Yeast_FLO8_MSS11_Deletions_LowN
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE40530 | GEO | 2012/09/04
SECONDARY ACCESSION(S): PRJNA174254
REPOSITORIES: GEO
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Project description:In filamentous strains of S. cerevisiae, nitrogen stress elicits a complex morphogenetic program resulting in the transition to a filamentous form of growth. The transcription factors Flo8p and Mss11p are both required for this filamentous growth transition, in that homozygous diploid flo8(delta/delta) and mss11(delta/delta) strains do not undergo filamentation under conditions of nitrogen stress. To identify genes that are regulated either directly or indirectly by the Flo8p and Mss11p transcription factors, we have implemented DNA microarray analysis to profile changes in mRNA levels in homozygous diploid strains of the filamentous (Sigma)1278b genetic background deleted for FLO8 and MSS11, respectively, under conditions of nitrogen stress. The transcriptional profiles will identify genes with altered transcriptional levels in the deletion mutants, serving as a means to identify cellular processes and signaling pathways regulated by Flo8p and Mss11p function. Deletion mutants were constructed using a one-step polymerase chain reaction (PCR)-based gene disruption strategy with the G418 resistance cassette from plasmid pFA6a-KanMX6. After the haploid mutants were confirmed via PCR, the strains were allowed to mate on YPD+G418 plates for approximately 20 hours at 30 degrees Celsius. Mated cells were then streaked on SC-Trp-Leu plates to select for Y825 x HLY337 diploids. Yeast transformations were performed according to standard lithium acetate-mediated protocols with modifications. Yeast strains (wild-type and deletion mutants) were cultured under conditions of nitrogen stress (low ammonium media), and RNA was prepared using the Poly(A) Purist kit (Ambion, Austin, TX). RNA concentration and purity were determined spectrophotometrically and by gel electrophoresis. Microarray hybridization was performed with the Yeast Genome S98 Array using standard protocols (Affymetrix, Inc, Santa Clara, CA). Differentially expressed genes were identified by significance analysis of microarrays (SAM). SAM computed a nonparametric score for each gene by dividing the between-group difference of (normalized log) gene expression levels and the within-group difference of gene expression levels. The score was then compared with random permutation scores. The random permutation scores for a gene were computed in the same manner as the original score, but based on randomly sampled gene expressions. If the difference between the original score and the random permutation score was larger than a chosen threshold value, the corresponding change in gene expression was claimed to be significant. Each threshold value corresponded to a false discovery rate (the percentage of genes identified as being significant by chance alone). For all deletion comparisons in this study, we used SAM’s two-class unpaired analysis function, with significance thresholds selected so that the corresponding false discovery rate was 0.
2012-09-04 | E-GEOD-40530 | biostudies-arrayexpress
Project description:Effect of FLO8 or MSS11 deletion and -overexpression on yeast transcript profiles compared to wild type in laboratory yeast strains Σ1278b and S288c.
2011-09-21 | GSE17716 | GEO
Project description:Pseudohyphal growth is a developmental pathway seen in some strains of yeast, in which cells elongate and form multicellular filaments in response to environmental stresses. Regulation of this process is complex and involves multiple signaling pathways and a large number of transcription factors (TFs). We used multiplexed transposon “Calling Cards” to simultaneously record the genome-wide binding patterns of 28 TFs in nitrogen-starved yeast. We were able to identify TF targets relevant for pseudohyphal growth, producing a detailed map of the regulatory network that governs this process. Using tools from graph theory, we identified 8 transcription factors that lie at the center of this network, including Flo8, Mss11, and Mfg1, which bind as a complex. Surprisingly, the DNA binding preferences for these key transcription factors were not known. Using Calling Card data, we predicted the in vivo DNA binding motif for the Flo8-Mss11-Mfg1 complex and validated it using a reporter assay. We found that this complex binds several important targets, including FLO11, at both their promoter and termination sequences. We demonstrated that this binding pattern is the result of DNA-looping, which regulates the transcription of these targets and is stabilized by an interaction with the nuclear pore complex. This looping provides yeast cells with a transcriptional memory, enabling them to more rapidly execute the filamentous growth program when nitrogen-starved if they have been previously exposed to this condition.
2014-02-11 | GSE54831 | GEO
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).
