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Calorie restriction reduces rDNA recombination independently of rDNA silencing.

Riesen M, Morgan A - Aging Cell (2009)

Bottom Line: Although accumulation of extrachromosomal rDNA circles is specific to yeast aging, it is thought that Sirtuin activation represents a conserved longevity mechanism through which the beneficial effects of CR are mediated in various species.We show here that growing yeast on 0.05 or 0.5% glucose (severe and moderate CR, respectively) does not increase silencing at either sub-telomeric or rDNA loci compared with standard (2% glucose) media.In contrast, CR and deletion of the FOB1, HXK2, SCH9 and TOR1 genes, all significantly reduced rDNA recombination.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool, UK.

ABSTRACT
Calorie restriction (CR) extends lifespan in yeast, worms, flies and mammals, suggesting that it acts via a conserved mechanism. In yeast, activation of the NAD-dependent histone deacetylase, Sir2, by CR is thought to increase silencing at the ribosomal DNA, thereby reducing the recombination-induced generation of extrachromosomal rDNA circles, hence increasing replicative lifespan. Although accumulation of extrachromosomal rDNA circles is specific to yeast aging, it is thought that Sirtuin activation represents a conserved longevity mechanism through which the beneficial effects of CR are mediated in various species. We show here that growing yeast on 0.05 or 0.5% glucose (severe and moderate CR, respectively) does not increase silencing at either sub-telomeric or rDNA loci compared with standard (2% glucose) media. Furthermore, rDNA silencing was unaffected in the hxk2Delta, sch9Delta and tor1Delta genetic mimics of CR, but inhibited by FOB1 deletion. All these interventions extend lifespan in multiple yeast backgrounds, revealing a poor correlation between rDNA silencing and longevity. In contrast, CR and deletion of the FOB1, HXK2, SCH9 and TOR1 genes, all significantly reduced rDNA recombination. This silencing-independent mechanism for suppressing rDNA recombination may therefore contribute to CR-mediated lifespan extension.

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Calorie restriction does not affect subtelomeric gene silencing. (A) Qualitative silencing assay. The telomeric silencing reporter parent strain, AEY1017, and its isogenic sir2Δ strain were grown up in YPD, then diluted in H2O and serially spotted out on synthetic complete media (SC) and SC + FOA media (FOA). Spots correspond to OD600 = 1 (left) then serial 1 in 5 dilutions to the right. Plates were imaged after 2 (0.5% and 2% glucose) and 3 days (0.05% glucose) at 30 °C. Reduced growth on FOA compared with SC indicates reduced silencing. (B) Quantitative silencing assay. Serial dilutions of overnight cultures were spread on plates and colonies were counted after 2 (0.5% and 2% glucose) or 3 days (0.05% glucose). Silencing is proportional to viability on FOA media, which was calculated by dividing the numbers of colonies on FOA plates by the total numbers of colonies on −ura and FOA plates combined. 62 000 colonies were counted in total. The graph shows pooled data normalized to the 2% glucose control value, expressed as mean + standard error of the mean. No statistically significant difference between the conditions was found.
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fig02: Calorie restriction does not affect subtelomeric gene silencing. (A) Qualitative silencing assay. The telomeric silencing reporter parent strain, AEY1017, and its isogenic sir2Δ strain were grown up in YPD, then diluted in H2O and serially spotted out on synthetic complete media (SC) and SC + FOA media (FOA). Spots correspond to OD600 = 1 (left) then serial 1 in 5 dilutions to the right. Plates were imaged after 2 (0.5% and 2% glucose) and 3 days (0.05% glucose) at 30 °C. Reduced growth on FOA compared with SC indicates reduced silencing. (B) Quantitative silencing assay. Serial dilutions of overnight cultures were spread on plates and colonies were counted after 2 (0.5% and 2% glucose) or 3 days (0.05% glucose). Silencing is proportional to viability on FOA media, which was calculated by dividing the numbers of colonies on FOA plates by the total numbers of colonies on −ura and FOA plates combined. 62 000 colonies were counted in total. The graph shows pooled data normalized to the 2% glucose control value, expressed as mean + standard error of the mean. No statistically significant difference between the conditions was found.

Mentions: If CR acts via Sir2 activation (Fig. 1, models 1 and 2), then this would be predicted to result in increased silencing at subtelomeric loci. However, it has recently been claimed that severe CR imposed by growth on 0.05% glucose has no effect on telomeric silencing in the PSY316 yeast strain (Kaeberlein et al., 2005c). We therefore investigated the effects of both moderate (0.5% glucose) and severe (0.05% glucose) CR on telomeric silencing using a well established reporter strain, AEY1017, which has a URA3 marker integrated into a subtelomeric region of chromosome VII (Meijsing & Ehrenhofer-Murray, 2001). Expression of the URA3 gene from nonsilenced genomic loci enables growth on media lacking uracil, but prevents growth on media containing FOA, because of conversion to the toxic product, 5-fluorouracil. However, integration of URA3 into subtelomeric regions results in partial silencing of the reporter gene and hence the unusual ability to grow both on media lacking uracil and on media containing FOA. Deletion of the SIR2 gene inhibits telomeric silencing, resulting in increased expression of the URA3 reporter gene, which is routinely visualized as decreased growth on FOA. Indeed, no growth was observed on FOA plates for the sir2 deletion strain (Fig. 2A), thus confirming the validity of this approach for assaying in vivo Sir2 activity. In contrast to the profound effect of sir2 deletion on silencing, no obvious difference in growth on FOA plates was seen between 2%, 0.5% and 0.05% glucose (Fig. 2A).


