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Cells undergoing developmental processes are characterized by persistent non-genetic alterations in chromatin, termed epigenetic changes, represented by distinct patterns of DNA methylation and histone post-translational modifications. Sirtuins, a group of conserved NAD+-dependent deacetylases or ADP-ribosyltransferases, promote longevity in diverse organisms; however, their molecular mechanisms in ageing regulation remain poorly understood. Yeast Sir2, the first member of the family to be found, establishes and maintains chromatin silencing by removing histone H4 lysine 16 acetylation and bringing in other silencing proteins. Here we report an age-associated decrease in Sir2 protein abundance accompanied by an increase in H4 lysine 16 acetylation and loss of histones at specific subtelomeric regions in replicatively old yeast cells, which results in compromised transcriptional silencing at these loci. Antagonizing activities of Sir2 and Sas2, a histone acetyltransferase, regulate the replicative lifespan through histone H4 lysine 16 at subtelomeric regions. This pathway, distinct from existing ageing models for yeast, may represent an evolutionarily conserved function of sirtuins in regulation of replicative ageing by maintenance of intact telomeric chromatin.

Gene silencing is essential for regulating cell fate in eukaryotes. Altered chromatin architectures contribute to maintaining the silenced state in a variety of species. The silent information regulator (Sir) proteins regulate mating type in Saccharomyces cerevisiae. One of these proteins, Sir3, interacts directly with the nucleosome to help generate silenced domains. We determined the crystal structure of a complex of the yeast Sir3 BAH (bromo-associated homology) domain and the nucleosome core particle at 3.0 angstrom resolution. We see multiple molecular interactions between the protein surfaces of the nucleosome and the BAH domain that explain numerous genetic mutations. These interactions are accompanied by structural rearrangements in both the nucleosome and the BAH domain. The structure explains how covalent modifications on H4K16 and H3K79 regulate formation of a silencing complex that contains the nucleosome as a central component.

Cohesin mediates central features of chromosome architecture. The protein complex governs sister chromatid cohesion and organizes genomes into loops and domains. In budding yeast, cohesin accumulates at discrete sites on chromosome arms, as well as at domains of heterochromatin. The molecular basis for the distribution at these sites is not yet resolved, although the heterochromatin protein Sir2 has been implicated. If cohesin were to bind a recurring feature of heterochromatin, then size of the cohesin domain would match the size of the heterochromatin domain. To test this hypothesis, the span of heterochromatin at the HMR silent mating-type locus was truncated with artificial barrier elements built from bacterial DNA binding proteins. We found that the most effective barrier reduced the footprint of Sir3 and Smc3, representative components of yeast heterochromatin and cohesin. These results show that reducing the span of heterochromatin at HMR constrains the size of the cohesin bound domain.

Gene silencing at the mating-type loci in budding yeast ( HMRa and HMLα ) depends on the Sir proteins. Sir2, Sir3 and Sir4 are indispensable, whereas Sir1 has a limited role. Sir proteins are also involved in repression at telomeres and rDNA repeats. Sir proteins may mediate silencing by limiting access to DNA. Using an inducible DNA methyltransferase expression system, we showed previously that the silenced mating-type loci are methylated at a much slower rate than the rest of the genome in vivo. Here, we show that Sir2, Sir3 and Sir4 are all required to impede access to the mating-type loci and telomeric X-elements. rDNA access is impeded by Sir2 and Sir3, but not Sir4. Methylation rates at adjacent rDNA repeats are not strongly correlated, suggesting that Sir proteins silence rDNA repeats randomly. Sir1 is required to impede access to HMRa , but not to HMLα , telomeres or rDNA. Since silenced DNA is accessible in vivo, albeit at a slower rate than elsewhere in the genome, steric occlusion is unlikely to be the primary mechanism of silencing.

Aging is characterized by the accumulation of damaged cellular macromolecules caused by declining repair and elimination pathways. An integral component employed by cells to counter toxic protein aggregates is the conserved ubiquitin/proteasome system (UPS). Previous studies have described an age-dependent decline of proteasomal function and increased longevity correlates with sustained proteasome capacity in centenarians and in naked mole rats, a long-lived rodent. Proof for a direct impact of enhanced proteasome function on longevity, however, is still lacking. To determine the importance of proteasome function in yeast aging, we established a method to modulate UPS capacity by manipulating levels of the UPS-related transcription factor Rpn4. While cells lacking RPN4 exhibit a decreased non-adaptable proteasome pool, loss of UBR2, an ubiquitin ligase that regulates Rpn4 turnover, results in elevated Rpn4 levels, which upregulates UPS components. Increased UPS capacity significantly enhances replicative lifespan (RLS) and resistance to proteotoxic stress, while reduced UPS capacity has opposing consequences. Despite tight transcriptional co-regulation of the UPS and oxidative detoxification systems, the impact of proteasome capacity on lifespan is independent of the latter, since elimination of Yap1, a key regulator of the oxidative stress response, does not affect lifespan extension of cells with higher proteasome capacity. Moreover, since elevated proteasome capacity results in improved clearance of toxic huntingtin fragments in a yeast model for neurodegenerative diseases, we speculate that the observed lifespan extension originates from prolonged elimination of damaged proteins in old mother cells. Epistasis analyses indicate that proteasome-mediated modulation of lifespan is at least partially distinct from dietary restriction, Tor1, and Sir2. These findings demonstrate that UPS capacity determines yeast RLS by a mechanism that is distinct from known longevity pathways and raise the possibility that interventions to promote enhanced proteasome function will have beneficial effects on longevity and age-related disease in humans.

