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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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Confirmation of Ace2 target genes.mRNA levels of the genes CTS1, EXG1 and YHB1 from WT and Degron-strains were measured using qPCR and normalized against the WT level. qPCR levels are compared to the levels determined in the corresponding microarray assays. The experiments were performed in biological triplicates, and error bars represent the standard deviation. P-values where calculated using Student’s t-test. * Indicates p-value < 0.05, ** indicates p-value <0.01.
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pone-0073137-g004: Confirmation of Ace2 target genes.mRNA levels of the genes CTS1, EXG1 and YHB1 from WT and Degron-strains were measured using qPCR and normalized against the WT level. qPCR levels are compared to the levels determined in the corresponding microarray assays. The experiments were performed in biological triplicates, and error bars represent the standard deviation. P-values where calculated using Student’s t-test. * Indicates p-value < 0.05, ** indicates p-value <0.01.

Mentions: DSE1 and SCW11, which are specifically down regulated in the med5/med15 double Degron strain (Table 1), have both been identified as two of a total of 22 targets genes for the transcriptional activator Ace2 [33], [34]. In order to study the potential involvement of Ace2 in the synthetic lethality, we examined our microarray data in more detail. Remarkably, we found that 11 of the 22 previously identified Ace2 target genes were significantly down regulated in both the med5/med15 and med15/med16 double Degron mutants when compared only to the wild-type strain (Table 2). Several of these genes (i.e. CLN3, CTS1, AMN1, EGT2, DSE1, SCW11) have been shown to be induced in G1 and are required for normal progression through G1 [35]–[40]. The reason why these genes were not initially scored as uniquely regulated in the double Degron strains is that several genes that were significantly down regulated in the double Degron strains were down regulated to some extent already in the med15 single Degron strain. Down-regulation of three of the Ace2-target genes was verified using qPCR (Figure 4). One obvious explanation for this finding is that Ace2 is itself affected by loss of Med15. However, we found no effects of changes in Ace2 expression in any of our Degron strains and we thus conclude that the observed effects are most likely caused by a requirement of Med15 for the ability of Ace2 to affect its target promoters, rather than a requirement of Med15 for expression of ACE2. Irrespective of mechanism, our results indicate that the Med15 subunit is required for activation of genes in the Ace2 pathway.


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)

Confirmation of Ace2 target genes.mRNA levels of the genes CTS1, EXG1 and YHB1 from WT and Degron-strains were measured using qPCR and normalized against the WT level. qPCR levels are compared to the levels determined in the corresponding microarray assays. The experiments were performed in biological triplicates, and error bars represent the standard deviation. P-values where calculated using Student’s t-test. * Indicates p-value < 0.05, ** indicates p-value <0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073137-g004: Confirmation of Ace2 target genes.mRNA levels of the genes CTS1, EXG1 and YHB1 from WT and Degron-strains were measured using qPCR and normalized against the WT level. qPCR levels are compared to the levels determined in the corresponding microarray assays. The experiments were performed in biological triplicates, and error bars represent the standard deviation. P-values where calculated using Student’s t-test. * Indicates p-value < 0.05, ** indicates p-value <0.01.
Mentions: DSE1 and SCW11, which are specifically down regulated in the med5/med15 double Degron strain (Table 1), have both been identified as two of a total of 22 targets genes for the transcriptional activator Ace2 [33], [34]. In order to study the potential involvement of Ace2 in the synthetic lethality, we examined our microarray data in more detail. Remarkably, we found that 11 of the 22 previously identified Ace2 target genes were significantly down regulated in both the med5/med15 and med15/med16 double Degron mutants when compared only to the wild-type strain (Table 2). Several of these genes (i.e. CLN3, CTS1, AMN1, EGT2, DSE1, SCW11) have been shown to be induced in G1 and are required for normal progression through G1 [35]–[40]. The reason why these genes were not initially scored as uniquely regulated in the double Degron strains is that several genes that were significantly down regulated in the double Degron strains were down regulated to some extent already in the med15 single Degron strain. Down-regulation of three of the Ace2-target genes was verified using qPCR (Figure 4). One obvious explanation for this finding is that Ace2 is itself affected by loss of Med15. However, we found no effects of changes in Ace2 expression in any of our Degron strains and we thus conclude that the observed effects are most likely caused by a requirement of Med15 for the ability of Ace2 to affect its target promoters, rather than a requirement of Med15 for expression of ACE2. Irrespective of mechanism, our results indicate that the Med15 subunit is required for activation of genes in the Ace2 pathway.

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