2011-09-20 | E-GEOD-17716 | biostudies-arrayexpress
Project description:Yeast filamentous growth is a stress response to conditions of nitrogen deprivation, wherein yeast colonies form pseudohyphal filaments of elongated and connected cells. As proteins mediating adhesion and transport are required for this growth transition, the protein complement at the yeast cell periphery plays a critical and tightly regulated role in enabling pseudohyphal filamentation. To identify proteins differentially abundant at the yeast cell periphery during pseudohyphal growth, we generated quantitative proteomic profiles of plasma membrane protein preparations under conditions of vegetative growth and filamentation. By iTRAQ chemistry and two-dimensional LC-MS/MS, we profiled 2,463 peptides and 356 proteins, from which we identified eleven differentially abundant proteins that localize to the yeast cell periphery. This protein set includes Ylr414cp, herein renamed Pun1p, a previously uncharacterized protein localized to the plasma membrane compartment of Can1 (MCC). Pun1p abundance is increased two-fold under conditions of nitrogen stress, and deletion of PUN1 abolishes filamentous growth in haploids and diploids; pun1D mutants are non-invasive, lack surface-spread filamentation, grow slowly, and exhibit impaired cell adhesion. Conversely, overexpression of PUN1 results in exaggerated cell elongation under conditions of nitrogen stress. PUN1 contributes to yeast nitrogen signaling, as pun1D mutants misregulate amino acid biosynthetic genes during nitrogen deprivation. By chromatin immunoprecipitation and RT-PCR, we find that the filamentous growth factor Mss11p directly binds to the PUN1 promoter and regulates its transcription. In total, this study provides the first profile of protein abundance during pseudohyphal growth, identifying a previously uncharacterized MCC protein required for wild-type nitrogen signaling and filamentous growth. For this study, we constructed a homozygous diploid strain in the filamentous ?1278b background deleted for PUN1; a wild-type diploid strain of the same background served as the control. Both strains were grown under conditions of low nitrogen, and RNA was extracted from three biological replicates of each strain after identical culturing. The RNA samples were analyzed using affymerix DNA microarrays.
2010-02-17 | E-GEOD-20351 | biostudies-arrayexpress
Project description:Morphogenetic transitions are prevalent in the fungal kingdom. For a leading human fungal pathogen, Candida albicans, the capacity to transition between yeast and filaments is key for virulence. For the model yeast Saccharomyces cerevisiae, filamentation enables nutrient acquisition. A recent functional genomic screen in S. cerevisiae identified Mfg1 as a regulator of morphogenesis that acts in complex with Flo8 and Mss11 to enable transcriptional responses crucial for filamentation. In C. albicans, Mfg1 also interacts physically with Flo8 and Mss11 and is critical for filamentation in response to diverse cues, but the mechanisms through which it regulates morphogenesis remained elusive. Here, we explored the consequences of perturbation of Mfg1, Flo8, and Mss11 on C. albicans morphogenesis, and identified functional divergence of complex members. We observed that C. albicans Mss11 was dispensable for filamentation, and that overexpression of FLO8 caused constitutive filamentation even in the absence of Mfg1. Epistasis experiments suggested that Mfg1 acts downstream of protein kinase A, as does Flo8. Harnessing transcriptional profiling and chromatin immunoprecipitation coupled to microarray analysis, we identified divergence between transcriptional targets of Mfg1 and Flo8 in C. albicans. A key direct transcriptional target of Mfg1 is TEC1, for which overexpression was sufficient to restore filamentation in the absence of Mfg1. To identify additional Mfg1 downstream effectors, we employed a novel strategy to select for mutations that restore filamentation in the absence of Mfg1. Whole genome sequencing of filamentation-competent mutants revealed chromosome 6 amplification as a conserved adaptive mechanism. A key determinant of the amplification on chromosome 6 is FLO8, as deletion of one allele blocked morphogenesis, and chromosome 6 was not amplified in evolved lineages for which FLO8 was re-located to a different chromosome. Thus, this work highlights rewiring of key morphogenetic regulators over evolutionary time and aneuploidy as an adaptive mechanism driving fungal morphogenesis.