The formation of heterochromatin at HML , HMR , and telomeres in Saccharomyces cerevisiae involves two main steps: the recruitment of Sir proteins to silencers and their spread throughout the silenced domain. We developed a method to study these two processes at single basepair resolution. Using a fusion protein between the heterochromatin protein Sir3 and the nonsite-specific bacterial adenine methyltransferase M.EcoGII, we mapped sites of Sir3–chromatin interactions genome-wide using long-read Nanopore sequencing to detect adenines methylated by the fusion protein and by ChIP-seq to map the distribution of Sir3–M.EcoGII. A silencing-deficient mutant of Sir3 lacking its Bromo-Adjacent Homology (BAH) domain, sir3-bah∆ , was still recruited to HML , HMR , and telomeres. However, in the absence of the BAH domain, it was unable to spread away from those recruitment sites. Overexpression of Sir3 did not lead to further spreading at HML , HMR , and most telomeres. A few exceptional telomeres, like 6R, exhibited a small amount of Sir3 spreading, suggesting that boundaries at telomeres responded variably to Sir3-M.EcoGII overexpression. Finally, by using a temperature-sensitive allele of SIR3 fused to M.ECOGII , we tracked the positions first methylated after induction and found that repression of genes at HML and HMR began before Sir3 occupied the entire locus.

The Origin Recognition Complex (ORC) is essential for replication, heterochromatin formation, telomere maintenance and genome stability in eukaryotes. Here we present the structure of the yeast Orc1 BAH domain bound to the nucleosome core particle. Our data reveal that Orc1, unlike its close homolog Sir3 involved in gene silencing, does not appear to discriminate between acetylated and non-acetylated lysine 16, modification states of the histone H4 tail that specify open and closed chromatin respectively. We elucidate the mechanism for this unique feature of Orc1 and hypothesize that its ability to interact with nucleosomes regardless of K16 modification state enables it to perform critical functions in both hetero- and euchromatin. We also show that direct interactions with nucleosomes are essential for Orc1 to maintain the integrity of rDNA borders during meiosis, a process distinct and independent from its known roles in silencing and replication. The Origin Recognition Complex (ORC) plays conserved and diverse roles in eukaryotes. Here the authors present the structure of a chromatin interacting domain of yeast Orc1 in complex with the nucleosome core particle, revealing that Orc1 interacts with the histone H4 tail irrespective of K16 acetylation; a modification that regulates accessibility to chromatin.

Saccharomyces cerevisiae telomeres have been a paradigm for studying telomere position effects on gene expression. Telomere position effect was first described in yeast by its effect on the expression of reporter genes inserted adjacent to truncated telomeres. The reporter genes showed variable silencing that depended on the Sir2/3/4 complex. Later studies examining subtelomeric reporter genes inserted at natural telomeres hinted that telomere position effects were less pervasive than previously thought. Additionally, more recent data using the sensitive technology of chromatin immunoprecipitation and massively parallel sequencing (ChIP-Seq) revealed a discrete and noncontinuous pattern of coenrichment for all three Sir proteins at a few telomeres, calling the generality of these conclusions into question. Here we combined the ChIP-Seq of the Sir proteins with RNA sequencing (RNA-Seq) of messenger RNAs (mRNAs) in wild-type and in SIR2, SIR3, and SIR4 deletion mutants to characterize the chromatin and transcriptional landscape of all native S. cerevisiae telomeres at the highest achievable resolution. Most S. cerevisiae chromosomes had subtelomeric genes that were expressed, with only ∼6% of subtelomeric genes silenced in a SIR-dependent manner. In addition, we uncovered 29 genes with previously unknown cell-type-specific patterns of expression. These detailed data provided a comprehensive assessment of the chromatin and transcriptional landscape of the subtelomeric domains of a eukaryotic genome.

In yeast, heterochromatin silencing is reported to decline in aging mother cells, causing sterility in old cells. This process is thought to reflect a decrease in the activity of the NAD⁺ (oxidized nicotinamide adenine dinucleotide)–dependent deacetylase Sir2. We tested whether Sir2 becomes nonfunctional gradually or precipitously during aging. Unexpectedly, silencing of the heterochromatic HML and HMR loci was not lost during aging. Old cells could initiate a mating response; however, they were less sensitive to mating pheromone than were young cells because of age-dependent aggregation of Whi3, an RNA-binding protein controlling S-phase entry. Removing the polyglutamine domain of Whi3 restored the pheromone sensitivity of old cells. We propose that aging phenotypes previously attributed to loss of heterochromatin silencing are instead caused by aggregation of the Whi3 cell cycle regulator

In diverse organisms, calorie restriction slows the pace of ageing and increases maximum lifespan. In the budding yeast Saccharomyces cerevisia e, calorie restriction extends lifespan by increasing the activity of Sir2 (ref. 1 ), a member of the conserved sirtuin family of NAD + -dependent protein deacetylases 2 , 3 , 4 , 5 , 6 . Included in this family are SIR-2.1, a Caenorhabditis elegans enzyme that regulates lifespan 7 , and SIRT1, a human deacetylase that promotes cell survival by negatively regulating the p53 tumour suppressor 8 , 9 , 10 . Here we report the discovery of three classes of small molecules that activate sirtuins. We show that the potent activator resveratrol, a polyphenol found in red wine, lowers the Michaelis constant of SIRT1 for both the acetylated substrate and NAD + , and increases cell survival by stimulating SIRT1-dependent deacetylation of p53. In yeast, resveratrol mimics calorie restriction by stimulating Sir2, increasing DNA stability and extending lifespan by 70%. We discuss possible evolutionary origins of this phenomenon and suggest new lines of research into the therapeutic use of sirtuin activators.