphorylations on the transcriptional activator Ndd1 that prevent its chromatin recruitment via interactions with the forkhead factor Fkh2. Relevant modification sites on Ndd1 were identified by mass spectrometry, and corresponding alanine substitutions were able to suppress a methyl methanesulfonate-induced block in Ndd1 chromatin recruitment. Whereas effective suppression by these Ndd1 mutants is achieved for DNA damage, this is not the case under replication stress conditions, suggesting that additional mechanisms must operate under such conditions. We propose that budding yeast cells prevent the normal transcription of G(2)/M-specific genes upon genotoxic stress to precisely coordinate the timing of mitotic and postmitotic events with respect to S phase.
Project description:Variegated expression of genes contributes to phenotypic variation within populations of genetically identical cells. Such variation plays a role in development and host pathogen interaction and can be important in adaptation to harsh environments. The expression state of genes placed near telomeres shows a variegated pattern of inheritance due to heterochromatin formation, a phenomenon that is called telomere position effect (TPE). We show that in budding yeast, TPE is controlled by the a1/alpha2 developmental repressor, which dictates developmental decisions in response to environmental changes. Two a1/alpha2 repressed genes, STE5, a MAPK scaffold and HOG1, a stress-activated MAPK, are the targets of this heterochromatin regulation pathway. We provide new evidence that link MAPK signaling and heterochromatin formation in yeast. Our results show that the same components that regulate gene expression states in euchromatic regions regulate heterochromatic expression states and that stress can play a part in turning on or off genes placed in heterochromatic regions.
Project description:In response to starvation, cells undergo increased levels of autophagy and cell cycle arrest but the role of autophagy in starvation-induced cell cycle arrest is not fully understood. Here we show that autophagy genes regulate cell cycle arrest in the budding yeast Saccharomyces cerevisiae during nitrogen starvation. While exponentially growing wild-type yeasts preferentially arrest in G?/G? in response to starvation, yeasts carrying null mutations in autophagy genes show a significantly higher percentage of cells in G?/M. In these autophagy-deficient yeast strains, starvation elicits physiological properties associated with quiescence, such as Snf1 activation, glycogen and trehalose accumulation as well as heat-shock resistance. However, while nutrient-starved wild-type yeasts finish the G?/M transition and arrest in G?/G 0? autophagy-deficient yeasts arrest in telophase. Our results suggest that autophagy is crucial for mitotic exit during starvation and appropriate entry into a G?/G? quiescent state.