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Functional studies of the yeast med5, med15 and med16 mediator tail subunits.

Larsson M, Uvell H, Sandström J, Rydén P, Selth LA, Björklund S - PLoS ONE (2013)

Bottom Line: We also find that all strains in which MED15 is inactivated show down-regulation of genes that have been identified as targets for the Ace2 transcriptional activator protein, which is important for progression through the G1 phase of the cell cycle.Supporting this observation, we demonstrate that loss of Med15 leads to a G1 arrest phenotype.Collectively, these findings provide insight into the function of the Mediator Tail module.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

ABSTRACT
The yeast Mediator complex can be divided into three modules, designated Head, Middle and Tail. Tail comprises the Med2, Med3, Med5, Med15 and Med16 protein subunits, which are all encoded by genes that are individually non-essential for viability. In cells lacking Med16, Tail is displaced from Head and Middle. However, inactivation of MED5/MED15 and MED15/MED16 are synthetically lethal, indicating that Tail performs essential functions as a separate complex even when it is not bound to Middle and Head. We have used the N-Degron method to create temperature-sensitive (ts) mutants in the Mediator tail subunits Med5, Med15 and Med16 to study the immediate effects on global gene expression when each subunit is individually inactivated, and when Med5/15 or Med15/16 are inactivated together. We identify 25 genes in each double mutant that show a significant change in expression when compared to the corresponding single mutants and to the wild type strain. Importantly, 13 of the 25 identified genes are common for both double mutants. We also find that all strains in which MED15 is inactivated show down-regulation of genes that have been identified as targets for the Ace2 transcriptional activator protein, which is important for progression through the G1 phase of the cell cycle. Supporting this observation, we demonstrate that loss of Med15 leads to a G1 arrest phenotype. Collectively, these findings provide insight into the function of the Mediator Tail module.

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Transcription profile analysis using AffymetrixsYeast Genome 2.0 Array.25 genes were differently regulated in both of the double-Degron strains (med5/med15 (A) and med15/med16 (B)), 45 minutes after induction of degradation, compared to the single Degron (med5, med15 and med16) and wild type (Wt) strains, as shown in the Venn diagrams (FDR<0.05 and FC>abs(log2(1.5)).
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pone-0073137-g003: Transcription profile analysis using AffymetrixsYeast Genome 2.0 Array.25 genes were differently regulated in both of the double-Degron strains (med5/med15 (A) and med15/med16 (B)), 45 minutes after induction of degradation, compared to the single Degron (med5, med15 and med16) and wild type (Wt) strains, as shown in the Venn diagrams (FDR<0.05 and FC>abs(log2(1.5)).

Mentions: In order to identify genes that are differentially regulated and therefore could contribute to the synthetic lethality of cells lacking both Med5/Med15 or Med15/Med16, we used Affymetrix microarrays to compare global gene expression in wild type cells and the different Degron strains. We isolated RNA from each strain 45 minutes after changing from the permissive to the restrictive growth conditions in order to minimize possible secondary effects on gene expression that are not directly related to the Degron construct(s). This time point was chosen based on the experiments shown in Figure 1B, where the levels of each Degron-tagged protein was undetectable at 45 minutes after switching to the restrictive growth conditions. The results from these experiments including all possible combinations of comparisons of expression between wild type cells, single Degron mutants and double Degron mutants are presented in Tables 1, and S1. Genes that were differentially expressed in the med5/med15 strain compared to any individual control strain (WT, med5, and med15 respectively) are depicted as a Venn diagram in Figure 3A. Interestingly, the microarray results identified a set of only 25 genes that were uniquely differentially expressed in the med5/med15 double Degron strain, i.e. genes that were differentially expressed when compared to the wild type, single med5 and single med15 Degron strains (Figure 3A; Table 1). Considering that the med15 Degron strain shows the most severe growth defects of all the single Degron mutant strains, it is interesting to note that only 33 genes (25+5+1+2) were uniquely expressed in the med5/med15 double Degron strain compared to the med15 single Degron strain. In contrast, 481 genes (25+2+119+335) are differentially expressed when comparing the med5/med15 double Degron strain with the med5 single Degron strain and 655 (335+290+25+5) genes are differently expressed when comparing the med5/med15 double Degron strain with the wild type strain. These results provide an explanation why the single med15 Degron strain shows a more profound growth defect than the single med5 strain.


