H3K36 methylation promotes longevity by enhancing transcriptional fidelity.

Epigenetic mechanisms, including histone post-translational modifications, control longevity in diverse organisms. Relatedly, loss of proper transcriptional regulation on a global scale is an emerging phenomenon of shortened life span, but the specific mechanisms linking these observations remain to be uncovered. Here, we describe a life span screen in Saccharomyces cerevisiae that is designed to identify amino acid residues of histones that regulate yeast replicative aging. Our results reveal that lack of sustained histone H3K36 methylation is commensurate with increased cryptic transcription in a subset of genes in old cells and with shorter life span. In contrast, deletion of the K36me2/3 demethylase Rph1 increases H3K36me3 within these genes, suppresses cryptic transcript initiation, and extends life span. We show that this aging phenomenon is conserved, as cryptic transcription also increases in old worms. We propose that epigenetic misregulation in aging cells leads to loss of transcriptional precision that is detrimental to life span, and, importantly, this acceleration in aging can be reversed by restoring transcriptional fidelity.

Defining the impact of mutation accumulation on replicative lifespan in yeast using cancer-associated mutator phenotypes.

Mutations accumulate within somatic cells and have been proposed to contribute to aging. It is unclear what level of mutation burden may be required to consistently reduce cellular lifespan. Human cancers driven by a mutator phenotype represent an intriguing model to test this hypothesis, since they carry the highest mutation burdens of any human cell. However, it remains technically challenging to measure the replicative lifespan of individual mammalian cells. Here, we modeled the consequences of cancer-related mutator phenotypes on lifespan using yeast defective for mismatch repair (MMR) and/or leading strand (Polε) or lagging strand (Polδ) DNA polymerase proofreading. Only haploid mutator cells with significant lifetime mutation accumulation (MA) exhibited shorter lifespans. Diploid strains, derived by mating haploids of various genotypes, carried variable numbers of fixed mutations and a range of mutator phenotypes. Some diploid strains with fewer than two mutations per megabase displayed a 25% decrease in lifespan, suggesting that moderate numbers of random heterozygous mutations can increase mortality rate. As mutation rates and burdens climbed, lifespan steadily eroded. Strong diploid mutator phenotypes produced a form of genetic anticipation with regard to aging, where the longer a lineage persisted, the shorter lived cells became. Using MA lines, we established a relationship between mutation burden and lifespan, as well as population doubling time. Our observations define a threshold of random mutation burden that consistently decreases cellular longevity in diploid yeast cells. Many human cancers carry comparable mutation burdens, suggesting that while cancers appear immortal, individual cancer cells may suffer diminished lifespan due to accrued mutation burden.

Genetic screen identifies adaptive aneuploidy as a key mediator of ER stress resistance in yeast.

The yeast genome becomes unstable during stress, which often results in adaptive aneuploidy, allowing rapid activation of protective mechanisms that restore cellular homeostasis. In this study, we performed a genetic screen in Saccharomyces cerevisiae to identify genome adaptations that confer resistance to tunicamycin-induced endoplasmic reticulum (ER) stress. Whole-genome sequencing of tunicamycin-resistant mutants revealed that ER stress resistance correlated significantly with gains of chromosomes II and XIII. We found that chromosome duplications allow adaptation of yeast cells to ER stress independently of the unfolded protein response, and that the gain of an extra copy of chromosome II alone is sufficient to induce protection from tunicamycin. Moreover, the protective effect of disomic chromosomes can be recapitulated by overexpression of several genes located on chromosome II. Among these genes, overexpression of UDP-N-acetylglucosamine-1-P transferase (ALG7), a subunit of the 20S proteasome (PRE7), and YBR085C-A induced tunicamycin resistance in wild-type cells, whereas deletion of all three genes completely reversed the tunicamycin-resistance phenotype. Together, our data demonstrate that aneuploidy plays a critical role in adaptation to ER stress by increasing the copy number of ER stress protective genes. While aneuploidy itself leads to proteotoxic stress, the gene-specific effects of chromosome II aneuploidy counteract the negative effect resulting in improved protein folding.

Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in Caenorhabditis elegans.

Mitochondrial dysfunction can increase oxidative stress and extend lifespan in Caenorhabditis elegans. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPRmt) and extends lifespan. Here we report that transaldolase (tald-1) deficiency impairs mitochondrial function in vivo, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H2O2 levels. Lifespan extension from knockdown of tald-1 is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (fmo-2). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.

Tether mutations that restore function and suppress pleiotropic phenotypes of the C. elegans isp-1(qm150) Rieske iron-sulfur protein.

Mitochondria play an important role in numerous diseases as well as normative aging. Severe reduction in mitochondrial function contributes to childhood disorders such as Leigh Syndrome, whereas mild disruption can extend the lifespan of model organisms. The Caenorhabditis elegans isp-1 gene encodes the Rieske iron-sulfur protein subunit of cytochrome c oxidoreductase (complex III of the electron transport chain). The partial loss of function allele, isp-1(qm150), leads to several pleiotropic phenotypes. To better understand the molecular mechanisms of ISP-1 function, we sought to identify genetic suppressors of the delayed development of isp-1(qm150) animals. Here we report a series of intragenic suppressors, all located within a highly conserved six amino acid tether region of ISP-1. These intragenic mutations suppress all of the evaluated isp-1(qm150) phenotypes, including developmental rate, pharyngeal pumping rate, brood size, body movement, activation of the mitochondrial unfolded protein response reporter, CO2 production, mitochondrial oxidative phosphorylation, and lifespan extension. Furthermore, analogous mutations show a similar effect when engineered into the budding yeast Rieske iron-sulfur protein Rip1, revealing remarkable conservation of the structure-function relationship of these residues across highly divergent species. The focus on a single subunit as causal both in generation and in suppression of diverse pleiotropic phenotypes points to a common underlying molecular mechanism, for which we propose a "spring-loaded" model. These observations provide insights into how gating and control processes influence the function of ISP-1 in mediating pleiotropic phenotypes including developmental rate, movement, sensitivity to stress, and longevity.

Yeast replicative aging: a paradigm for defining conserved longevity interventions.

The finite replicative life span of budding yeast mother cells was demonstrated as early as 1959, but the idea that budding yeast could be used to model aging of multicellular eukaryotes did not enter the scientific mainstream until relatively recently. Despite continued skepticism by some, there are now abundant data that several interventions capable of extending yeast replicative life span have a similar effect in multicellular eukaryotes including nematode worms, fruit flies, and rodents. In particular, dietary restriction, mTOR signaling, and sirtuins are among the most studied longevity interventions in the field. Here, we describe key conserved longevity pathways in yeast and discuss relationships that may help explain how such broad conservation of aging processes could have evolved.

Quantitative determination of isopentenyl diphosphate in cultured mammalian cells.

Isopentenyl diphosphate (IPP), an intermediate of the isoprenoid biosynthetic pathway (IBP), has several important biological functions, yet a method to determine its basal level has not been described. Here, we describe a nonradioactive and sensitive analytical method to isolate and specifically quantify IPP from cultured mammalian cells. This method applies an enzymatic coupling reaction to determine intracellular concentrations of IPP. In this reaction, geranylgeranyl diphosphate synthase catalyzes the formation of geranylgeranyl diphosphate (GGPP) from IPP and farnesyl diphosphate (FPP). Subsequently, geranylgeranyl protein transferase I conjugates GGPP with a fluorescently labeled peptide. The geranylgeranylated peptide can be quantified by high-performance liquid chromatography (HPLC) with a fluorescence detector, thereby allowing for IPP quantification. The detection lower limit of the fluorescence-labeled geranylgeranyl peptide is approximately 5 pg (~0.017 pmol). This method was used to examine the effects of IBP inhibitors such as lovastatin and zoledronate on intracellular levels of IPP. Inhibition of hydroxymethylglutaryl coenzyme A reductase (HMGCR) by lovastatin (50 nM) decreases IPP levels by 78% and 53% in K562 and MCF-7 cells, respectively. Whereas zoledronic acid (10 µM) increased IPP levels 12.6-fold when compared with untreated cells in the K562 cell line, an astonishing 960-fold increase was observed in the MCF-7 cells.

