Modulation of life-span by histone deacetylase genes in Saccharomyces cerevisiae - PubMed (original) (raw)
Modulation of life-span by histone deacetylase genes in Saccharomyces cerevisiae
S Kim et al. Mol Biol Cell. 1999 Oct.
Free PMC article
Abstract
The yeast Saccharomyces cerevisiae has a limited life-span, which is measured by the number of divisions that individual cells complete. Among the many changes that occur as yeasts age are alterations in chromatin-dependent transcriptional silencing. We have genetically manipulated histone deacetylases to modify chromatin, and we have examined the effect on yeast longevity. Deletion of the histone deacetylase gene RPD3 extended life-span. Its effects on chromatin functional state were evidenced by enhanced silencing at the three known heterochromatic regions of the genome, the silent mating type (HM), subtelomeric, and rDNA loci, which occurred even in the absence of SIR3. Similarly, the effect of the rpd3Delta on life-span did not depend on an intact Sir silencing complex. In fact, deletion of SIR3 itself had little effect on life-span, although it markedly accelerated the increase in cell generation time that is observed during yeast aging. Deletion of HDA1, another histone deacetylase gene, did not result in life-span extension, unless it was combined with deletion of SIR3. The hda1Delta sir3Delta resulted in an increase in silencing, but only at the rDNA locus. Deletion of RPD3 suppressed the loss of silencing in rDNA in a sir2 mutant; however, the silencing did not reach the level found in the rpd3Delta single mutant, and RPD3 deletion did not overcome the life-span shortening seen in the sir2 mutant. Deletion of both RPD3 and HDA1 caused a decrease in life-span, which resulted from a substantial increase in initial mortality of the population. The expression of both of these genes declines with age, providing one possible explanation for the increase in mortality during the life-span. Our results are consistent with the loss of rDNA silencing leading to aging in yeast. The functions of RPD3 and HDA1 do not overlap entirely. RPD3 exerts its effect on chromatin at additional sites in the genome, raising the possibility that events at loci other than rDNA play a role in the aging process.
Figures
Figure 1
Effects of histone deacetylase mutants on life-span and colony-forming ability. (A) Survival curves of wild-type (YSK663), hda1Δ (YSK664), rpd3Δ (YSK661), and hda1Δ rpd3Δ (YSK662) segregants of YSK631. The percentages of live cells are plotted as a function of age in generations. Mean life-span (and the number of cells analyzed) was 19.8 (50) for the wild-type, 18.9 (50) for the hda1Δ, 27.9 (49) for the rpd3Δ, and 14.8 (48) for the hda1Δ rpd3Δ strains. The rpd3Δ and hda1Δ rpd3Δ strains were significantly different from the wild-type in life-span (the P values were 0.000001 and 0.0078, respectively). (B) Tetrads obtained from sporulated YSK631 [+/rpd3Δ:: URA3, +/_hda1Δ:: HIS3_] were dissected on a YPD plate. After 3-d incubation at 30°C, the germinated colonies were replica-plated onto complete synthetic medium lacking uracil (SC-URA) or histidine (SC-HIS). All of the segregants that formed smaller colonies on the YPD plate were Ura+ and His+, indicating that the hda1Δ rpd3Δ double-mutant segregants had difficulty in normal colony formation.
Figure 2
Effects of SIR3 deletion on life-span and senescence. (A) Survival curves of wild-type (YSK712), rpd3Δ (YSK713), sir3Δ (YSK711), and rpd3Δ sir3Δ (YSK710) segregants of YSK668. Mean life-span (and the number of cells analyzed) was 19.1 (40) for the wild-type, 27.9 (40) for the rpd3Δ, 17.8 (40) for the sir3Δ, and 28.4 (40) for the rpd3Δ sir3Δ strains. The rpd3Δ and rpd3Δ sir3Δ strains differed from wild-type in life-span _(_P < 0.00001). (B) Survival curves of wild-type (YSK663), _hda1Δ_ (YSK664), _sir3Δ_ (YSK770), and _hda1Δ sir3Δ_ (YSK771) strains. Mean life-span (and the number of cells analyzed) was 18.6 (50) for the wild-type, 18.3 (50) for the _hda1Δ_, 19.0 (50) for the _sir3Δ_, and 25.6 (50) for the _hda1Δ sir3Δ_ strains. The life-span of the _hda1Δ sir3Δ_ strain was significantly longer than that of the control, the _sir3Δ,_ or the _hda1Δ_ strains (P = 0.000002, 0.000028, and 0.000002, respectively). (C) Change in generation time of the _hda1Δ_, _sir3Δ_, or _hda1Δ sir3Δ_ mother cells relative to the wild-type control during the life-spans shown in B. From the day life-span determination was started, the total number of buds generated by the wild-type mother cells was divided by the total number of buds generated by the _hda1Δ, sir3Δ,_ or _hda1Δ sir3Δ_ mother cells. The resulting number corresponds to an estimate of the average generation time of each mutant strain relative to that of the wild type. The relative generation times were calculated until day 10. By this time, all of the strains completed >96% of their life-spans.
