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Chromatin instability and protein homeostasis (proteostasis) stress are two well-established hallmarks of aging, which have been considered largely independent of each other. Using microfluidics and single-cell imaging approaches, we observed that, during the replicative aging of Saccharomyces cerevisiae, a challenge to proteostasis occurs specifically in the fraction of cells with decreased stability within the ribosomal DNA (rDNA). A screen of 170 yeast RNA-binding proteins identified ribosomal RNA (rRNA)-binding proteins as the most enriched group that aggregate upon a decrease in rDNA stability induced by inhibition of a conserved lysine deacetylase Sir2. Further, loss of rDNA stability induces age-dependent aggregation of rRNA-binding proteins through aberrant overproduction of rRNAs. These aggregates contribute to age-induced proteostasis decline and limit cellular lifespan. Our findings reveal a mechanism underlying the interconnection between chromatin instability and proteostasis stress and highlight the importance of cell-to-cell variability in aging processes.

Pyridine nucleotides are essential coenzymes in many cellular redox reactions in all living systems. In addition to functioning as a redox carrier, NAD(+) is also a required co-substrate for the conserved sirtuin deacetylases. Sirtuins regulate transcription, genome maintenance and metabolism and function as molecular links between cells and their environment. Maintaining NAD(+) homeostasis is essential for proper cellular function and aberrant NAD(+) metabolism has been implicated in a number of metabolic- and age-associated diseases. Recently, NAD(+) metabolism has been linked to the phosphate-responsive signaling pathway (PHO pathway) in the budding yeast Saccharomyces cerevisiae. Activation of the PHO pathway is associated with the production and mobilization of the NAD(+) metabolite nicotinamide riboside (NR), which is mediated in part by PHO-regulated nucleotidases. Cross-regulation between NAD(+) metabolism and the PHO pathway has also been reported; however, detailed mechanisms remain to be elucidated. The PHO pathway also appears to modulate the activities of common downstream effectors of multiple nutrient-sensing pathways (Ras-PKA, TOR, Sch9/AKT). These signaling pathways were suggested to play a role in calorie restriction-mediated beneficial effects, which have also been linked to Sir2 function and NAD(+) metabolism. Here, we discuss the interactions of these pathways and their potential roles in regulating NAD(+) metabolism. In eukaryotic cells, intracellular compartmentalization facilitates the regulation of enzymatic functions and also concentrates or sequesters specific metabolites. Various NAD(+)-mediated cellular functions such as mitochondrial oxidative phosphorylation are compartmentalized. Therefore, we also discuss several key players functioning in mitochondrial, cytosolic and vacuolar compartmentalization of NAD(+) intermediates, and their potential roles in NAD(+) homeostasis. To date, it remains unclear how NAD(+) and NAD(+) intermediates shuttle between different cellular compartments. Together, these studies provide a molecular basis for how NAD(+) homeostasis factors and the interacting signaling pathways confer metabolic flexibility and contribute to maintaining cell fitness and genome stability.

The conserved protein kinase Rio1 localizes to the cytoplasm and nucleus of eukaryotic cells. While the roles of Rio1 in the cytoplasm are well characterized, its nuclear function remains unknown. Here we show that nuclear Rio1 promotes rDNA array stability and segregation in Saccharomyces cerevisiae. During rDNA replication in S phase, Rio1 downregulates RNA polymerase I (PolI) and recruits the histone deacetylase Sir2. Both interventions ensure rDNA copy-number homeostasis and prevent the formation of extrachromosomal rDNA circles, which are linked to accelerated ageing in yeast. During anaphase, Rio1 downregulates PolI by targeting its subunit Rpa43, causing PolI to dissociate from the rDNA. By stimulating the processing of PolI-generated transcripts at the rDNA, Rio1 allows for rDNA condensation and segregation in late anaphase. These events finalize the genome transmission process. We identify Rio1 as an essential nucleolar housekeeper that integrates rDNA replication and segregation with ribosome biogenesis.