End-of-life cell cycle arrest contributes to stochasticity of yeast replicative aging.

There is growing evidence that stochastic events play an important role in determining individual longevity. Studies in model organisms have demonstrated that genetically identical populations maintained under apparently equivalent environmental conditions display individual variation in life span that can be modeled by the Gompertz-Makeham law of mortality. Here, we report that within genetically identical haploid and diploid wild-type populations, shorter-lived cells tend to arrest in a budded state, while cells that arrest in an unbudded state are significantly longer-lived. This relationship is particularly notable in diploid BY4743 cells, where mother cells that arrest in a budded state have a shorter mean life span (25.6 vs. 35.6) and larger coefficient of variance with respect to individual life span (0.42 vs. 0.32) than cells that arrest in an unbudded state. Mutations that cause genomic instability tend to shorten life span and increase the proportion of the population that arrest in a budded state. These observations suggest that randomly occurring damage may contribute to stochasticity during replicative aging by causing a subset of the population to terminally arrest prematurely in the S or G2 phase of the cell cycle.

A simple and accessible CRISPR genome editing laboratory exercise using yeast.

CRISPR is a revolutionary tool to engineer the genome. Herein, we describe a laboratory exercise designed to introduce students to CRISPR by editing the genome of yeast to disrupt the ADE2 gene, which results in yeast that form red colored colonies. The experiment was constructed to be performed in a single laboratory session and to be accessible to students and instructors without experience working with yeast.

A novel bisphosphonate inhibitor of squalene synthase combined with a statin or a nitrogenous bisphosphonate in vitro.

Statins and nitrogenous bisphosphonates (NBP) inhibit 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR) and farnesyl diphosphate synthase (FDPS), respectively, leading to depletion of farnesyl diphosphate (FPP) and disruption of protein prenylation. Squalene synthase (SQS) utilizes FPP in the first committed step from the mevalonate pathway toward cholesterol biosynthesis. Herein, we have identified novel bisphosphonates as potent and specific inhibitors of SQS, including the tetrasodium salt of 9-biphenyl-4,8-dimethyl-nona-3,7-dienyl-1,1-bisphosphonic acid (compound 5). Compound 5 reduced cholesterol biosynthesis and lead to a substantial intracellular accumulation of FPP without reducing cell viability in HepG2 cells. At high concentrations, lovastatin and zoledronate impaired protein prenylation and decreased cell viability, which limits their potential use for cholesterol depletion. When combined with lovastatin, compound 5 prevented lovastatin-induced FPP depletion and impairment of protein farnesylation. Compound 5 in combination with the NBP zoledronate completely prevented zoledronate-induced impairment of both protein farnesylation and geranylgeranylation. Cotreatment of cells with compound 5 and either lovastatin or zoledronate was able to significantly prevent the reduction of cell viability caused by lovastatin or zoledronate alone. The combination of an SQS inhibitor with an HMGCR or FDPS inhibitor provides a rational approach for reducing cholesterol synthesis while preventing nonsterol isoprenoid depletion.

Bisphosphonates induce autophagy by depleting geranylgeranyl diphosphate.

