Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Glucose-depletion time-course experiment in Saccharomyces cerevisiae wild-type cells.

ABSTRACT: To investigate the glucose regulatory system in Saccharomyces cerevisiae, we conducted a time-course in which glucose-depleted wildtype (WT) cells were inoculated into fresh media (SC, 2% glucose). Their subsequent transcriptional output was monitored over a period of five hours by DNA microarrays: samples for gene expression profiling were taken immediately after, as well as 3, 7.5, 15, 30, 60, 110, 150, and 300 minutes after inoculation into fresh medium. Transcripts upregulated are involved in translational processes such as the GO biological processes “ribosome biogenesis” and “ribosome localization”. Transcripts downregulated are enriched for the GO biological processes “cellular respiration” and various metabolism related processes. The time-course was used to verify the physiological relevance of gene expression profiles determined for individual deletions of glucose regulatory system components. Importantly, transcripts up- or downregulated in WT cells upon the addition of glucose are similarly up- or downregulated in deletion mutants that each lack a component of the glucose regulatory system. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used for each separate hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Two overnight WT cultures were used to inoculate 50 ml cultures at an OD600 of 0.15. These were depleted of glucose by growing for 24 hours and were used the next day to inoculate 500 ml cultures in fresh medium to an OD600 of 0.15. Samples for expression profiling were taken immediately after, as well as 3, 7.5, 15, 30, 60, 110, 150, and 300 minutes after inoculation into fresh medium.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Patrick Kemmeren

PROVIDER: E-GEOD-33098 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Yeast glucose pathways converge on the transcriptional regulation of trehalose biosynthesis.

Apweiler Eva E Sameith Katrin K Margaritis Thanasis T Brabers Nathalie N van de Pasch Loes L Bakker Linda V LV van Leenen Dik D Holstege Frank Cp FC Kemmeren Patrick P

BMC genomics 20120614

<h4>Background</h4>Cellular glucose availability is crucial for the functioning of most biological processes. Our understanding of the glucose regulatory system has been greatly advanced by studying the model organism Saccharomyces cerevisiae, but many aspects of this system remain elusive. To understand the organisation of the glucose regulatory system, we analysed 91 deletion mutants of the different glucose signalling and metabolic pathways in Saccharomyces cerevisiae using DNA microarrays.<h ...[more]

PMID: 22697265

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Project description:To understand the organisation of the glucose regulatory system, we analysed 91 deletion mutants of established glucose signalling and metabolic pathway members in Saccharomyces cerevisiae by DNA microarrays. These deletion mutants do not induce pathway-specific transcriptional responses reflecting the tight interconnection between pathways of the glucose regulatory system. Instead, one main transcriptional response is discerned, which varies in direction to mimic either a high or a low glucose response. The study reveals both known and unknown relationships within and between individual pathways and their members. Metabolic components of the glucose regulatory system are most frequently affected at the transcriptional level. A new network approach is applied that exposes the hierarchical organisation of the glucose regulatory system. Tps2 and Tsl1, two enzymes involved in trehalose biosynthesis, are predicted to be the most downstream transcriptional components. This prediction is further validated by epistasis analysis of Tps2 double mutants. Taken together, this suggests that changes in perceived glucose levels ultimately lead to a shift in trehalose biosynthesis. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used for each separate hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Up to five deletion strains were grown on a single day. Wt cultures were grown parallel to the deletion mutants to assess day-to-day variance.