Double-strand-break repair proteins interact with and recruit Sir proteins to ectopic sites in the genome. Recruitment results in gene silencing, which depends on Sir proteins, as well as on histone H2A modification. Silencing also results in the localization of the locus to the nuclear periphery., Heterochromatin formation and nuclear organization are important in gene regulation and genome fidelity. Proteins involved in gene silencing localize to sites of damage and some DNA repair proteins localize to heterochromatin, but the biological importance of these correlations remains unclear. In this study, we examined the role of double-strand-break repair proteins in gene silencing and nuclear organization. We find that the ATM kinase Tel1 and the proteins Mre11 and Esc2 can silence a reporter gene dependent on the Sir, as well as on other repair proteins. Furthermore, these proteins aid in the localization of silenced domains to specific compartments in the nucleus. We identify two distinct mechanisms for repair protein–mediated silencing—via direct and indirect interactions with Sir proteins, as well as by tethering loci to the nuclear periphery. This study reveals previously unknown interactions between repair proteins and silencing proteins and suggests insights into the mechanism underlying genome integrity.

The yeast silent information regulator (Sir)2 protein links cellular metabolism and transcriptional silencing through its nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase activity. We report that mitochondria from mammalian cells contain intrinsic NAD-dependent deacetylase activity. This activity is inhibited by the NAD hydrolysis product nicotinamide, but not by trichostatin A, consistent with a class III deacetylase. We identify this deacetylase as the nuclear-encoded human Sir2 homologue hSIRT3, and show that hSIRT3 is located within the mitochondrial matrix. Mitochondrial import of hSIRT3 is dependent on an NH2-terminal amphipathic α-helix rich in basic residues. hSIRT3 is proteolytically processed in the mitochondrial matrix to a 28-kD product. This processing can be reconstituted in vitro with recombinant mitochondrial matrix processing peptidase (MPP) and is inhibited by mutation of arginines 99 and 100. The unprocessed form of hSIRT3 is enzymatically inactive and becomes fully activated in vitro after cleavage by MPP. These observations demonstrate the existence of a latent class III deacetylase that becomes catalytically activated upon import into the human mitochondria.

We have analyzed the yeast replicative lifespan of a large number of open reading frame (ORF) deletions. Here, we report that strains lacking genes SGF73, SGF11, and UBP8 encoding SAGA/SLIK complex histone deubiquitinase module (DUBm) components are exceptionally long lived. Strains lacking other SAGA/SALSA components, including the acetyltransferase encoded by GCN5, are not long lived; however, these genes are required for the lifespan extension observed in DUBm deletions. Moreover, the SIR2-encoded histone deacetylase is required, and we document both a genetic and physical interaction between DUBm and Sir2. A series of studies assessing Sir2-dependent functions lead us to propose that DUBm strains are exceptionally long lived because they promote multiple prolongevity events, including reduced rDNA recombination and altered silencing of telomere-proximal genes. Given that ataxin-7, the human Sgf73 ortholog, causes the neurodegenerative disease spinocerebellar ataxia type 7, our findings indicate that the genetic and epigenetic interactions between DUBm and SIR2 will be relevant to neurodegeneration and aging. © 2014 The Authors.

We have developed a novel technique for combined immunofluorescence/in situ hybridization on fixed budding yeast cells that maintains the three-dimensional structure of the nucleus as monitored by focal sections of cells labeled with fluorescent probes and by staining with a nuclear pore antibody. Within the resolution of these immunodetection techniques, we show that proteins encoded by the SIR3, SIR4, and RAP1 genes colocalize in a statistically significant manner with Y' telomere-associated DNA sequences. In wild-type cells the Y' in situ hybridization signals can be resolved by light microscopy into fewer than ten foci per diploid nucleus. This suggests that telomeres are clustered in vegetatively growing cells, and that proteins essential for telomeric silencing are concentrated at their sites of action, i.e., at telomeres and/or subtelomeric regions. As observed for Rap1, the Sir4p staining is diffuse in a sir3- strain, and similarly, Sir3p staining is no longer punctate in a sir4- strain, although the derivatized Y' probe continues to label discrete sites in these strains. Nonetheless, the Y' FISH is altered in a qualitative manner in sir3 and sir4 mutant strains, consistent with the previously reported phenotypes of shortened telomeric repeats and loss of telomeric silencing.

