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Rapamycin increases rDNA stability by enhancing association of Sir2 with rDNA in Saccharomyces cerevisiae.

Ha CW, Huh WK - Nucleic Acids Res. (2010)

Bottom Line: TORC1 inhibition increases transcriptional silencing of RNA polymerase II-transcribed gene integrated at the rDNA locus and reduces homologous recombination between rDNA repeats that causes formation of toxic extrachromosomal rDNA circles.In addition, TORC1 inhibition induces deacetylation of histones at rDNA.Taken together, our findings suggest that inhibition of TORC1 signaling stabilizes the rDNA locus by enhancing association of Sir2 with rDNA, thereby leading to extension of replicative lifespan in S. cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Research Center for Functional Cellulomics, Institute of Microbiology, Seoul National University, Seoul 151-747, Republic of Korea.

ABSTRACT
The target of rapamycin (TOR) kinase is an evolutionarily conserved key regulator of eukaryotic cell growth and proliferation. Recently, it has been reported that inhibition of TOR signaling pathway can delay aging and extend lifespan in several eukaryotic organisms, but how lifespan extension is mediated by inhibition of TOR signaling is poorly understood. Here we report that rapamycin treatment and nitrogen starvation, both of which cause inactivation of TOR complex 1 (TORC1), lead to enhanced association of Sir2 with ribosomal DNA (rDNA) in Saccharomyces cerevisiae. TORC1 inhibition increases transcriptional silencing of RNA polymerase II-transcribed gene integrated at the rDNA locus and reduces homologous recombination between rDNA repeats that causes formation of toxic extrachromosomal rDNA circles. In addition, TORC1 inhibition induces deacetylation of histones at rDNA. We also found that Pnc1 and Net1 are required for enhancement of association of Sir2 with rDNA under TORC1 inhibition. Taken together, our findings suggest that inhibition of TORC1 signaling stabilizes the rDNA locus by enhancing association of Sir2 with rDNA, thereby leading to extension of replicative lifespan in S. cerevisiae.

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Pnc1 is required for enhancement of Sir2 binding to rDNA under TORC1 inhibition. (A) PNC1 overexpression enhances association of Sir2 with rDNA. The degree of Sir2 binding to rDNA was measured using the ChIP assay in wild-type, pnc1Δ, pnc1Δ cells containing an empty vector and pnc1Δ cells expressing Pnc1-GFP on the p416GPD vector. (B) PNC1 overexpression lowers the H3 acetylation level at rDNA. The acetylation level of histone H3 at the rDNA regions was measured using the ChIP assay in wild-type, pnc1Δ, pnc1Δ cells containing an empty vector and pnc1Δ cells expressing Pnc1-GFP on the p416GPD vector. (C) SIR2 overexpression enhances association of Sir2 with rDNA. The degree of Sir2 binding to rDNA was measured using the ChIP assay in wild-type and sir2Δ cells expressing Sir2-TAP on the p416GPD vector. (D) SIR2 overexpression lowers the H3 acetylation level at rDNA. The acetylation level of histone H3 at the rDNA regions was measured using the ChIP assay in wild-type and sir2Δ cells expressing Sir2-TAP on the p416GPD vector. (E) Rapamycin does not enhance association of Sir2 with rDNA in pnc1Δ cells. The degree of Sir2 binding to rDNA was measured using the ChIP assay in wild-type and pnc1Δ cells after treatment with or without 200 ng/ml rapamycin for 1 h. (F) Rapamycin does not lower the H3 acetylation level at rDNA in pnc1Δ cells. The acetylation level of histone H3 at the rDNA regions was measured using the ChIP assay in wild-type and pnc1Δ cells after treatment with or without 200 ng/ml rapamycin for 1 h. Values represent the average of three independent experiments and error bars indicate standard deviations.
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Figure 4: Pnc1 is required for enhancement of Sir2 binding to rDNA under TORC1 inhibition. (A) PNC1 overexpression enhances association of Sir2 with rDNA. The degree of Sir2 binding to rDNA was measured using the ChIP assay in wild-type, pnc1Δ, pnc1Δ cells containing an empty vector and pnc1Δ cells expressing Pnc1-GFP on the p416GPD vector. (B) PNC1 overexpression lowers the H3 acetylation level at rDNA. The acetylation level of histone H3 at the rDNA regions was measured using the ChIP assay in wild-type, pnc1Δ, pnc1Δ cells containing an empty vector and pnc1Δ cells expressing Pnc1-GFP on the p416GPD vector. (C) SIR2 overexpression enhances association of Sir2 with rDNA. The degree of Sir2 binding to rDNA was measured using the ChIP assay in wild-type and sir2Δ cells expressing Sir2-TAP on the p416GPD vector. (D) SIR2 overexpression lowers the H3 acetylation level at rDNA. The acetylation level of histone H3 at the rDNA regions was measured using the ChIP assay in wild-type and sir2Δ cells expressing Sir2-TAP on the p416GPD vector. (E) Rapamycin does not enhance association of Sir2 with rDNA in pnc1Δ cells. The degree of Sir2 binding to rDNA was measured using the ChIP assay in wild-type and pnc1Δ cells after treatment with or without 200 ng/ml rapamycin for 1 h. (F) Rapamycin does not lower the H3 acetylation level at rDNA in pnc1Δ cells. The acetylation level of histone H3 at the rDNA regions was measured using the ChIP assay in wild-type and pnc1Δ cells after treatment with or without 200 ng/ml rapamycin for 1 h. Values represent the average of three independent experiments and error bars indicate standard deviations.

