The polyHIS Tract of Yeast AMPK Coordinates Carbon Metabolism with Iron Availability.

Energy status in all eukaryotic cells is sensed by AMP-kinases. We have previously found that the poly-histidine tract at the N-terminus of S. cerevisiae AMPK (Snf1) inhibits its function in the presence of glucose via a pH-regulated mechanism. We show here that in the absence of glucose, the poly-histidine tract has a second function, linking together carbon and iron metabolism. Under conditions of iron deprivation, when different iron-intense cellular systems compete for this scarce resource, Snf1 is inhibited. The inhibition is via an interaction of the poly-histidine tract with the low-iron transcription factor Aft1. Aft1 inhibition of Snf1 occurs in the nucleus at the nuclear membrane, and only inhibits nuclear Snf1, without affecting cytosolic Snf1 activities. Thus, the temporal and spatial regulation of Snf1 activity enables a differential response to iron depending upon the type of carbon source. The linkage of nuclear Snf1 activity to iron sufficiency ensures that sufficient clusters are available to support respiratory enzymatic activity and tests mitochondrial competency prior to activation of nuclear Snf1.

A comparative analysis of telomere length maintenance circuits in fission and budding yeast.

The natural ends of the linear eukaryotic chromosomes are protected by telomeres, which also play an important role in aging and cancer development. Telomere length varies between species, but it is strictly controlled in all organisms. The process of Telomere Length Maintenance (TLM) involves many pathways, protein complexes and interactions that were first discovered in budding and fission yeast model organisms (Saccharomyces cerevisiae, Schizosaccharomyces pombe). In particular, large-scale systematic genetic screens in budding yeast uncovered a network of ≈ 500 genes that, when mutated, cause telomeres to lengthen or to shorten. In contrast, the TLM network in fission yeast remains largely unknown and systematic data is still lacking. In this work we try to close this gap and develop a unified interpretable machine learning framework for TLM gene discovery and phenotype prediction in both species. We demonstrate the utility of our framework in pinpointing the pathways by which TLM homeostasis is maintained and predicting novel TLM genes in fission yeast. The results of this study could be used for better understanding of telomere biology and serve as a step towards the adaptation of computational methods based on telomeric data for human prognosis.

Telomeres and stress in yeast cells: When genes and environment interact.

Telomeres are structures composed of simple DNA repeats and specific proteins that protect the eukaryotic chromosomal ends from degradation, and facilitate the replication of the genome. They are central to the maintenance of the genome integrity, and play important roles in the development of cancer and in the process of aging in humans. The yeast Saccharomyces cerevisiae has greatly contributed to our understanding of basic telomere biology. Our laboratory has carried out systematic screen for mutants that affect telomere length, and identified ∼500 genes that, when mutated, affect telomere length. Remarkably, all ∼500 TLM (Telomere Length Maintenance) genes participate in a very tight homeostatic process, and it is enough to mutate one of them to change the steady-state telomere length. Despite this complex network of balances, it is also possible to change telomere length in yeast by applying several types of external stresses. We summarize our insights about the molecular mechanisms by which genes and environment interact to affect telomere length.

The Yeast PCNA Unloader Elg1 RFC-Like Complex Plays a Role in Eliciting the DNA Damage Checkpoint.

The PCNA (proliferating cell nuclear antigen) ring plays central roles during DNA replication and repair. The yeast Elg1 RFC-like complex (RLC) is the principal unloader of chromatin-bound PCNA and thus plays a central role in maintaining genome stability. Here we identify a role for Elg1 in the unloading of PCNA during DNA damage. Using DNA damage checkpoint (DC)-inducible and replication checkpoint (RC)-inducible strains, we show that Elg1 is essential for eliciting the signal in the DC branch. In the absence of Elg1 activity, the Rad9 (53BP1) and Dpb11 (TopBP1) adaptor proteins are recruited but fail to be phosphorylated by Mec1 (ATR), resulting in a lack of checkpoint activation. The chromatin immunoprecipitation of PCNA at the Lac operator sites reveals that accumulated local PCNA influences the checkpoint activation process in elg1 mutants. Our data suggest that Elg1 participates in a mechanism that may coordinate PCNA unloading during DNA repair with DNA damage checkpoint induction.IMPORTANCE The Elg1protein forms an RFC-like complex in charge of unloading PCNA from chromatin during DNA replication and repair. Mutations in the ELG1 gene caused genomic instability in all organisms tested and cancer in mammals. Here we show that Elg1 plays a role in the induction of the DNA damage checkpoint, a cellular response to DNA damage. We show that this defect is due to a defect in the signal amplification process during induction. Thus, cells coordinate the cell's response and the PCNA unloading through the activity of Elg1.

