MSN2 and MSN4 link calorie restriction and TOR to sirtuin-mediated lifespan extension in Saccharomyces cerevisiae

Abstract

Calorie restriction (CR) robustly extends the lifespan of numerous species. In the yeast Saccharomyces cerevisiae, CR has been proposed to extend lifespan by boosting the activity of sirtuin deacetylases, thereby suppressing the formation of toxic repetitive ribosomal DNA (rDNA) circles. An alternative theory is that CR works by suppressing the TOR (target of rapamycin) signaling pathway, which extends lifespan via mechanisms that are unknown but thought to be independent of sirtuins. Here we show that TOR inhibition extends lifespan by the same mechanism as CR: by increasing Sir2p activity and stabilizing the rDNA locus. Further, we show that rDNA stabilization and lifespan extension by both CR and TOR signaling is due to the relocalization of the transcription factors Msn2p and Msn4p from the cytoplasm to the nucleus, where they increase expression of the nicotinamidase gene PNC1. These findings suggest that TOR and sirtuins may be part of the same longevity pathway in higher organisms, and that they may promote genomic stability during aging.

Figure 1. MSN2/4 Are Required for Lifespan Extension by CR and Rapamycin

(A) MSN2/4 are required for lifespan extension by CR (growth on 0.5% glucose). Average lifespans: W303 (wild-type [wt]) on 2% glucose, 25.0 divisions; W303 on CR diet, 30.8; msn2Δ/4Δ on 2% glucose, 24.2; and msn2Δ/4Δ on CR diet, 23.9. (B) The lifespans of msn2Δ and msn4Δ single mutants can be extended by CR. Average lifespans: msn2Δ on 2% glucose, 22.5 divisions; msn2Δ on CR diet, 29.9; msn4Δ on 2% glucose, 24.7; and msn4Δ on CR diet, 33.2. (C) MSN2/4 are required for lifespan extension by rapamycin (Rap). Average lifespans: wild-type, 23.3 divisions; wild-type + 1 nM rapamycin, 26.9; msn2Δ/4Δ, 23.8; and msn2Δ/4Δ + 1 nM rapamycin, 22.5.

Figure 2. Effect of TOR Signaling on Sir2p Functions Requires MSN2/4 and PNC1

(A) Rapamycin and CR suppress rDNA recombination in an MSN2/4-dependent manner. (B) Deletion of PNC1 or treatment with NAM blocks the ability of CR or rapamycin treatment to lower rDNA recombination. (C) CR and rapamycin treatment do not increase Sir2p levels. (D) Deletion of PNC1 blocks the ability of rapamycin to extend lifespan. Average lifespans: wild-type (wt), 23.3 divisions; wild-type + 1 nM rapamycin, 26.9; pnc1Δ, 20.9; and pnc1Δ + 1 nM rapamycin, 21.4. (E) Overexpression (o/e) of PNC1 suppresses rDNA recombination in both wild-type and msn2Δ/4Δ strains. (F) Expression of PNC1 can extend the lifespan of a strain lacking MSN2/4. Average lifespans: wild-type, 23.2 divisions; wild-type + PNC1, 28.0; msn2Δ/4Δ, 22.0; and msn2Δ/4Δ + PNC1, 27.3. Glu, glucose; Rap, rapamycin.

Figure 3. MSN2/4 Regulate the Expression of PNC1

(A) Expression of Pnc1p-GFP is induced by CR and by rapamycin. (B) Western blotting shows that CR and rapamycin induce high levels of Pnc1p in an MSN2/4-dependent manner. (C) A cdc25–10 mutant has increased expression of PNC1 but not if MSN2/4 are deleted. (D) Increased expression of PNC1 in response to salt stress, osmotic stress, and amino acid restriction requires MSN2/4, but increased expression of PNC1 in response to heat shock does not. (E) MSN2/4 are not required for lifespan extension by heat shock. Average lifespans: wild-type at 30 °C, 22.2 divisions; wild-type at 37 °C, 26.8; msn2Δ/4Δ at 30 °C, 20.7; and msn2Δ/4Δ at 37 °C, 25.8. (F) Heat shock induction of PNC1 in an msn2Δ/4Δ strain is dependent upon HSF1. 2/4Δ, msn2Δ/4Δ; dox, doxycycline; Glu, glucose; low a.a., low amino acids; rap, rapamycin; Sorb, sorbitol; wt, wild-type.

Figure 4. Msn2p Localizes to the Nucleus during CR

(A) CR promotes increased nuclear migration of Msn2p-GFP in a dose-dependent manner. (B) Quantification of the percent of cells with predominately nuclear Msn2p-GFP or Msn4p-GFP. (C) Deletion of TOR1 promotes nuclear localization of Msn2p-GFP. (D) A cdc25–10 mutant has increased nuclear localization of Msn2p-GFP.

Figure 5. CR Induces Nucleo-Cytoplasmic Oscillations of Msn2p-GFP

During CR, Msn2p-GFP oscillates between the cytoplasm and nucleus. (A and B) Cells grown at 2% glucose concentrations (A) exhibit exclusively cytoplasmic localization of Msn2p-GFP. Cells placed in medium containing 0% glucose (B) exhibit exclusively nuclear localization of Msn2p-GFP. For cells in 2% glucose or 0% glucose, a plot of the ratio of average nuclear intensity versus average cytoplasmic intensity for a single representative cell, labeled with an arrowhead in the respective photomicrographs, is shown. (C) During CR conditions, cells exhibit nucleo-cytoplasmic oscillations of Msn2p-GFP with a periodicity of ∼2–3 min. A representative population of cells is shown at 0.1% glucose concentrations. The ratios of average nuclear intensity versus average cytoplasmic intensity for three representative cells, labeled a, b, and c, are shown plotted over the course of 20 min beneath the photomicrographs.

Figure 6. Deletion of MSN2/4 Abrogates Expression of LacZ Reporter Constructs Driven by the PNC1 Promoter

(A) Reporter constructs for analysis of STRE and Msn2p/4p function. (B) β-galactosidase activity of reporter constructs in wild-type (wt) cells grown in 2% YPD (Glu) (white bars), 0.5% YPD (black bars), or 2%YPD at 37 °C (grey bars). (C–E) Activity of reporter constructs in (C) an msn2Δ/4Δ strain, (D) an msn2Δ strain, or (E) an msn4Δ strain. Activity is listed as nanomoles of cleaved o-nitrophenol-β-D-galactopyranoside substrate per minute per milligram total protein.

Figure 7. Msn2p Directly Regulates PNC1

(A) Nuclear localization of Msn2p is sufficient to induce PNC1. (B) Chromatin immunoprecipitation analysis of Msn2p-HA binding to the PNC1 promoter. IN, input; IP, immunoprecipitation. (C) Relative enrichment of Msn2p-HA at the PNC1 promoter shows increased promoter binding during CR.

Figure 8. Model of How TOR Signaling and CR Regulate Yeast Replicative Lifespan

CR and TOR signaling regulate the nuclear localization of Msn2p/4p. When localized to the nucleus, Msn2p/4p promote the transcription of PNC1, a nicotinamidase. The removal of NAM by Pnc1p increases the activity of sirtuins, including Sir2p and Hst2p, which promote longevity by stabilizing the yeast rDNA array and preventing the formation of ERCs. TOR signaling may also regulate lifespan by sirtuin-independent pathways, such as the regulation of ribosomal biogenesis.