Mentions: The PNC1 gene encodes a nicotinamidase that converts nicotinamide to nicotinic acid as part of the NAD+ salvage pathway (47). Therefore, deletion of PNC1 results in an elevated level of nicotinamide in cells that can inhibit the Sir2 activity. It has been shown that calorie restriction and other mild stresses extend yeast lifespan by increasing expression of PNC1 (46,48). A recent study has reported that inhibition of TORC1 signaling by rapamycin also promotes expression of PNC1 (30). Our results showing that rapamycin enhances association of Sir2 with rDNA and lowers the H3 acetylation level at rDNA raised a possibility that overexpression of PNC1 would have the same effect of rapamycin on rDNA. To check this possibility, we analyzed the degree of Sir2 binding to rDNA and the H3 acetylation level at rDNA in cells where PNC1 was deleted or overexpressed. In pnc1Δ cells, the degree of Sir2 binding to rDNA was essentially unchanged (Figure 4A), but the H3 acetylation level at rDNA increased considerably compared to wild-type cells (Figure 4B). In contrast, overexpression of PNC1 increased binding of Sir2 to rDNA (Figure 4A) and lowered the H3 acetylation level at rDNA (Figure 4B). Whether PNC1 was deleted or overexpressed, the protein level of Sir2 was not changed (data not shown). This result demonstrates that overexpression of PNC1 and rapamycin treatment exert the same effect on rDNA and suggests that enhancement of association of Sir2 with rDNA is a common mechanism underlying extension of lifespan in yeast by calorie restriction, other mild stresses, or TORC1 inhibition.Figure 4.


Rapamycin increases rDNA stability by enhancing association of Sir2 with rDNA in Saccharomyces cerevisiae.

Ha CW, Huh WK - Nucleic Acids Res. (2010)