Functional studies of the yeast med5, med15 and med16 mediator tail subunits.

Larsson M, Uvell H, Sandström J, Rydén P, Selth LA, Björklund S - PLoS ONE (2013)

Transcription profile analysis using AffymetrixsYeast Genome 2.0 Array.25 genes were differently regulated in both of the double-Degron strains (med5/med15 (A) and med15/med16 (B)), 45 minutes after induction of degradation, compared to the single Degron (med5, med15 and med16) and wild type (Wt) strains, as shown in the Venn diagrams (FDR<0.05 and FC>abs(log2(1.5)).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750046&req=5

pone-0073137-g003: Transcription profile analysis using AffymetrixsYeast Genome 2.0 Array.25 genes were differently regulated in both of the double-Degron strains (med5/med15 (A) and med15/med16 (B)), 45 minutes after induction of degradation, compared to the single Degron (med5, med15 and med16) and wild type (Wt) strains, as shown in the Venn diagrams (FDR<0.05 and FC>abs(log2(1.5)).
Mentions: In order to identify genes that are differentially regulated and therefore could contribute to the synthetic lethality of cells lacking both Med5/Med15 or Med15/Med16, we used Affymetrix microarrays to compare global gene expression in wild type cells and the different Degron strains. We isolated RNA from each strain 45 minutes after changing from the permissive to the restrictive growth conditions in order to minimize possible secondary effects on gene expression that are not directly related to the Degron construct(s). This time point was chosen based on the experiments shown in Figure 1B, where the levels of each Degron-tagged protein was undetectable at 45 minutes after switching to the restrictive growth conditions. The results from these experiments including all possible combinations of comparisons of expression between wild type cells, single Degron mutants and double Degron mutants are presented in Tables 1, and S1. Genes that were differentially expressed in the med5/med15 strain compared to any individual control strain (WT, med5, and med15 respectively) are depicted as a Venn diagram in Figure 3A. Interestingly, the microarray results identified a set of only 25 genes that were uniquely differentially expressed in the med5/med15 double Degron strain, i.e. genes that were differentially expressed when compared to the wild type, single med5 and single med15 Degron strains (Figure 3A; Table 1). Considering that the med15 Degron strain shows the most severe growth defects of all the single Degron mutant strains, it is interesting to note that only 33 genes (25+5+1+2) were uniquely expressed in the med5/med15 double Degron strain compared to the med15 single Degron strain. In contrast, 481 genes (25+2+119+335) are differentially expressed when comparing the med5/med15 double Degron strain with the med5 single Degron strain and 655 (335+290+25+5) genes are differently expressed when comparing the med5/med15 double Degron strain with the wild type strain. These results provide an explanation why the single med15 Degron strain shows a more profound growth defect than the single med5 strain.

Bottom Line: We also find that all strains in which MED15 is inactivated show down-regulation of genes that have been identified as targets for the Ace2 transcriptional activator protein, which is important for progression through the G1 phase of the cell cycle.Supporting this observation, we demonstrate that loss of Med15 leads to a G1 arrest phenotype.Collectively, these findings provide insight into the function of the Mediator Tail module.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

ABSTRACT
The yeast Mediator complex can be divided into three modules, designated Head, Middle and Tail. Tail comprises the Med2, Med3, Med5, Med15 and Med16 protein subunits, which are all encoded by genes that are individually non-essential for viability. In cells lacking Med16, Tail is displaced from Head and Middle. However, inactivation of MED5/MED15 and MED15/MED16 are synthetically lethal, indicating that Tail performs essential functions as a separate complex even when it is not bound to Middle and Head. We have used the N-Degron method to create temperature-sensitive (ts) mutants in the Mediator tail subunits Med5, Med15 and Med16 to study the immediate effects on global gene expression when each subunit is individually inactivated, and when Med5/15 or Med15/16 are inactivated together. We identify 25 genes in each double mutant that show a significant change in expression when compared to the corresponding single mutants and to the wild type strain. Importantly, 13 of the 25 identified genes are common for both double mutants. We also find that all strains in which MED15 is inactivated show down-regulation of genes that have been identified as targets for the Ace2 transcriptional activator protein, which is important for progression through the G1 phase of the cell cycle. Supporting this observation, we demonstrate that loss of Med15 leads to a G1 arrest phenotype. Collectively, these findings provide insight into the function of the Mediator Tail module.

Show MeSH
Related in: MedlinePlus