The Silent Information Regulatory proteins, Sir3 and Sir4, and the telomeric repeat-binding protein RAP1 are required for the chromatin-mediated gene repression observed at yeast telomeric regions. All three proteins are localized by immunofluorescence staining to foci near the nuclear periphery suggesting a relationship between subnuclear localization and silencing. We present several lines of immunological and biochemical evidence that Sir3, Sir4, and RAP1 interact in intact yeast cells. First, immunolocalization of Sir3 to foci at the yeast nuclear periphery is lost in rap1 mutants carrying deletions for either the terminal 28 or 165 amino acids of RAP1. Second, the perinuclear localization of both Sir3 and RAP1 is disrupted by overproduction of the COOH terminus of Sir4. Third, overproduction of the Sir4 COOH terminus alters the solubility properties of both Sir3 and full-length Sir4. Finally, we demonstrate that RAP1 and Sir4 coprecipitate in immune complexes using either anti-RAP1 or anti-Sir4 antibodies. We propose that the integrity of a tertiary complex between Sir4, Sir3, and RAP1 is involved in both the maintenance of telomeric repression and the clustering of telomeres in foci near the nuclear periphery.

The silent mating loci and chromosomal regions adjacent to telomeres of S. cerevisiae have features similar to heterochromatin of more complex eukaryotes. Transcriptional repression at these sites depends on the silent information regulators SIR3 and SIR4 as well as histones H3 and H4. We show here that the SIR3 and SIR4 proteins interact with specific silencing domains of the H3 and H4 N-termini in vitro. Certain mutations in these factors, which affect their silencing functions in vivo, also disrupt their interactions in vitro. Immunofluorescence studies with antibodies against RAP1 and SIR3 demonstrate that the H3 and H4 N-termini are required for the association of SIR3 with telomeric chromatin and the perinuclear positioning of yeast telomeres. Based on these interactions, we propose a model for heterochromatin-mediated transcriptional silencing in yeast, which may serve as a paradigm for other eukaryotic organisms as well.

The establishment of silencing in Saccharomyces cerevisiae is similar to heterochromatin formation in multi-cellular eukaryotes. Previous batch culture studies determined that the de novo establishment of silencing initiates during S phase and continues for up to 5 cell divisions for completion. To track silencing phenotypically, we developed an assay that introduces Sir3 protein into individual sir3Δ mutant cells synchronously and then detects the onset of silencing with single-cell resolution. Silencing was completed within the first one to two cell divisions in most cells queried. Moreover, we uncovered unexpected complexity in the contributions of a histone acetyltransferase (Sas2), two histone methytransferases (Dot1 and Set1), and one histone demethylase (Jhd2) to the dynamics of silencing. Our findings revealed that removal of methyl modifications at H3 K4 and H3 K79 were important steps in silent chromatin formation, and that Jhd2 and Set1 played competing roles in the process.

In budding yeast genes integrated near telomeres succumb to a variegated pattern of gene repression that requires the silent information regulatory proteins Sir2p, Sir3p and Sir4p, which form a nucleosome-binding complex. Immunolocalization shows that the Sir proteins co-localize with the telomeric repeat binding protein Rap1p and with telomeric DNA in a limited number of foci near the periphery of interphase nuclei. All conditions tested so far that disrupt telomere proximal repression result in a dispersed staining pattern for Sir2p, Sir3p and Sir4p. Although the focal organization is clearly not sufficient for establishing repression, genetic studies suggest that the high local concentration of Sir proteins at telomeric foci facilitates the formation of repressed chromatin. In addition to its telomeric localization, Sir2p is shown by immunostaining and cross-linking to bind a subdomain of the nucleolus. In strains lacking an intact Sir4p, Sir3p also becomes concentrated in the nucleolus by a pathway requiring SIR2 and UTH4. This unexpected localization correlates with observed effects of sir mutations on rDNA stability and longevity, defining a new site of action for silent information regulatory factors. We report a novel WD40 repeat-containing factor, Sif2p, that binds specifically to the Sir4p N-terminus. Like Sir1p and Uth4p, Sif2p antagonizes telomeric silencing by regulating an equilibrium between alternative assembly pathways at different subnuclear loci.

