Dataset Information

Expression data from Saccharomyces cerevisiae Dnup60, Dada2, and Dnup60 Dada2 cells

ABSTRACT: In Saccharomyces cerevisiae, specific genes physically relocate from the nucleoplasm to the nuclear periphery concomitant with transcriptional activation, where they associate with the nuclear pore complex (NPC). We took a genomics approach in order to gain insight into the universality and physiological relevance of the interaction between active genes and the NPC. Using synthetic genetic array (SGA) approach, we identified interactions between components of the SAGA histone acetyltransferase complex and the Mlp and Nup60 subunits of the NPC. Cells lacking these SAGA and NPC components display growth defects under optimal growth conditions, in which cells are grown on rich medium containing the preferred sugar glucose as a carbon source and incubated at their optimal temperature. That growth defects were observed under these non-stress conditions suggests that these interactions are indicative of defects in normal cell physiology. These results are consistent with a model where physical interactions between the NPC and SAGA are important for transcription of constitutively expressed genes. To test this hypothesis, we used microarray analysis to assess changes global transcript levels in the absence of Nup60, Ada2, or Nup60 and Ada2. Microarray analysis reveals that the growth defect in these double mutants is correlated with a synthetic reduction in steady-state transcript levels for numerous genes that are strongly expressed in wildtype cells. These results suggest that SAGA and Nup60 cooperate in transcriptional regulation, and are consistent with a model for global regulation of SAGA-dependent transcription at the NPC.

ORGANISM(S): Saccharomyces cerevisiae Schizosaccharomyces pombe

PROVIDER: GSE21870 | GEO | 2013/05/13

SECONDARY ACCESSION(S): PRJNA127059

REPOSITORIES: GEO

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Project description:In Saccharomyces cerevisiae, specific genes physically relocate from the nucleoplasm to the nuclear periphery concomitant with transcriptional activation, where they associate with the nuclear pore complex (NPC). We took a genomics approach in order to gain insight into the universality and physiological relevance of the interaction between active genes and the NPC. Using synthetic genetic array (SGA) approach, we identified interactions between components of the SAGA histone acetyltransferase complex and the Mlp and Nup60 subunits of the NPC. Cells lacking these SAGA and NPC components display growth defects under optimal growth conditions, in which cells are grown on rich medium containing the preferred sugar glucose as a carbon source and incubated at their optimal temperature. That growth defects were observed under these non-stress conditions suggests that these interactions are indicative of defects in normal cell physiology. These results are consistent with a model where physical interactions between the NPC and SAGA are important for transcription of constitutively expressed genes. To test this hypothesis, we used microarray analysis to assess changes global transcript levels in the absence of Nup60, Ada2, or Nup60 and Ada2. Microarray analysis reveals that the growth defect in these double mutants is correlated with a synthetic reduction in steady-state transcript levels for numerous genes that are strongly expressed in wildtype cells. These results suggest that SAGA and Nup60 cooperate in transcriptional regulation, and are consistent with a model for global regulation of SAGA-dependent transcription at the NPC. Wildype, single deletion, and double deletion cells were inoculated to equal starting ODs in 50 mL YEPD media and grown at 30C with shaking. Cells were collected at mid-log phase, washed with chilled water, and stored at -80C for later processing. RNA was isolated using TRIzol reagent according to the manufacturer's protocol, with modification for yeast cells.

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

Expression data from Saccharomyces cerevisiae Dnup60, Dada2, and Dnup60 Dada2 cells

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