Gis1 and Rph1 Regulate Glycerol and Acetate Metabolism in Glucose Depleted Yeast Cells

Aging in organisms as diverse as yeast, nematodes, and mammals is delayed by caloric restriction, an effect mediated by the nutrient sensing TOR, RAS/cAMP, and AKT/Sch9 pathways. The transcription factor Gis1 functions downstream of these pathways in extending the lifespan of nutrient restricted yeast cells, but the mechanisms involved are still poorly understood. We have used gene expression microarrays to study the targets of Gis1 and the related protein Rph1 in different growth phases. Our results show that Gis1 and Rph1 act both as repressors and activators, on overlapping sets of genes as well as on distinct targets. Interestingly, both the activities and the target specificities of Gis1 and Rph1 depend on the growth phase. Thus, both proteins are associated with repression during exponential growth, targeting genes with STRE or PDS motifs in their promoters. After the diauxic shift, both become involved in activation, with Gis1 acting primarily on genes with PDS motifs, and Rph1 on genes with STRE motifs. Significantly, Gis1 and Rph1 control a number of genes involved in acetate and glycerol formation, metabolites that have been implicated in aging. Furthermore, several genes involved in acetyl-CoA metabolism are downregulated by Gis1

Functional studies of RNA polymerase II-dependent transcription in yeast Saccharomyces cerevisiae

Nutrient availability is one of the environmental conditions that have the largest effect on cell growth. The nutrient status is transmitted by several signal transduction pathways, providing an essential framework for the coordination of cell growth. These pathways have been shown to be remarkably well conserved among eukaryotes. Transcriptional initiation is the endpoint for many signal transduction pathways and thus the final answer to an environmental change. The control of gene expression through transcription initiation is therefore one of the most fundamental processes in biology, containing many layers of complexity. I have used the model organism Saccharomyces cerevisiae to study different mechanisms involved in the control of gene expression. The mediator protein complex is required for regulated transcription of nearly all RNA polymerase II-dependent genes. Studies of the Mediator subunit Med21 revealed that it genetically and physically interacts with two other Mediator subunits, Med7 and Med10. A strong Med21 self-interaction was also identified, suggesting a possible dimerisation of Med21. The second part of the study focused on elucidating the function of the split jumonji domain, comprising the jmjN and jmjC subdomains, that has been identified in many transcription factors, by studying its physical interactions. I identified 19 different yeast proteins that could interact either directly or indirectly with the jumonji domain of the zinc finger protein Gis1 in a complex interaction pattern. Gis1 is known to be involved in nutrient dependent regulation of gene expression. Genome-wide microarray analysis was used to investigate the role of Gis1 and the related Rph1 protein in the growth phase-dependent gene regulation. This resulted in the identification of both redundant and unique roles of Gis1 and Rph1 in regulation of genes with an enrichment of STRE and PDS elements in their promoters. One of the proteins that interact directly with the jumonji domain of Gis1 is the unconvential prefoldin-like protein Bud27. Genetic and biochemical methods were used to study the roles of Bud27 and Gis1 in nutrient dependent regulation of gene expression by the TOR signaling pathway

Effects of Gis1 and Rph1 on gene expression.

<p>(A) Clustering of differential gene expression profiles at each growth stage. The heat maps show the log ratios in the following contrasts: <i>gis1 vs.</i> wild type (g), <i>rph1 vs.</i> wild type (r) and <i>gis1 rph1 vs.</i> wild type (gr). Only genes that had a significant differential expression in any of the pairwise strain comparisons were included. Selected clusters are highlighted, with details given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031577#pone-0031577-t001" target="_blank">Tables 1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031577#pone-0031577-t002" target="_blank"></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031577#pone-0031577-t003" target="_blank">3</a>. (B) The number of differentially expressed genes in comparisons between different strains for each growth stage. Only 26 genes are differentially expressed at all three time points, and most genes are only differentially expressed at one time point.</p

Linear model of the effects of STRE and PDS motifs in different orientations on Gis1/Rph1-dependent gene expression.

<p>For each contrast, the effect of each orientation of the STRE and PDS motifs on gene expression (log ratios) in different contrasts are shown, along with ANOVA p-values. Statistically significant values (at the 0.001 level) are highlighted in bold face.</p

Model for gene regulation by Gis1 and Rph1 in different growth phases.

<p>Activation of transcription acting through a STRE or PDS motif is shown as an arrow, and repression as a cross-bar. A dashed line indicates a minor effect of Gis1 on STRE-mediated activation after the diauxic shift, which is only visible in an <i>rph1</i> mutant background.</p

Average orientations of STRE and PDS motifs in the promoters of different sets of genes.

<p>For each group of genes, the average number of forward and reverse oriented STRE and PDS motifs per promoter is shown, as well as the ratio between forward and reverse oriented motifs, with p-values for the deviation from the ratio in all ORFs. Deviations that are significant at p<0.05 are shown in bold face.</p>a<p>Up in gis1-wt or gis1rph1-rph1.</p>b<p>Up in rph1-wt or gis1rph1-gis1.</p>c<p>Down in gis1-wt or gis1rph1-rph1.</p>d<p>Down in rph1-wt or gis1rph1-gis1.</p

Growth curves and <i>SSA3</i> gene expression.

<p>(A) Growth curve for yeast strain BY4742 grown in batch culture for three days. (B) <i>SSA3</i> expression over time. Samples were harvested at an OD₆₀₀ of 0.4 (time zero) and at different later time points as indicated in the figure. The amount of <i>SSA3</i> mRNA at each time point was measured using reverse transcriptase-PCR with specific oligonucleotide primers. The induction of <i>SSA3</i> expression coincides with the diauxic shift-associated decrease in the growth rate. Expression of the <i>ACT1</i> gene encoding actin was included as a control.</p

Effects of Gis1 and Rph1 on glycerol, acetate and acetyl-CoA metabolism.

<p>(A) Effects of <i>gis1</i> and <i>rph1</i> mutations on gene expression after the diauxic shift. The columns show fold changes and p-values for different genetic contrasts in PDS cells and after 3 days of culture. Genes that are significantly (p<0.02) downregulated in a mutant are shown in green and those that are upregulated in red. Genes are shown in red/green are upregulated in the <i>gis1</i> mutant and downregulated in the <i>rph1</i> mutant. Also shown are the number of STRE and PDS motifs in each promoter. (B) Glycerol and acetate metabolic pathways in yeast. The effects of Gis1 and Rph1 on genes encoding some key enzymes are shown. It should be noted that regulation in several cases is likely to be indirect, being mediated by the repression or activation of some other transcription factor.</p

Expression details for the 6 log phase (L) gene clusters identified in Figure 4A.

<p>Strongly enriched functions and genes are shown in bold face.</p>a<p>In the group of all promoters, there are on average 0.47 STRE motifs/promoter. Enrichments significant at p<0.05 are shown in bold face.</p>b<p>In the group of all promoters, there are on average 0.15 PDS motifs/promoter. Enrichments significant at p<0.05 are shown in bold face.</p>c<p>Significantly lower occurrence of the motif than in the reference group of all promoters.</p

Enrichment of STRE and PDS motifs in differentially expressed promoters.

<p>For each group of differentially expressed genes, the average number of STRE and PDS motifs per promoter, along with p-values for the enrichment of STRE and PDS motifs is given. For comparison, among all yeast promoters, there is on average 0.47 STRE motifs and 0.15 PDS motifs per promoter. Enrichments that are significant at p<0.05 are shown in bold face.</p