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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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Flow cytometry analyses of Degron constructs.DNA content of cells carrying the indicated Degron constructs was analyzed by flow cytometry at 3 hours after switching from the permissive to the restrictive growth conditions. Numbers below each histogram indicate the percentage of cells in the G1-, the S-, and the G2+M-phases, respectively.
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pone-0073137-g005: Flow cytometry analyses of Degron constructs.DNA content of cells carrying the indicated Degron constructs was analyzed by flow cytometry at 3 hours after switching from the permissive to the restrictive growth conditions. Numbers below each histogram indicate the percentage of cells in the G1-, the S-, and the G2+M-phases, respectively.

Mentions: Several of the genes that we identified as down regulated in the med15 single Degron strain and the med5/med15 and med15/med16 double Degron strains, encode proteins that function in cell wall organization, cell wall biogenesis and cell division (Tables 1and S1). Notably, the Ace2 transcription factor described above is well known for its involvement in cell cycle regulation [41]. We therefore used flow cytometry analyses to study if these strains showed any cell cycle defects. At 3 hours after switching from the permissive conditions to the restrictive conditions, we found that all three strains expressing the MED15 Degron construct behaved differently from the strains that do not contain the MED15 Degron (Figure 5). More specifically, in the three strains that contain the MED15 Degron the fraction of cells in the S/G2 phases was only 22–24%, compared to more than 50% in the strains with normal Med15 expression. This indicates that loss of Med15 leads to a delay in cell cycle progression by arresting in G1 phase [42]. Interestingly, this correlates well with a previous report demonstrating that Ace2 activates transcription of G1 specific genes [34]. These results suggest that the requirement of Med15 for Ace2-mediated transcriptional activation may be reflected by the observed cell cycle defects in the strains expressing Degron-tagged Med15.


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)

Flow cytometry analyses of Degron constructs.DNA content of cells carrying the indicated Degron constructs was analyzed by flow cytometry at 3 hours after switching from the permissive to the restrictive growth conditions. Numbers below each histogram indicate the percentage of cells in the G1-, the S-, and the G2+M-phases, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073137-g005: Flow cytometry analyses of Degron constructs.DNA content of cells carrying the indicated Degron constructs was analyzed by flow cytometry at 3 hours after switching from the permissive to the restrictive growth conditions. Numbers below each histogram indicate the percentage of cells in the G1-, the S-, and the G2+M-phases, respectively.
Mentions: Several of the genes that we identified as down regulated in the med15 single Degron strain and the med5/med15 and med15/med16 double Degron strains, encode proteins that function in cell wall organization, cell wall biogenesis and cell division (Tables 1and S1). Notably, the Ace2 transcription factor described above is well known for its involvement in cell cycle regulation [41]. We therefore used flow cytometry analyses to study if these strains showed any cell cycle defects. At 3 hours after switching from the permissive conditions to the restrictive conditions, we found that all three strains expressing the MED15 Degron construct behaved differently from the strains that do not contain the MED15 Degron (Figure 5). More specifically, in the three strains that contain the MED15 Degron the fraction of cells in the S/G2 phases was only 22–24%, compared to more than 50% in the strains with normal Med15 expression. This indicates that loss of Med15 leads to a delay in cell cycle progression by arresting in G1 phase [42]. Interestingly, this correlates well with a previous report demonstrating that Ace2 activates transcription of G1 specific genes [34]. These results suggest that the requirement of Med15 for Ace2-mediated transcriptional activation may be reflected by the observed cell cycle defects in the strains expressing Degron-tagged Med15.

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