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. 2012;8(8):e1002834.
doi: 10.1371/journal.pgen.1002834. Epub 2012 Aug 9.

Dissecting the Gene Network of Dietary Restriction to Identify Evolutionarily Conserved Pathways and New Functional Genes

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Free PMC article

Dissecting the Gene Network of Dietary Restriction to Identify Evolutionarily Conserved Pathways and New Functional Genes

Daniel Wuttke et al. PLoS Genet. .
Free PMC article

Abstract

Dietary restriction (DR), limiting nutrient intake from diet without causing malnutrition, delays the aging process and extends lifespan in multiple organisms. The conserved life-extending effect of DR suggests the involvement of fundamental mechanisms, although these remain a subject of debate. To help decipher the life-extending mechanisms of DR, we first compiled a list of genes that if genetically altered disrupt or prevent the life-extending effects of DR. We called these DR-essential genes and identified more than 100 in model organisms such as yeast, worms, flies, and mice. In order for other researchers to benefit from this first curated list of genes essential for DR, we established an online database called GenDR (http://genomics.senescence.info/diet/). To dissect the interactions of DR-essential genes and discover the underlying lifespan-extending mechanisms, we then used a variety of network and systems biology approaches to analyze the gene network of DR. We show that DR-essential genes are more conserved at the molecular level and have more molecular interactions than expected by chance. Furthermore, we employed a guilt-by-association method to predict novel DR-essential genes. In budding yeast, we predicted nine genes related to vacuolar functions; we show experimentally that mutations deleting eight of those genes prevent the life-extending effects of DR. Three of these mutants (OPT2, FRE6, and RCR2) had extended lifespan under ad libitum, indicating that the lack of further longevity under DR is not caused by a general compromise of fitness. These results demonstrate how network analyses of DR using GenDR can be used to make phenotypically relevant predictions. Moreover, gene-regulatory circuits reveal that the DR-induced transcriptional signature in yeast involves nutrient-sensing, stress responses and meiotic transcription factors. Finally, comparing the influence of gene expression changes during DR on the interactomes of multiple organisms led us to suggest that DR commonly suppresses translation, while stimulating an ancient reproduction-related process.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. DR–essential genes are molecularly conserved and form a tight interaction network.
A, The proportion of genes (shown as percentages) with orthologs from multiple species in the specified organisms is in all cases higher (by 10–30%) for DR-essential genes than expected by chance. B, The average degree (number of interactions) for DR-essential genes and DR-essential gene orthologs (the ortholog-complemented set) is higher than the interactome-wide average which serves as control. C, The average percentage of specificity in interconnectivity (100× specific interactions with seed genes/all interactions) of DR-essential genes to each other is higher than for other genes in interactomes, and complementation with orthologs of DR-essential genes from other species further increases the specificity of interconnectivity. ns, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001.
Figure 2
Figure 2. DR–essential vacuole-associated genes are required for DR lifespan extension and normal vacuolar morphology.
A, Survival curves of wild-type and rcr2Δ mutant strains measured on AL/YEPD and DR media. The units of age (X axis) are generations. Mean and maximum lifespans as well as sample sizes are listed in Table 2. B, Vacuolar morphology on AL and DR media of wild-type and rcr2Δ. Left panel: DIC images showing the cells; middle panel: TRITC-fluorescent images showing the vacuoles; right panel: overlay of cell and vacuole images.
Figure 3
Figure 3. Comparing the influence of gene-expression changes upon DR, in multiple interactomes.
The interactomes of yeast, worm, and fly were integrated with gene expression information upon DR and condensed interaction networks generated via the ExprEssence algorithm (Step 1). Interaction networks restricted to either suppressed or stimulated interactions for each species were created (Step 2). The suppressed and stimulated networks were then separately compared for common significant functional enrichments (Step 3). Functional terms which were common to suppressed and induced interactions upon DR in yeast, worm, and fly are listed with their respective p-values (Tables 5 and 6).
Figure 4
Figure 4. Nuclear phosphorylation network of yeast.
DR–upregulated genes are shown in red and downregulated genes in green. Kinases are marked by a golden halo.
Figure 5
Figure 5. Localization of vacuole-related DR–essential proteins and lipids in yeast cells.
In response to environmental triggers such as DR, membrane transporters (Opt2 and others) on the plasma membrane are endocytosed through lipid rafts (ergosterol-sphingolipids) after ubiquitinylation. On early endosomes, proteins and lipids are sorted to late endosomes, Golgi, or other organelles. In late endosomes, different endosomal sorting complexes required for transport (ESCRT) push the target proteins with their membranes into the lumen of late-endosome, or multivesicular bodies (MVB). Matured MVB can fuse with vacuoles, within which proteases and lipases (Atg15) degrade target proteins and lipids. In mammalian cells, maturation of late endosomes is essential for activation of the master regulator for growth, the mammalian target of rapamycin 1 (mTORC1). In yeast cells, TORC1 suppresses meiotic transcription factors which are normally only active during gametogenesis . The inhibitory effects of DR on TORC1 thus activate these rejuvenation genes. EE: early endosome. LE: late endosome. MVB: multivesicular body. Black arrows indicate movement of vesicles. DR-essential proteins are labelled in their localized organelles. Localization data were extracted from the SGD database. Ypt7 localization on late endosomes was reported by . Green arrows indicate activation and a red line indicates an inhibitory effect.

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