Calorie restriction reduces rDNA recombination independently of rDNA silencing.

Riesen M, Morgan A - Aging Cell (2009)

Calorie restriction does not affect subtelomeric gene silencing. (A) Qualitative silencing assay. The telomeric silencing reporter parent strain, AEY1017, and its isogenic sir2Δ strain were grown up in YPD, then diluted in H2O and serially spotted out on synthetic complete media (SC) and SC + FOA media (FOA). Spots correspond to OD600 = 1 (left) then serial 1 in 5 dilutions to the right. Plates were imaged after 2 (0.5% and 2% glucose) and 3 days (0.05% glucose) at 30 °C. Reduced growth on FOA compared with SC indicates reduced silencing. (B) Quantitative silencing assay. Serial dilutions of overnight cultures were spread on plates and colonies were counted after 2 (0.5% and 2% glucose) or 3 days (0.05% glucose). Silencing is proportional to viability on FOA media, which was calculated by dividing the numbers of colonies on FOA plates by the total numbers of colonies on −ura and FOA plates combined. 62 000 colonies were counted in total. The graph shows pooled data normalized to the 2% glucose control value, expressed as mean + standard error of the mean. No statistically significant difference between the conditions was found.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2805862&req=5

fig02: Calorie restriction does not affect subtelomeric gene silencing. (A) Qualitative silencing assay. The telomeric silencing reporter parent strain, AEY1017, and its isogenic sir2Δ strain were grown up in YPD, then diluted in H2O and serially spotted out on synthetic complete media (SC) and SC + FOA media (FOA). Spots correspond to OD600 = 1 (left) then serial 1 in 5 dilutions to the right. Plates were imaged after 2 (0.5% and 2% glucose) and 3 days (0.05% glucose) at 30 °C. Reduced growth on FOA compared with SC indicates reduced silencing. (B) Quantitative silencing assay. Serial dilutions of overnight cultures were spread on plates and colonies were counted after 2 (0.5% and 2% glucose) or 3 days (0.05% glucose). Silencing is proportional to viability on FOA media, which was calculated by dividing the numbers of colonies on FOA plates by the total numbers of colonies on −ura and FOA plates combined. 62 000 colonies were counted in total. The graph shows pooled data normalized to the 2% glucose control value, expressed as mean + standard error of the mean. No statistically significant difference between the conditions was found.
Mentions: If CR acts via Sir2 activation (Fig. 1, models 1 and 2), then this would be predicted to result in increased silencing at subtelomeric loci. However, it has recently been claimed that severe CR imposed by growth on 0.05% glucose has no effect on telomeric silencing in the PSY316 yeast strain (Kaeberlein et al., 2005c). We therefore investigated the effects of both moderate (0.5% glucose) and severe (0.05% glucose) CR on telomeric silencing using a well established reporter strain, AEY1017, which has a URA3 marker integrated into a subtelomeric region of chromosome VII (Meijsing & Ehrenhofer-Murray, 2001). Expression of the URA3 gene from nonsilenced genomic loci enables growth on media lacking uracil, but prevents growth on media containing FOA, because of conversion to the toxic product, 5-fluorouracil. However, integration of URA3 into subtelomeric regions results in partial silencing of the reporter gene and hence the unusual ability to grow both on media lacking uracil and on media containing FOA. Deletion of the SIR2 gene inhibits telomeric silencing, resulting in increased expression of the URA3 reporter gene, which is routinely visualized as decreased growth on FOA. Indeed, no growth was observed on FOA plates for the sir2 deletion strain (Fig. 2A), thus confirming the validity of this approach for assaying in vivo Sir2 activity. In contrast to the profound effect of sir2 deletion on silencing, no obvious difference in growth on FOA plates was seen between 2%, 0.5% and 0.05% glucose (Fig. 2A).

Bottom Line: Although accumulation of extrachromosomal rDNA circles is specific to yeast aging, it is thought that Sirtuin activation represents a conserved longevity mechanism through which the beneficial effects of CR are mediated in various species.We show here that growing yeast on 0.05 or 0.5% glucose (severe and moderate CR, respectively) does not increase silencing at either sub-telomeric or rDNA loci compared with standard (2% glucose) media.In contrast, CR and deletion of the FOB1, HXK2, SCH9 and TOR1 genes, all significantly reduced rDNA recombination.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool, UK.

ABSTRACT
Calorie restriction (CR) extends lifespan in yeast, worms, flies and mammals, suggesting that it acts via a conserved mechanism. In yeast, activation of the NAD-dependent histone deacetylase, Sir2, by CR is thought to increase silencing at the ribosomal DNA, thereby reducing the recombination-induced generation of extrachromosomal rDNA circles, hence increasing replicative lifespan. Although accumulation of extrachromosomal rDNA circles is specific to yeast aging, it is thought that Sirtuin activation represents a conserved longevity mechanism through which the beneficial effects of CR are mediated in various species. We show here that growing yeast on 0.05 or 0.5% glucose (severe and moderate CR, respectively) does not increase silencing at either sub-telomeric or rDNA loci compared with standard (2% glucose) media. Furthermore, rDNA silencing was unaffected in the hxk2Delta, sch9Delta and tor1Delta genetic mimics of CR, but inhibited by FOB1 deletion. All these interventions extend lifespan in multiple yeast backgrounds, revealing a poor correlation between rDNA silencing and longevity. In contrast, CR and deletion of the FOB1, HXK2, SCH9 and TOR1 genes, all significantly reduced rDNA recombination. This silencing-independent mechanism for suppressing rDNA recombination may therefore contribute to CR-mediated lifespan extension.

Show MeSH
Related in: MedlinePlus