Figure 3
Effect of various deletions on rDNA silencing. Cells from (A) M9 (Ty1_-MET15_ inserted in a non-rDNA locus), (B) M1 (_RDN1::Ty1_-MET15), (C) JS218 (M1 with sir2:: HIS3), (D) YSK757 (M1 with sir3Δ:: LEU2), (E) YSK753 (M1 with hda1Δ:: LEU2), (F) YSK781 (M1 with sir3Δ:: LEU2, hda1Δ:: HIS3), (G) YSK755 (M1 with rpd3Δ:: TRP1), (H) YSK783 (M1 with sir3Δ:: LEU2, rpd3Δ:: URA3), (I) YSK779 (M1 with rpd3Δ:: URA3, sir2:: HIS3), and (J) YCYL11 (M9 with rpd3Δ:: TRP1) were streaked on modified YPD agar medium containing Pb2+. The plates were incubated for 1 wk at 30°C. MET15+ cells grown on Pb2+ medium form white colonies (A), but met15 mutant cells develop dark brown colonies on the same medium. In the control M1 (B), the Ty1-MET15 is located upstream of the 5S rDNA in the RDN1 locus (Smith and Boeke, 1997).
Figure 4
Effects of SIR2 deletion on life-span and ERC generation. (A) Survival curves of wild-type control (YPK9), sir2 (YAB11), rpd3Δ (YAB12), and rpd3Δ sir2 (YAB13). Mean life-span (and the number of cells analyzed) was 18.7 (39) for the wild-type, 25.8 (40) for the rpd3Δ, 12.5 (39) for the sir2, and 11.6 (39) for the rpd3Δ sir2 strains, respectively. The rpd3Δ, sir2, and rpd3Δ sir2 strain life-spans differed from wild-type (P ≪ 0.0001). There is no statistical difference between the life-spans of the sir2 and rpd3Δ sir2 strains (P = 0.8). (B) ERC generation in the wild-type and sir2 strains used in A. The large arrowhead indicates genomic rDNA, whereas the smaller arrows point to extrachromosomal rDNA.
Figure 5
SIR3 and HDA1 mRNA levels in age-synchronized cells. (A) Northern blot. RNA was prepared from 2, 5, 8, 11, 14, and 17 generation-old cells. Total RNA (10 μg) from each RNA sample was loaded in each lane of a gel. The same blot was used repeatedly by hybridizing with one DNA probe at a time and stripping it. The probe DNA to detect 5.8S rRNA (158 bp) was prepared by labeling the 5′ end of an oligonucleotide (5′-CATTTCGCTGCGTTCTTCATC-3′) with 32P. The DNA probe to detect TLC1 RNA (1.3 kb) was the 1.28-kb _Xho_I fragment isolated from pBlue61 (Singer and Gottschling, 1994). The DNA probe for HDA1 mRNA (2.5 kb) was the 1.8-kb _Xho_I– _Xba_I fragment isolated from pskB93 (Rundlett et al., 1996). The DNA probe specific to SIR3 mRNA (3 kb) was the 2.3-kb _Cla_I–_Xho_I fragment isolated from pJR517 (provided by J. Rine, University of California, Berkeley, CA). (B) Quantitation of mRNA. The amount of mRNA present in each lane was quantitated by phosphorimaging and normalized to the amount of TLC1 RNA present in the same lane to obtain age-specific relative amounts of individual mRNA species. The amount of TLC1 RNA, which is a component of the yeast telomerase, remains relatively constant compared with 5.8S rRNA.
Figure 6
RT-PCR analysis of the RPD3 mRNA levels in age-synchronized cells. (A) Gel electrophoresis of PCR products. RNA was prepared from young (2-generation old, g2), middle-aged (8-generation old, g8), old (17-generation old, g17), and mixed-age cells from a stationary culture (St). After equalization of the amount of first-strand cDNA synthesized from each of these RNA samples, twofold serial dilutions of each were prepared and subjected to PCR (see MATERIALS AND METHODS). (B) Quantitation of PCR products. The amount of the PCR amplification products obtained for each dilution was quantitated in each lane in A by phosphorimaging. This was plotted against the template concentration. The slopes of the linear regressions obtained are plotted as a function of age to portray the change in mRNA levels during the life-span. The r2 values for the linear regressions were 0.948, 0.981, and 0.958 for the 2-, 8-, and 17-generation–old cells, respectively.
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