2018-07-24 | GSE117477 | GEO
Project description:In response to limited nitrogen and abundant carbon sources, diploid Saccharomyces cerevisiae strains undergo a filamentous transition in cell growth as part of pseudohyphal differentiation. Use of the disaccharide maltose as the principal carbon source, in contrast to the preferred nutrient monosaccharide glucose, has been shown to induce a hyper-filamentous growth phenotype in a strain deficient for GPA2 which codes for a Galpha protein component that interacts with the glucose-sensing receptor Gpr1p to regulate filamentous growth. In this report, we compare the global transcript and proteomic profiles of wild-type and Gpa2p deficient diploid yeast strains grown on both rich and nitrogen starved maltose media. We find that deletion of GPA2 results in significantly different transcript and protein profiles when switching from rich to nitrogen starvation media. The results are discussed with a focus on the genes associated with carbon utilization, or regulation thereof, and a model for the contribution of carbon sensing/metabolism-based signal transduction to pseudohyphal differentiation is proposed. Keywords: Saccharomyces cerevisiae, nitrogen starvation, maltose, pseudohyphal differentiation, yeast, expression profiling
2007-12-21 | GSE7820 | GEO
Project description:Pseudohyphal growth is a developmental pathway seen in some strains of yeast, in which cells elongate and form multicellular filaments in response to environmental stresses. Regulation of this process is complex and involves multiple signaling pathways and a large number of transcription factors (TFs). We used multiplexed transposon M-bM-^@M-^\Calling CardsM-bM-^@M-^] to simultaneously record the genome-wide binding patterns of 28 TFs in nitrogen-starved yeast. We were able to identify TF targets relevant for pseudohyphal growth, producing a detailed map of the regulatory network that governs this process. Using tools from graph theory, we identified 8 transcription factors that lie at the center of this network, including Flo8, Mss11, and Mfg1, which bind as a complex. Surprisingly, the DNA binding preferences for these key transcription factors were not known. Using Calling Card data, we predicted the in vivo DNA binding motif for the Flo8-Mss11-Mfg1 complex and validated it using a reporter assay. We found that this complex binds several important targets, including FLO11, at both their promoter and termination sequences. We demonstrated that this binding pattern is the result of DNA-looping, which regulates the transcription of these targets and is stabilized by an interaction with the nuclear pore complex. This looping provides yeast cells with a transcriptional memory, enabling them to more rapidly execute the filamentous growth program when nitrogen-starved if they have been previously exposed to this condition. These data contain mapped insertion sites from Ty5 transposon "Calling Cards" in M-NM-#1278b yeast sequenced using an Illumina Hiseq. Other sequencing data present are RNA-seq reads.
2014-02-11 | E-GEOD-54831 | biostudies-arrayexpress
Project description:Candida albicans is an important fungal pathogen in humans. Several virulence factors of C. albicans have been reported, including a morphological transition from yeast to filamentous forms (hyphae and pseudohyphae). Mss11 is a transcriptional activator required for hyphal formation. To reveal the potential target genes of Mss11, DNA microarray analysis was performed to compare wild type and mss11-deleted mutant.
2016-09-24 | GSE51168 | GEO
Project description:In response to limited nitrogen and abundant carbon sources, diploid Saccharomyces cerevisiae strains undergo a filamentous transition in cell growth as part of pseudohyphal differentiation. Use of the disaccharide maltose as the principal carbon source, in contrast to the preferred nutrient monosaccharide glucose, has been shown to induce a hyper-filamentous growth phenotype in a strain deficient for GPA2 which codes for a Gï¡ protein component that interacts with the glucose-sensing receptor Gpr1p to regulate filamentous growth. In this report, we compare the global transcript and proteomic profiles of wild-type and Gpa2p deficient diploid yeast strains grown on both rich and nitrogen starved maltose media. We find that deletion of GPA2 results in significantly different transcript and protein profiles when switching from rich to nitrogen starvation media. The results are discussed with a focus on the genes associated with carbon utilization, or regulation thereof, and a model for the contribution of carbon sensing/metabolism-based signal transduction to pseudohyphal differentiation is proposed. Experiment Overall Design: For transcriptome profiling, there were 12 Affymetrix Yeast S98 microarrays total. There were four conditions: wildtype MLY61 and gpa2 deletion mutant MLY132 grown in YPM media or transferred to low nitrogen media SLAM. Each condition was done in triplicate, starting with triplicate yeast cultures. Four conditions done in triplicates resulted in 12 samples that went onto 12 microarrays.
2008-06-15 | E-GEOD-7820 | biostudies-arrayexpress