Multiple studies have implicated the depletion of isoprenoid biosynthetic pathway intermediates in the induction of autophagy. However, the exact mechanism by which isoprenoid biosynthesis inhibitors induce autophagy has not been well established. We hypothesized that inhibition of farnesyl diphosphate synthase (FDPS) and geranylgeranyl diphosphate synthase (GGDPS) by bisphosphonates would induce autophagy by depleting cellular geranylgeranyl diphosphate (GGPP) and impairing protein geranylgeranylation. Herein, we show that an inhibitor of FDPS (zoledronate) and an inhibitor of GGDPS (digeranyl bisphosphonate, DGBP) induce autophagy in PC3 prostate cancer and MDA-MB-231 breast cancer cells as measured by accumulation of the autophagic marker LC3-II. Treatment of cells with lysosomal protease inhibitors [(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (E-64d) and pepstatin A] in combination with zoledronate or digeranyl bisphosphonate further enhances the formation of LC3-II, indicating that these compounds induce autophagic flux. It is noteworthy that the addition of exogenous GGPP prevented the accumulation of LC3-II and impairment of Rab6 (a GGTase II substrate) geranylgeranylation by isoprenoid pathway inhibitors (lovastatin, zoledronate, and DGBP). However, exogenous GGPP did not restore isoprenoid pathway inhibitor-induced impairment of Rap1a (a GGTase I substrate) geranylgeranylation. In addition, specific inhibitors of farnesyl transferase and geranylgeranyl transferase I are unable to induce autophagy in our system. Furthermore, the addition of bafilomycin A1 (an inhibitor of autophagy processing) enhanced the antiproliferative effects of digeranyl bisphosphonate. These results are the first to demonstrate that bisphosphonates induce autophagy. Our study suggests that induction of autophagy in PC3 cells with these agents is probably dependent upon impairment of geranylgeranylation of GGTase II substrates.

Inactivating histone deacetylase HDA promotes longevity by mobilizing trehalose metabolism.

Histone acetylations are important epigenetic markers for transcriptional activation in response to metabolic changes and various stresses. Using the high-throughput SEquencing-Based Yeast replicative Lifespan screen method and the yeast knockout collection, we demonstrate that the HDA complex, a class-II histone deacetylase (HDAC), regulates aging through its target of acetylated H3K18 at storage carbohydrate genes. We find that, in addition to longer lifespan, disruption of HDA results in resistance to DNA damage and osmotic stresses. We show that these effects are due to increased promoter H3K18 acetylation and transcriptional activation in the trehalose metabolic pathway in the absence of HDA. Furthermore, we determine that the longevity effect of HDA is independent of the Cyc8-Tup1 repressor complex known to interact with HDA and coordinate transcriptional repression. Silencing the HDA homologs in C. elegans and Drosophila increases their lifespan and delays aging-associated physical declines in adult flies. Hence, we demonstrate that this HDAC controls an evolutionarily conserved longevity pathway.

Suppression of telomere capping defects of Saccharomyces cerevisiae yku70 and yku80 mutants by telomerase.

The Ku complex performs multiple functions inside eukaryotic cells, including protection of chromosomal DNA ends from degradation and fusion events, recruitment of telomerase, and repair of double-strand breaks (DSBs). Inactivation of Ku complex genes YKU70 or YKU80 in cells of the yeast Saccharomyces cerevisiae gives rise to mutants that exhibit shortened telomeres and temperature-sensitive growth. In this study, we have investigated the mechanism by which overexpression of telomerase suppresses the temperature sensitivity of yku mutants. Viability of yku cells was restored by overexpression of the Est2 reverse transcriptase and TLC1 RNA template subunits of telomerase, but not the Est1 or Est3 proteins. Overexpression of other telomerase- and telomere-associated proteins (Cdc13, Stn1, Ten1, Rif1, Rif2, Sir3, and Sir4) did not suppress the growth defects of yku70 cells. Mechanistic features of suppression were assessed using several TLC1 RNA deletion derivatives and Est2 enzyme mutants. Supraphysiological levels of three catalytically inactive reverse transcriptase mutants (Est2-D530A, Est2-D670A, and Est2-D671A) suppressed the loss of viability as efficiently as the wild-type Est2 protein, without inducing cell senescence. Roles of proteins regulating telomere length were also determined. The results support a model in which chromosomes in yku mutants are stabilized via a replication-independent mechanism involving structural reinforcement of protective telomere cap structures.

Evolution of natural lifespan variation and molecular strategies of extended lifespan in yeast.