Project description:TATA-binding protein (TBP) is central to the regulation of transcription initiation. Recruitment of TBP to target genes can be positively regulated by one of two basal transcription factor complexes: SAGA or TFIID. Negative regulation of TBP promoter association can be performed by Mot1 or the NC2 complex. Recent evidence suggest that Mot1, NC2, and TBP form a DNA-dependent protein complex. Here, we compare the functions of Mot1 and NC2beta during basal and activated transcription using the anchor-away technique for conditional nuclear depletion. Genome-wide expression analysis indicates that both proteins regulate a highly similar set of genes (r2=0.8). Upregulated genes were enriched for SAGA occupancy, while downregulated genes preferred TFIID binding. Mot1p and NC2beta depletion during heat shock resulted in a failure to downregulate gene expression after initial activation, which was accompanied by increased TBP and RNA pol II promoter occupancies. Depletion of Mot1p or NC2beta displayed preferential synthetic lethality with the TBP-interaction module of SAGA. Our results suggest that Mot1 and NC2beta cooperate in vivo to regulate TBP function, and that they are involved in maintaining basal expression levels as well as in resetting gene expression after induction by stress. In this study 3 S. cerevisiae strains were grown with and without addition of rapamycin. For each experimental factor two independent colonies were inoculated in Complete Synthetic Medium (CSM) with 2% glucose. Overnight cultures were diluted to 0.3 OD600 in 50 ml medium and grown to 1 OD600. Cultures were then grown for 60 minutes in the absence or presence of rapamycin at 1ug/ml (LC laboratories). Cells were harvested by centrifugation at 4000 rpm for 3 minutes and were frozen in liquid nitrogen. Amplified cRNA samples were labeled with either cy3 or cy5 and put on microarray together with an oppositely labeled common reference sample consisting of cRNA of untreated wildtype strain.

Project description:The Saccharomyces cerevisiae Slx5/8 complex is the founding member of a recently defined class of ubiquitin ligases (STUbLs) that target sumoylated substrates. Slx5/8 has been implicated in genome stability and transcription, but the precise contribution to these processes is not clear. To characterize Slx5/8 function, we determined genome-wide changes in gene expression upon loss of either subunit. The majority of mRNA changes are part of a general stress response, also exhibited by mutants of other genome integrity pathways and therefore indicative of an indirect effect on transcription. Genome-wide binding analysis reveals a uniquely centromeric location for Slx5. Detailed phenotype analyses of slx5D and slx8D mutants show severe mitotic defects that include aneuploidy, spindle mispositioning, fish hook spindles and aberrant spindle kinetics. This is associated with accumulation of the PP2A regulatory subunit Rts1 at centromeres prior to entry into anaphase. Knockdown of the human STUbL orthologue RNF4 also results in chromosome segregation defects due to formation of chromosome bridges. The study shows that STUbLs have a conserved role in maintenance of chromosomal stability and the results link SUMO-dependent ubiquitination to a centromere-specific function during mitosis. Two channel microarrays were used. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used in one of the channels for each hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Using the Erlenmeyer growth protocol up to five deletion strains were grown on a single day. In the tecan platereader, up to eleven deletion strains could be grown on a single day. Wt cultures were grown parallel to the deletion mutants to assess day-to-day variance.

Project description:Evidence indicates that transcription and mRNA export are linked processes. The molecular mechanisms of this coordination are not clear however. Sus1 (hENY2) participates in this coordination as part of two protein complexes: SAGA, a transcriptional co-activator and TREX-2 that functions in mRNA biogenesis and export. Here we investigate the coordinated action of SAGA and TREX-2 that is required for gene expression. We demonstrate that the TREX-2/proteasomal subunit Sem1, influences Sus1 role in mRNA export and TREX-2 stability. Wide analyses of gene expression reveal that Sem1 and Sus1 have also overlapping functions in transcription. In the absence of Sem1, expression of some SAGA-dependent genes is compromised with a concomitant decrease of RNAP II recruitment to promoters. Notably, ChIP experiments revealed a distinct dependency for SAGA subunits recruitment on Sem1. While absence of Sem1 lowers Ada2 and Taf9 recruitment to GAL1 promoter upon activation, association of the deubiquitylation module remains intact. However, H2B deubiquitylation activity is dramatically decreased. These results unveil a new role for Sem1 in influencing activation of SAGA-dependent H2B deubiquitylation likely mediated by stabilization of TREX-2 complex and SAGA modular assembly. Our work gives insights in how modular architecture of SAGA is determinant for its function in gene expression. Two channel microarrays were used. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used in one of the channels for each hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Using the Erlenmeyer growth protocol up to five deletion strains were grown on a single day. In the tecan platereader, up to eleven deletion strains could be grown on a single day. Wt cultures were grown parallel to the deletion mutants to assess day-to-day variance.

Dataset Information

Glucose-depletion time-course experiment in Saccharomyces cerevisiae wild-type cells.

Publications

Yeast glucose pathways converge on the transcriptional regulation of trehalose biosynthesis.

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