Frequent ploidy changes in growing yeast cultures.

Ploidy is considered a very stable cellular characteristic. Although rare, changes in ploidy play important roles in the acquisition of long-term adaptations. Since these duplications allow the subsequent loss of individual chromosomes and accumulation of mutations, changes in ploidy can also cause genomic instability, and have been found to promote cancer. Despite the importance of the subject, measuring the rate of whole-genome duplications has proven extremely challenging. We have recently measured the rate of diploidization in yeast using long-term, in-lab experiments. We found that spontaneous diploidization occurs frequently, by two different mechanisms: endoreduplication and mating type switching. Despite its common occurrence, spontaneous diploidization is usually selected against, although it can be advantageous under some stressful conditions. Our results have implications for the understanding of evolutionary processes, as well as for the use of yeast cells in biotechnological applications.

Spontaneous Changes in Ploidy Are Common in Yeast.

Changes in ploidy are relatively rare, but play important roles in the development of cancer and the acquisition of long-term adaptations. Genome duplications occur across the tree of life, and can alter the rate of adaptive evolution. Moreover, by allowing the subsequent loss of individual chromosomes and the accumulation of mutations, changes in ploidy can promote genomic instability and/or adaptation. Although many studies have been published in the last years about changes in chromosome number and their evolutionary consequences, tracking and measuring the rate of whole-genome duplications have been extremely challenging. We have systematically studied the appearance of diploid cells among haploid yeast cultures evolving for over 100 generations in different media. We find that spontaneous diploidization is a relatively common event, which is usually selected against, but under certain stressful conditions may become advantageous. Furthermore, we were able to detect and distinguish between two different mechanisms of diploidization, one that requires whole-genome duplication (endoreduplication) and a second that involves mating-type switching despite the use of heterothallic strains. Our results have important implications for our understanding of evolution and adaptation in fungal pathogens and the development of cancer, and for the use of yeast cells in biotechnological applications.

Do long telomeres affect cellular fitness?

Telomeres protect the chromosome ends and maintain the genome stability; they, therefore, play important roles in aging and cancer. Despite the wide variability in telomere length among eukaryotes, in all telomerase-expressing cells telomere length is strictly controlled within a very narrow range. In humans, telomeres shorten with age, and it has been proposed that telomere shortening may play a causal role in aging. Using yeast strains with genetically or physiologically generated differences in telomere length, we have explored the question of whether having long telomeres affects telomere function and fitness or cellular lifespan. We found no effect of long telomeres on vegetative cell division, meiosis, or in cellular lifespan. No positive or negative effect on fitness was observed either under stressful conditions.

Rnr1's role in telomere elongation cannot be replaced by Rnr3: a role beyond dNTPs?

Telomeres, the nucleoprotein complexes at the end of eukaryotic chromosomes, protect them from degradation and ensure the replicative capacity of cells. In most human tumors and in budding yeast, telomere length is maintained by the activity of telomerase, an enzyme that adds dNTPs according to an internal RNA template. The dNTPs are generated with the help of the ribonucleotide reductase (RNR) complex. We have recently generated strains lacking the large subunit of RNR, Rnr1, which were kept viable by the expression of RNR complexes containing the Rnr1 homolog, Rnr3. Interestingly, we found that these Rnr1-deficient strains have short telomeres that are stably maintained, but cannot become efficiently elongated by telomerase. Thus, a basic maintenance of short telomeres is possible under conditions, where Rnr1 activity is absent, but a sustained elongation of short telomeres fully depends on Rnr1 activity. We show that Rnr3 cannot compensate for this telomeric function of Rnr1 even when overall cellular dNTP values are restored. This suggests that Rnr1 plays a role in telomere elongation beyond increasing cellular dNTP levels. Furthermore, our data indicate that telomerase may act in two different modes, one that is capable of coping with the "end-replication problem" and is functional even in the absence of Rnr1 and another required for the sustained elongation of short telomeres, which fully depends on the presence of Rnr1. Supply of dNTPs for telomere elongation is provided by the Mec1ATR checkpoint, both during regular DNA replication and upon replication fork stalling. We discuss the implications of these results on telomere maintenance in yeast and cancer cells.