To understand the genetic basis and selective forces acting on longevity, it is useful to examine lifespan variation among closely related species, or ecologically diverse isolates of the same species, within a controlled environment. In particular, this approach may lead to understanding mechanisms underlying natural variation in lifespan. Here, we analyzed 76 ecologically diverse wild yeast isolates and discovered a wide diversity of replicative lifespan (RLS). Phylogenetic analyses pointed to genes and environmental factors that strongly interact to modulate the observed aging patterns. We then identified genetic networks causally associated with natural variation in RLS across wild yeast isolates, as well as genes, metabolites, and pathways, many of which have never been associated with yeast lifespan in laboratory settings. In addition, a combined analysis of lifespan-associated metabolic and transcriptomic changes revealed unique adaptations to interconnected amino acid biosynthesis, glutamate metabolism, and mitochondrial function in long-lived strains. Overall, our multiomic and lifespan analyses across diverse isolates of the same species shows how gene-environment interactions shape cellular processes involved in phenotypic variation such as lifespan.

Pterocarpus marsupium extract extends replicative lifespan in budding yeast.

As the molecular mechanisms of biological aging become better understood, there is growing interest in identifying interventions that target those mechanisms to promote extended health and longevity. The budding yeast Saccharomyces cerevisiae has served as a premier model organism for identifying genetic and molecular factors that modulate cellular aging and is a powerful system in which to evaluate candidate longevity interventions. Here we screened a collection of natural products and natural product mixtures for effects on the growth rate, mTOR-mediated growth inhibition, and replicative lifespan. No mTOR inhibitory activity was detected, but several of the treatments affected growth rate and lifespan. The strongest lifespan shortening effects were observed for green tea extract and berberine. The most robust lifespan extension was detected from an extract of Pterocarpus marsupium and another mixture containing Pterocarpus marsupium extract. These findings illustrate the utility of the yeast system for longevity intervention discovery and identify Pterocarpus marsupium extract as a potentially fruitful longevity intervention for testing in higher eukaryotes.

Inhibition of DNA double-strand break repair by the Ku heterodimer in mrx mutants of Saccharomyces cerevisiae.

Yeast rad50 and mre11 nuclease mutants are hypersensitive to physical and chemical agents that induce DNA double-strand breaks (DSBs). This sensitivity was suppressed by elevating intracellular levels of TLC1, the RNA subunit of telomerase. Suppression required proteins linked to homologous recombination, including Rad51, Rad52, Rad59 and Exo1, but not genes of the nonhomologous end-joining (NHEJ) repair pathway. Deletion mutagenesis experiments demonstrated that the 5'-end of TLC1 RNA was essential and a segment containing a binding site for the Yku70/Yku80 complex was sufficient for suppression. A mutant TLC1 RNA unable to associate with Yku80 protein did not increase resistance. These and other genetic studies indicated that association of the Ku heterodimer with broken DNA ends inhibits recombination in mrx mutants, but not in repair-proficient cells or in other DNA repair single mutants. In support of this model, DNA damage resistance of mrx cells was enhanced when YKU70 was co-inactivated. Defective recombinational repair of DSBs in mrx cells thus arises from at least two separate processes: loss of Mrx nuclease-associated DNA end-processing and inhibition of the Exo1-mediated secondary recombination pathway by Ku.

Synthesis and biological evaluation of a series of aromatic bisphosphonates.

Geminal bisphosphonates display varied biological activity depending on the nature of the substituents on the central carbon atom. For example, the nitrogenous bisphosphonates zoledronate and risedronate inhibit the enzyme farnesyl diphosphate synthase while digeranyl bisphosphonate has been shown to inhibit the enzyme geranylgeranyl diphosphate synthase. We now have synthesized isoprenoid bisphosphonates where an aromatic ring has been used to replace one of the isoprenoid olefins in an isoprenoid bisphosphonate and investigated the ability of these new compounds to impair protein geranylgeranylation within cells. Several of these new compounds are potent inhibitors of the enzyme geranylgeranyl diphosphate synthase.

The antifungal plant defensin HsAFP1 induces autophagy, vacuolar dysfunction and cell cycle impairment in yeast.