Telomere length-variation in deletion strains of Saccharomyces cerevisiae was used to identify genes and pathways that regulate telomere length. We found 72 genes that when deleted confer short telomeres, and 80 genes that confer long telomeres relative to those of wild-type yeast. Among identified genes, 88 have not been previously implicated in telomere length control. Genes that regulate telomere length span a variety of functions that can be broadly separated into telomerase-dependent and telomerase-independent pathways. We also found 39 genes that have an important role in telomere maintenance or cell proliferation in the absence of telomerase, including genes that participate in deoxyribonucleotide biosynthesis, sister chromatid cohesion, and vacuolar protein sorting. Given the large number of loci identified, we investigated telomere lengths in 13 wild yeast strains and found substantial natural variation in telomere length among the isolates. Furthermore, we crossed a wild isolate to a laboratory strain and analyzed telomere length in 122 progeny. Genome-wide linkage analysis among these segregants revealed two loci that account for 30%–35% of telomere length-variation between the strains. These findings support a general model of telomere length-variation in outbred populations that results from polymorphisms at a large number of loci. Furthermore, our results laid the foundation for studying genetic determinants of telomere length-variation and their roles in human disease., Synopsis Telomere maintenance is of great importance to ensure genome stability in organisms with linear genomes. In humans, telomeres shorten as a function of age and serve as a marker of cell replication history. Understanding the genetic differences in telomere length-maintenance may help provide the insights into the basis for different rates of aging among individuals and differences in individuals' propensity for aging-associated diseases such as cancer. Studies in yeast and other model organisms have defined several pathways that ensure stability of chromosome ends. In order to capture full complement of genes that participate in telomere maintenance in yeast Saccharomyces cerevisiae, the authors undertook a comprehensive screen for genes that affect telomere length. Among 152 identified genes, the authors found 39 genes whose function is critical for telomere maintenance in the absence of telomerase. The authors extended their studies from laboratory yeast strains to outbred populations of yeast and discovered significant phenotypic variation in telomere length among the isolates. Telomere length-analysis of a cross between a wild yeast isolate and a laboratory strain support a general model of telomere length-variation in outbred populations that results from polymorphisms at a large number of loci. This finding provides a basis for genetic studies of telomere maintenance in human populations.

Sirtuins are a family of phylogenetically conserved nicotinamide adenine dinucleotide-dependent deacetylases that have a firmly established role in aging. Using a simple Saccharomyces cerevisiae yeast heterochromatic derepression assay, we tested a number of environmental chemicals to address the possibility that humans are exposed to sirtuin inhibitors. Here we show that dihydrocoumarin (DHC), a compound found in Melilotus officinalis (sweet clover) that is commonly added to food and cosmetics, disrupted heterochromatic silencing and inhibited yeast Sir2p as well as human SIRT1 deacetylase activity. DHC exposure in the human TK6 lymphoblastoid cell line also caused concentration-dependent increases in p53 acetylation and cytotoxicity. Flow cytometric analysis to detect annexin V binding to phosphatidylserine demonstrated that DHC increased apoptosis more than 3-fold over controls. Thus, DHC inhibits both yeast Sir2p and human SIRT1 deacetylases and increases p53 acetylation and apoptosis, a phenotype associated with senescence and aging. These findings demonstrate that humans are potentially exposed to epigenetic toxicants that inhibit sirtuin deacetylases., Synopsis The effects of chronic low-dose human exposure to environmental chemicals are difficult to study and poorly understood. Chemicals are routinely tested for the ability to induce DNA mutations, cause chromosome damage, or produce cell death, but are rarely tested for their ability to cause epigenetic changes, which can influence the behavior of a cell without directly changing the DNA sequence. Epigenetic changes have become the focus of intense research in an attempt to understand the mechanisms by which they function. The Sir2 family of deacetylases is one class of proteins that controls some epigenetic processes and, interestingly, has been implicated in extending the longevity of several organisms. Here the authors describe a novel assay based upon yeast Sir2p function to screen environmental chemicals for their ability to alter epigenetic silencing. From screening a relatively small number of agents, the authors found that dihydrocoumarin, a natural compound found in Melilotus officinalis (sweet clover) that is synthetically manufactured and frequently added to both food and cosmetics, disrupted epigenetic processes in the yeast Saccharomyces cerevisiae. Dihydrocoumarin also inhibited several human Sir2 family deacetylases (SIRT1 and SIRT2) and, when added to cells in culture, increased p53 tumor suppressor protein acetylation and caused elevated levels of apoptosis. The present study suggests that humans are exposed to a number of environmental chemicals that may be classified as epigenetic toxicants.