Long Telomeres Do Not Affect Cellular Fitness in Yeast.

Telomeres, the ends of the eukaryotic chromosomes, help to maintain the genome's integrity and thus play important roles in aging and cancer. Telomere length is strictly controlled in all organisms. In humans, telomeres shorten with age, and it has been proposed that telomere shortening may play a causal role in aging. We took advantage of the availability of yeast strains with genetically or physiologically generated differences in telomere length to measure the effect that telomere length may have on cellular growth. By comparing the growth rates affecting telomere length of various yeast mutants we show that there is no correlation between their telomere length and cellular fitness. We also show that wild-type yeast cells carrying extremely long telomeres (~5 times longer than the average) showed no signs of mitotic or meiotic defects, and competition experiments found no differences in growth between strains with normal telomeres and strains with long telomeres. No advantage or disadvantage of cells with long telomeres was detected under stress conditions either. Finally, telomere length had no effect in a chronological life span assay, which measures survival of post-mitotic-stage cells. We conclude that extreme telomere length has no effects (positive or negative) on the fitness of yeast cells.IMPORTANCE Telomeres protect the chromosomal ends from fusion, degradation, and unwanted repair. Therefore, telomeres preserve genome stability and cell viability. In humans, telomeres shorten with each cell duplication event and with age. It has thus been proposed that telomere shortening may be responsible for human aging and that elongation of telomeres may be a way to rejuvenate cells and to combat aging. However, it is difficult to prove this hypothesis in human cells. Yeasts are easy to manipulate and have telomeres whose length is strictly maintained. Here we show that yeast cells manipulated to have extremely long telomeres (~5-fold those of normal cells) did not show any improvement or reduction in fitness compared to otherwise identical cells with telomeres of normal length under all the conditions tested. Moreover, an assay that measures cell aging showed no effect of the presence of extremely long telomeres. We thus conclude that extreme telomere length, at least in yeast cells, does not affect cellular fitness, aging, or senescence.

Role of the ESCRT Complexes in Telomere Biology.

Eukaryotic chromosomal ends are protected by telomeres from fusion, degradation, and unwanted double-strand break repair events. Therefore, telomeres preserve genome stability and integrity. Telomere length can be maintained by telomerase, which is expressed in most human primary tumors but is not expressed in the majority of somatic cells. Thus, telomerase may be a highly relevant anticancer drug target. Genome-wide studies in the yeast Saccharomyces cerevisiae identified a set of genes associated with telomere length maintenance (TLM genes). Among the tlm mutants with short telomeres, we found a strong enrichment for those affecting vacuolar and endosomal traffic (particularly the endosomal sorting complex required for transport [ESCRT] pathway). Here, we present our results from investigating the surprising link between telomere shortening and the ESCRT machinery. Our data show that the whole ESCRT system is required to safeguard proper telomere length maintenance. We propose a model of impaired end resection resulting in too little telomeric overhang, such that Cdc13 binding is prevented, precluding either telomerase recruitment or telomeric overhang protection. IMPORTANCE: Telomeres are the ends of eukaryotic chromosomes. They are necessary for the proper replication of the genome and protect the chromosomes from degradation. In a large-scale systematic screen for mutants that affect telomere length in yeast, we found that mutations in any of the genes encoding the ESCRT complexes, required for the formation of transport vesicles within the cell, cause telomere shortening. We carried out an analysis of the mechanisms disrupted in these mutants and found that they are defective for the ability to elongate short telomeres, probably due to faulty end processing. We discuss the significance of these findings and how they could be relevant to anticancer therapies.

Mixed Integer Linear Programming based machine learning approach identifies regulators of telomerase in yeast.

Understanding telomere length maintenance mechanisms is central in cancer biology as their dysregulation is one of the hallmarks for immortalization of cancer cells. Important for this well-balanced control is the transcriptional regulation of the telomerase genes. We integrated Mixed Integer Linear Programming models into a comparative machine learning based approach to identify regulatory interactions that best explain the discrepancy of telomerase transcript levels in yeast mutants with deleted regulators showing aberrant telomere length, when compared to mutants with normal telomere length. We uncover novel regulators of telomerase expression, several of which affect histone levels or modifications. In particular, our results point to the transcription factors Sum1, Hst1 and Srb2 as being important for the regulation of EST1 transcription, and we validated the effect of Sum1 experimentally. We compiled our machine learning method leading to a user friendly package for R which can straightforwardly be applied to similar problems integrating gene regulator binding information and expression profiles of samples of e.g. different phenotypes, diseases or treatments.