The plant defensin HsAFP1 is characterized by broad-spectrum antifungal activity and induces apoptosis in Candida albicans. In this study, we performed a transcriptome analysis on C. albicans cultures treated with HsAFP1 to gain further insight in the antifungal mode of action of HsAFP1. Various genes coding for cell surface proteins, like glycosylphosphatidylinositol (GPI)-anchored proteins, and proteins involved in cation homeostasis, autophagy and in cell cycle were differentially expressed upon HsAFP1 treatment. The biological validation of these findings was performed in the model yeast Saccharomyces cerevisiae. To discriminate between events linked to HsAFP1's antifungal activity and those that are not, we additionally used an inactive HsAFP1 mutant. We demonstrated that (i) HsAFP1-resistent S. cerevisiae mutants that are characterized by a defect in processing GPI-anchors are unable to internalize HsAFP1, and (ii) moderate doses (FC50, fungicidal concentration resulting in 50% killing) of HsAFP1 induce autophagy in S. cerevisiae, while high HsAFP1 doses result in vacuolar dysfunction. Vacuolar function is an important determinant of replicative lifespan (RLS) under dietary restriction (DR). In line, HsAFP1 specifically reduces RLS under DR. Lastly, (iii) HsAFP1 affects S. cerevisiae cell cycle in the G2/M phase. However, the latter HsAFP1-induced event is not linked to its antifungal activity, as the inactive HsAFP1 mutant also impairs the G2/M phase. In conclusion, we demonstrated that GPI-anchored proteins are involved in HsAFP1's internalization, and that HsAFP1 induces autophagy, vacuolar dysfunction and impairment of the cell cycle. Collectively, all these data provide novel insights in the mode of action of HsAFP1 as well as in S. cerevisiae tolerance mechanisms against this peptide.

Loss of vacuolar acidity results in iron-sulfur cluster defects and divergent homeostatic responses during aging in Saccharomyces cerevisiae.

The loss of vacuolar/lysosomal acidity is an early event during aging that has been linked to mitochondrial dysfunction. However, it is unclear how loss of vacuolar acidity results in age-related dysfunction. Through unbiased genetic screens, we determined that increased iron uptake can suppress the mitochondrial respiratory deficiency phenotype of yeast vma mutants, which have lost vacuolar acidity due to genetic disruption of the vacuolar ATPase proton pump. Yeast vma mutants exhibited nuclear localization of Aft1, which turns on the iron regulon in response to iron-sulfur cluster (ISC) deficiency. This led us to find that loss of vacuolar acidity with age in wild-type yeast causes ISC defects and a DNA damage response. Using microfluidics to investigate aging at the single-cell level, we observe grossly divergent trajectories of iron homeostasis within an isogenic and environmentally homogeneous population. One subpopulation of cells fails to mount the expected compensatory iron regulon gene expression program, and suffers progressively severe ISC deficiency with little to no activation of the iron regulon. In contrast, other cells show robust iron regulon activity with limited ISC deficiency, which allows extended passage and survival through a period of genomic instability during aging. These divergent trajectories suggest that iron regulation and ISC homeostasis represent a possible target for aging interventions.

Pivaloyloxymethyl-modified isoprenoid bisphosphonates display enhanced inhibition of cellular geranylgeranylation.

Nitrogenous bisphosphonate inhibitors of farnesyl disphosphate synthase have been used clinically for treatment of bone disease. Because many of their effects may be mediated by depletion of geranylgeranyl diphosphate, our group has sought compounds that do this more directly through inhibition of geranylgeranyl diphosphate synthase and we have discovered a number of isoprenoid-containing bisphosphonates that selectively inhibit this enzyme. These compounds have a high negative charge at physiological pH which is necessary for inhibition of the enzyme but may limit their ability to enter cells. Therefore, chemical modifications that mask this charge may enhance their cellular potency. We now have synthesized novel pivaloyloxymethyl-modified isoprenoid bisphosphonates and investigated their ability to inhibit protein geranylgeranylation within cells. We have found that addition of pivaloyloxymethyl moieties to isoprenoid bisphosphonates increases their potency towards cellular geranylgeranylation even though this modification decreases their in vitro inhibition of geranylgeranyl diphosphate synthase. Pivaloyloxymethyl modifications more effectively increase the cellular activity of the more polar isoprenoid bisphosphonates. These results reveal structural relationships between in vitro and cellular activity which may serve as the basis for future development of more potent and/or drug-like inhibitors of geranylgeranyl diphosphate synthase.