Keywords: Silent Information Regulator Proteins ; Saccharomyces cerevisiae Reference EPFL-ARTICLE-134292 Record created on 2009-02-19, modified on 2017-05-12

A prior genetic study indicated that activity of Sir silencing proteins at a hypothetical AGE locus is essential for long life span. In this model, the SIR4-42 mutation would direct the Sir protein complex to the AGE locus, giving rise to a long life span. We show by indirect immunofluorescence that Sir3p and Sir4p are redirected to the nucleolus in the SIR4-42 mutant. Furthermore, this relocalization is dependent on both UTH4 a novel yeast gene that extends life span, and its homologue YGL023. Strikingly, the Sir complex is relocalized from telomeres to the nucleolus in old wild-type cells. We propose that the rDNA is the AGE locus and that nucleolar function is compromised in old yeast cells in a way that may be mitigated by targeting of Sir proteins to the nucleolus.

The mammalian Ku70 and Ku86 proteins form a heterodimer that binds to the ends of double-stranded DNA in vitro and is required for repair of radiation-induced strand breaks and V(D)J recombination [1,2]. Deletion of the Saccharomyces cerevisiae genes HDF1 and HDF2--encoding yKu70p and yKu80p, respectively--enhances radiation sensitivity in a rad52 background [3,4]. In addition to repair defects, the length of the TG-rich repeat on yeast telomere ends shortens dramatically [5,6]. We have shown previously that in yeast interphase nuclei, telomeres are clustered in a limited number of foci near the nuclear periphery [7], but the elements that mediate this localization remained unknown. We report here that deletion of the genes encoding yKu70p or its partner yKu80p altered the positioning of telomeric DNA in the yeast nucleus. These are the first mutants shown to affect the subnuclear localization of telomeres. Strains deficient for either yKu70p or yKu80p lost telomeric silencing, although they maintained repression at the silent mating-type loci. In addition, the telomere-associated silencing factors Sir3p and Sir4p and the TG-repeat-binding protein Rap1p lost their punctate pattern of staining and became dispersed throughout the nucleoplasm. Our results implicate the yeast Ku proteins directly in aspects of telomere organization, which in turn affects the repression of telomere-proximal genes.

Telomeric TG-rich repeats and their associated proteins protect the termini of eukaryotic chromosomes from end-to-end fusions. Associated with the cap structure at yeast telomeres is a subtelomeric domain of heterochromatin, containing the silent information regulator (SIR) complex. The Ku70/80 heterodimer (yKu) is associated both with the chromosome end and with subtelomeric chromatin. Surprisingly, both yKu and the chromatin-associated Rap1 and SIR proteins are released from telomeres in a RAD9-dependent response to DNA damage. yKu is recruited rapidly to double-strand cuts, while low levels of SIR proteins are detected near cleavage sites at later time points. Consistently, yKu- or SIR-deficient strains are hypersensitive to DNA-damaging agents. The release of yKu from telomeric chromatin may allow efficient scanning of the genome for DNA strand breaks.

Genes integrated near the telomeres of budding yeast have a variegated pattern of gene repression that is mediated by the silent information regulatory proteins Sir2p, Sir3p, and Sir4p. Immunolocalization and fluorescence in situ hybridization (FISH) reveal 6-10 perinuclear foci in which silencing proteins and subtelomeric sequences colocalize, suggesting that these are sites of Sir-mediated repression. Telomeres lacking subtelomeric repeat elements and the silent mating locus, HML, also localize to the periphery of the nucleus. Conditions that disrupt telomere proximal repression disrupt the focal staining pattern of Sir proteins, but not necessarily the localization of telomeric DNA. To monitor the telomere-associated pools of heterochromatin-binding proteins (Sir and Rap1 proteins) during mitotic cell division, we have performed immunofluorescence and telomeric FISH on populations of yeast cells synchronously traversing the cell cycle. We observe a partial release of Rap1p from telomeres in late G2/M, although telomeres appear to stay clustered during G2-phase and throughout mitosis. A partial release of Sir3p and Sir4p during mitosis also occurs. This is not observed upon HU arrest, although other types of DNA damage cause a dramatic relocalization of Sir and Rap1 proteins. The observed cell cycle dynamics were confirmed by direct epifluorescence of a GFP-Rap1p fusion. Using live GFP fluorescence we show that the diffuse mitotic distribution of GFP-Rap1p is restored to the interphase pattern of foci in early G1-phase.