Cross-Talk between Carbon Metabolism and the DNA Damage Response in S. cerevisiae.

Yeast cells with DNA damage avoid respiration, presumably because products of oxidative metabolism can be harmful to DNA. We show that DNA damage inhibits the activity of the Snf1 (AMP-activated) protein kinase (AMPK), which activates expression of genes required for respiration. Glucose and DNA damage upregulate SUMOylation of Snf1, catalyzed by the SUMO E3 ligase Mms21, which inhibits SNF1 activity. The DNA damage checkpoint kinases Mec1/ATR and Tel1/ATM, as well as the nutrient-sensing protein kinase A (PKA), regulate Mms21 activity toward Snf1. Mec1 and Tel1 are required for two SNF1-regulated processes-glucose sensing and ADH2 gene expression-even without exogenous genotoxic stress. Our results imply that inhibition of Snf1 by SUMOylation is a mechanism by which cells lower their respiration in response to DNA damage. This raises the possibility that activation of DNA damage checkpoint mechanisms could contribute to aerobic fermentation (Warburg effect), a hallmark of cancer cells.

Regulation of Elg1 activity by phosphorylation.

ELG1 is a conserved gene with important roles in the maintenance of genome stability. Elg1's activity prevents gross chromosomal rearrangements, maintains proper telomere length regulation, helps repairing DNA damage created by a number of genotoxins and participates in sister chromatid cohesion. Elg1 is evolutionarily conserved, and its Fanconi Anemia-related mammalian ortholog (also known as ATAD5) is embryonic lethal when lost in mice and acts as a tumor suppressor in mice and humans. Elg1 encodes a protein that forms an RFC-like complex that unloads the replicative clamp, PCNA, from DNA, mainly in its SUMOylated form. We have identified 2 different regions in yeast Elg1 that undergo phosphorylation. Phosphorylation of one of them, S112, is dependent on the ATR yeast ortholog, Mec1, and probably is a direct target of this kinase. We show that phosphorylation of Elg1 is important for its role at telomeres. Mutants unable to undergo phosphorylation suppress the DNA damage sensitivity of Δrad5 mutants, defective for an error-free post-replicational bypass pathway. This indicates a role of phosphorylation in the regulation of DNA repair. Our results open the way to investigate the mechanisms by which the activity of Elg1 is regulated during DNA replication and in response to DNA damage.

Fungal stress biology: a preface to the Fungal Stress Responses special edition.

There is currently an urgent need to increase global food security, reverse the trends of increasing cancer rates, protect environmental health, and mitigate climate change. Toward these ends, it is imperative to improve soil health and crop productivity, reduce food spoilage, reduce pesticide usage by increasing the use of biological control, optimize bioremediation of polluted sites, and generate energy from sustainable sources such as biofuels. This review focuses on fungi that can help provide solutions to such problems. We discuss key aspects of fungal stress biology in the context of the papers published in this Special Issue of Current Genetics. This area of biology has relevance to pure and applied research on fungal (and indeed other) systems, including biological control of insect pests, roles of saprotrophic fungi in agriculture and forestry, mycotoxin contamination of the food-supply chain, optimization of microbial fermentations including those used for bioethanol production, plant pathology, the limits of life on Earth, and astrobiology.

Elg1, a central player in genome stability.

ELG1 is a conserved gene uncovered in a number of genetic screens in yeast aimed at identifying factors important in the maintenance of genome stability. Elg1's activity prevents gross chromosomal rearrangements, maintains proper telomere length regulation, helps repairing DNA damage created by a number of genotoxins and participates in sister chromatid cohesion. Elg1 is evolutionarily conserved, and its mammalian ortholog (also known as ATAD5) is embryonic lethal when lost in mice, acts as a tumor suppressor in mice and humans, exhibits physical interactions with components of the human Fanconi Anemia pathway and may be responsible for some of the phenotypes associated with neurofibromatosis. In this review, we summarize the information available on Elg1-related activities in yeast and mammals, and present models to explain how the different phenotypes observed in the absence of Elg1 activity are related.

Glucose activates TORC2-Gad8 protein via positive regulation of the cAMP/cAMP-dependent protein kinase A (PKA) pathway and negative regulation of the Pmk1 protein-mitogen-activated protein kinase pathway.

The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.

Biology of telomeres: lessons from budding yeast.