Pnc1 is required for enhancement of Sir2 binding to rDNA under TORC1 inhibition. (A) PNC1 overexpression enhances association of Sir2 with rDNA. The degree of Sir2 binding to rDNA was measured using the ChIP assay in wild-type, pnc1Δ, pnc1Δ cells containing an empty vector and pnc1Δ cells expressing Pnc1-GFP on the p416GPD vector. (B) PNC1 overexpression lowers the H3 acetylation level at rDNA. The acetylation level of histone H3 at the rDNA regions was measured using the ChIP assay in wild-type, pnc1Δ, pnc1Δ cells containing an empty vector and pnc1Δ cells expressing Pnc1-GFP on the p416GPD vector. (C) SIR2 overexpression enhances association of Sir2 with rDNA. The degree of Sir2 binding to rDNA was measured using the ChIP assay in wild-type and sir2Δ cells expressing Sir2-TAP on the p416GPD vector. (D) SIR2 overexpression lowers the H3 acetylation level at rDNA. The acetylation level of histone H3 at the rDNA regions was measured using the ChIP assay in wild-type and sir2Δ cells expressing Sir2-TAP on the p416GPD vector. (E) Rapamycin does not enhance association of Sir2 with rDNA in pnc1Δ cells. The degree of Sir2 binding to rDNA was measured using the ChIP assay in wild-type and pnc1Δ cells after treatment with or without 200 ng/ml rapamycin for 1 h. (F) Rapamycin does not lower the H3 acetylation level at rDNA in pnc1Δ cells. The acetylation level of histone H3 at the rDNA regions was measured using the ChIP assay in wild-type and pnc1Δ cells after treatment with or without 200 ng/ml rapamycin for 1 h. Values represent the average of three independent experiments and error bars indicate standard deviations.
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Figure 4: Pnc1 is required for enhancement of Sir2 binding to rDNA under TORC1 inhibition. (A) PNC1 overexpression enhances association of Sir2 with rDNA. The degree of Sir2 binding to rDNA was measured using the ChIP assay in wild-type, pnc1Δ, pnc1Δ cells containing an empty vector and pnc1Δ cells expressing Pnc1-GFP on the p416GPD vector. (B) PNC1 overexpression lowers the H3 acetylation level at rDNA. The acetylation level of histone H3 at the rDNA regions was measured using the ChIP assay in wild-type, pnc1Δ, pnc1Δ cells containing an empty vector and pnc1Δ cells expressing Pnc1-GFP on the p416GPD vector. (C) SIR2 overexpression enhances association of Sir2 with rDNA. The degree of Sir2 binding to rDNA was measured using the ChIP assay in wild-type and sir2Δ cells expressing Sir2-TAP on the p416GPD vector. (D) SIR2 overexpression lowers the H3 acetylation level at rDNA. The acetylation level of histone H3 at the rDNA regions was measured using the ChIP assay in wild-type and sir2Δ cells expressing Sir2-TAP on the p416GPD vector. (E) Rapamycin does not enhance association of Sir2 with rDNA in pnc1Δ cells. The degree of Sir2 binding to rDNA was measured using the ChIP assay in wild-type and pnc1Δ cells after treatment with or without 200 ng/ml rapamycin for 1 h. (F) Rapamycin does not lower the H3 acetylation level at rDNA in pnc1Δ cells. The acetylation level of histone H3 at the rDNA regions was measured using the ChIP assay in wild-type and pnc1Δ cells after treatment with or without 200 ng/ml rapamycin for 1 h. Values represent the average of three independent experiments and error bars indicate standard deviations.
Mentions: The PNC1 gene encodes a nicotinamidase that converts nicotinamide to nicotinic acid as part of the NAD+ salvage pathway (47). Therefore, deletion of PNC1 results in an elevated level of nicotinamide in cells that can inhibit the Sir2 activity. It has been shown that calorie restriction and other mild stresses extend yeast lifespan by increasing expression of PNC1 (46,48). A recent study has reported that inhibition of TORC1 signaling by rapamycin also promotes expression of PNC1 (30). Our results showing that rapamycin enhances association of Sir2 with rDNA and lowers the H3 acetylation level at rDNA raised a possibility that overexpression of PNC1 would have the same effect of rapamycin on rDNA. To check this possibility, we analyzed the degree of Sir2 binding to rDNA and the H3 acetylation level at rDNA in cells where PNC1 was deleted or overexpressed. In pnc1Δ cells, the degree of Sir2 binding to rDNA was essentially unchanged (Figure 4A), but the H3 acetylation level at rDNA increased considerably compared to wild-type cells (Figure 4B). In contrast, overexpression of PNC1 increased binding of Sir2 to rDNA (Figure 4A) and lowered the H3 acetylation level at rDNA (Figure 4B). Whether PNC1 was deleted or overexpressed, the protein level of Sir2 was not changed (data not shown). This result demonstrates that overexpression of PNC1 and rapamycin treatment exert the same effect on rDNA and suggests that enhancement of association of Sir2 with rDNA is a common mechanism underlying extension of lifespan in yeast by calorie restriction, other mild stresses, or TORC1 inhibition.Figure 4.

Bottom Line: TORC1 inhibition increases transcriptional silencing of RNA polymerase II-transcribed gene integrated at the rDNA locus and reduces homologous recombination between rDNA repeats that causes formation of toxic extrachromosomal rDNA circles.In addition, TORC1 inhibition induces deacetylation of histones at rDNA.Taken together, our findings suggest that inhibition of TORC1 signaling stabilizes the rDNA locus by enhancing association of Sir2 with rDNA, thereby leading to extension of replicative lifespan in S. cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Research Center for Functional Cellulomics, Institute of Microbiology, Seoul National University, Seoul 151-747, Republic of Korea.

ABSTRACT
The target of rapamycin (TOR) kinase is an evolutionarily conserved key regulator of eukaryotic cell growth and proliferation. Recently, it has been reported that inhibition of TOR signaling pathway can delay aging and extend lifespan in several eukaryotic organisms, but how lifespan extension is mediated by inhibition of TOR signaling is poorly understood. Here we report that rapamycin treatment and nitrogen starvation, both of which cause inactivation of TOR complex 1 (TORC1), lead to enhanced association of Sir2 with ribosomal DNA (rDNA) in Saccharomyces cerevisiae. TORC1 inhibition increases transcriptional silencing of RNA polymerase II-transcribed gene integrated at the rDNA locus and reduces homologous recombination between rDNA repeats that causes formation of toxic extrachromosomal rDNA circles. In addition, TORC1 inhibition induces deacetylation of histones at rDNA. We also found that Pnc1 and Net1 are required for enhancement of association of Sir2 with rDNA under TORC1 inhibition. Taken together, our findings suggest that inhibition of TORC1 signaling stabilizes the rDNA locus by enhancing association of Sir2 with rDNA, thereby leading to extension of replicative lifespan in S. cerevisiae.

Show MeSH
Related in: MedlinePlus