Telomeres are nucleoprotein structures that cap the ends of the linear eukaryotic chromosomes and thereby protect their stability and integrity. Telomeres play central roles in maintaining the genome's integrity, distinguishing between the natural chromosomal ends and unwanted double-stranded breaks. In addition, telomeres are replicated by a special reverse transcriptase called telomerase, in a complex mechanism that is coordinated with the genome's replication. Telomeres also play an important role in tethering the chromosomes to the nuclear envelope, thus helping in positioning the chromosomes within the nucleus. The special chromatin configuration of telomeres affects the expression of nearby genes; nonetheless, telomeres are transcribed, creating noncoding RNA molecules that hybridize to the chromosomal ends and seem to play regulatory roles. The yeast Saccharomyces cerevisiae, with its sophisticated genetics and molecular biology, has provided many fundamental concepts in telomere biology, which were later found to be conserved in all organisms. Here, we present an overview of all the aspects of telomere biology investigated in yeast, which continues to provide new insights into this complex and important subject, which has significant medical implications, especially in the fields of aging and cancer.

A high-throughput chemical screen with FDA approved drugs reveals that the antihypertensive drug Spironolactone impairs cancer cell survival by inhibiting homology directed repair.

DNA double-strand breaks (DSBs) are the most severe type of DNA damage. DSBs are repaired by non-homologous end-joining or homology directed repair (HDR). Identifying novel small molecules that affect HDR is of great importance both for research use and therapy. Molecules that elevate HDR may improve gene targeting whereas inhibiting molecules can be used for chemotherapy, since some of the cancers are more sensitive to repair impairment. Here, we performed a high-throughput chemical screen for FDA approved drugs, which affect HDR in cancer cells. We found that HDR frequencies are increased by retinoic acid and Idoxuridine and reduced by the antihypertensive drug Spironolactone. We further revealed that Spironolactone impairs Rad51 foci formation, sensitizes cancer cells to DNA damaging agents, to Poly (ADP-ribose) polymerase (PARP) inhibitors and cross-linking agents and inhibits tumor growth in xenografts, in mice. This study suggests Spironolactone as a new candidate for chemotherapy.

Isp7 is a novel regulator of amino acid uptake in the TOR signaling pathway.

TOR proteins reside in two distinct complexes, TOR complexes 1 and 2 (TORC1 and TORC2), that are central for the regulation of cellular growth, proliferation, and survival. TOR is also the target for the immunosuppressive and anticancer drug rapamycin. In Schizosaccharomyces pombe, disruption of the TSC complex, mutations in which can lead to the tuberous sclerosis syndrome in humans, results in a rapamycin-sensitive phenotype under poor nitrogen conditions. We show here that the sensitivity to rapamycin is mediated via inhibition of TORC1 and suppressed by overexpression of isp7(+), a member of the family of 2-oxoglutarate-Fe(II)-dependent oxygenase genes. The transcript level of isp7(+) is negatively regulated by TORC1 but positively regulated by TORC2. Yet we find extensive similarity between the transcriptome of cells disrupted for isp7(+) and cells mutated in the catalytic subunit of TORC1. Moreover, Isp7 regulates amino acid permease expression in a fashion similar to that of TORC1 and opposite that of TORC2. Overexpression of isp7(+) induces TORC1-dependent phosphorylation of ribosomal protein Rps6 while inhibiting TORC2-dependent phosphorylation and activation of the AGC-like kinase Gad8. Taken together, our findings suggest a central role for Isp7 in amino acid homeostasis and the presence of isp7(+)-dependent regulatory loops that affect both TORC1 and TORC2.

Genome-wide studies of telomere biology in budding yeast.

Telomeres are specialized DNA-protein structures at the ends of eukaryotic chromosomes. Telomeres are essential for chromosomal stability and integrity, as they prevent chromosome ends from being recognized as double strand breaks. In rapidly proliferating cells, telomeric DNA is synthesized by the enzyme telomerase, which copies a short template sequence within its own RNA moiety, thus helping to solve the "end-replication problem", in which information is lost at the ends of chromosomes with each DNA replication cycle. The basic mechanisms of telomere length, structure and function maintenance are conserved among eukaryotes. Studies in the yeast Saccharomyces cerevisiae have been instrumental in deciphering the basic aspects of telomere biology. In the last decade, technical advances, such as the availability of mutant collections, have allowed carrying out systematic genome-wide screens for mutants affecting various aspects of telomere biology. In this review we summarize these efforts, and the insights that this Systems Biology approach has produced so far.