Eukaryotes typically maintain telomere length within a defined range. While short telomeres are known to activate DNA damage responses and limit cell proliferation, long telomeres are associated with extended proliferative capacity. The broader cellular consequences of long telomeres are comparatively less well understood. In budding yeast Saccharomyces cerevisiae, long telomeres have been shown to influence gene expression at specific loci, but whether long telomeres affect transcription genome-wide has not been reported. Here, we analysed transcriptomes in a lineage that inherited long telomeres (originally due to a rif2Δ mutation). Transcriptomes were assessed over two rounds of mitosis and meiosis in the absence of the rif2Δ mutation. We show that strains with long telomeres exhibit a distinct gene expression profile, including upregulation of membrane transporters and downregulation of a smaller subset of genes. Both up- and down-regulated genes were distributed across the genome, arguing against a purely telomereproximal effect on gene expression. Affected genes were enriched for Rap1 binding sites, consistent with a model in which long telomeres sequester telomereassociated transcriptional regulators, such as Rap1, and thereby affect gene expression at non-telomeric binding sites for these regulators. Accordingly, the magnitude of transcriptional changes was greatest in strains with the longest telomeres. Together, our findings demonstrate that long telomeres induce a genomewide transcriptional response that can accompany inherited long telomeres across generations. Similar effects of long telomeres are likely to occur in other eukaryotes, including humans, where long telomeres are associated with disease.

The ends of linear eukaryotic chromosomes are protected from being recognized as DNA double-strand breaks by telomeres, containing repetitive DNA sequences that bind specific proteins. In humans, mutations in telomere regulatory genes lead to short or long telomere syndromes. These syndromes often show genetic anticipation, where the disease has an earlier onset and a more severe manifestation in each new generation. Later generations inherit not only the mutation affecting telomere length, but also abnormal telomere length. Many aspects of telomere length homeostasis are conserved between mammals and yeast. Here, we explored telomere length inheritance patterns through the sexual cycle in yeast. Analysis of single telomeres, rather than bulk telomeres, shows that if haploid yeast with short telomeres mate with wild-type yeast, creating diploids, short telomere lengths rapidly normalize (within 30 cell divisions). However, long telomeres inherited from one parent can persist for more than 200 mitotic cell divisions. Long telomeres can also be transmitted through more than one round of meiosis, independently of mutations that cause long telomeres. These patterns, along with haploinsufficiency effects, show that even in yeast, there is a complex relationship between telomere length, telomere length inheritance, and mutations that affect telomere length. Our findings may have implications for families affected by telomere syndromes.

Saccharomyces cerevisiae is a commonly used model organism for understanding eukaryotic gene function. However, the close proximity between yeast genes can complicate the interpretation of yeast genetic data, particularly high-throughput data. In this study, we examined the interplay between genes encoding components of the PAF1 complex and VPS36, the gene located next to CDC73 on chromosome XII. The PAF1 complex (Cdc73, Paf1, Ctr9, Leo1, and Rtf1, in yeast) affects RNA levels by affecting transcription, histone modifications, and post-transcriptional RNA processing. The human PAF1 complex is linked to cancer, and in yeast, it has been reported to play a role in telomere biology. Vps36, part of the ESCRT-II complex, is involved in sorting proteins for vacuolar/lysosomal degradation. We document a complex set of genetic interactions, which include an adjacent gene effect between CDC73 and VPS36 and synthetic sickness between vps36Δ and cdc73Δ, paf1Δ, or ctr9Δ. Importantly, paf1Δ and ctr9Δ are synthetically lethal with deletions of other components of the ESCRT-II (SNF8 and VPS25), ESCRT-I (STP22), or ESCRT-III (SNF7) complexes. We found that RNA levels of VPS36, but not other ESCRT components, are positively regulated by all components of the PAF1 complex. Finally, we show that deletion of ESCRT components decreases the telomere length in the S288C yeast genetic background, but not in the W303 background. Together, our results outline complex interactions, in cis and in trans, between genes encoding PAF1 and ESCRT-II complex components that affect telomere function and cell viability in yeast.

The levels of telomeric proteins, such as telomerase, can have profound effects on telomere function, cell division and human disease. Here we demonstrate how levels of Stn1, a component of the conserved telomere capping CST (Cdc13, Stn1, Ten1) complex, are tightly regulated by an upstream overlapping open reading frame (oORF). In budding yeast inactivation of the STN1 oORF leads to a 10-fold increase in Stn1 levels, reduced telomere length, suppression of cdc13-1 and enhancement of yku70Δ growth defects. The STN1 oORF impedes translation of the main ORF and reduces STN1 mRNA via the nonsense mediated mRNA decay (NMD) pathway. Interestingly, the homologs of the translation re-initiation factors, MCT-1Tma20/DENRTma22 also reduce Stn1 levels via the oORF. Human STN1 also contains oORFs, which reduce expression, demonstrating that oORFs are a conserved mechanism for reducing Stn1 levels. Bioinformatic analyses of the yeast and human transcriptomes show that oORFs are more underrepresented than upstream ORFs (uORFs) and associated with lower protein abundance. We propose that oORFs are an important mechanism to control expression of a subset of the proteome.

The conserved PAF1 complex (Cdc73, Paf1, Ctr9, Leo1 and Rtf1, in yeast), binds RNA pol II, and affects levels of many RNAs. Although PAF1 is a complex, there is evidence that different components perform different functions. In yeast, Cdc73, Paf1 and Ctr9 maintain normal telomerase RNA (TLC1) levels and affect telomere length. Here we report a new connection between the PAF1 complex and telomere biology. We show that Paf1 and Ctr9 maintain low telomere repeat containing RNA (TERRA) levels while Cdc73, Leo1 and Rtf1 have lesser effects. Analysis of double mutants shows that Paf1 and Ctr9 can affect TERRA independently of Sir4, Rat1, and Trf4, previously identified regulators of TERRA. The data suggest that Paf1 and Ctr9 maintain low TERRA levels by affecting both transcription and degradation and that short telomeres in cdc73Δ, paf1Δ and ctr9Δ mutants do not induce TERRA. These data establish the PAF1 complex as a new regulator of TERRA, and are consistent with the model in which Paf1 and Ctr9, the core components of the PAF1 complex, affect transcript levels and cell fitness by numerous mechanisms.

Dna2 is a nuclease and helicase that functions redundantly with other proteins in Okazaki fragment processing, double-strand break resection, and checkpoint kinase activation. Dna2 is an essential enzyme, required for yeast and mammalian cell viability. Here, we report that numerous mutations affecting the DNA damage checkpoint suppress dna2∆ lethality in Saccharomyces cerevisiae. dna2∆ cells are also suppressed by deletion of helicases PIF1 and MPH1, and by deletion of POL32, a subunit of DNA polymerase δ. All dna2∆ cells are temperature sensitive, have telomere length defects, and low levels of telomeric 3′ single-stranded DNA (ssDNA). Interestingly, Rfa1, a subunit of the major ssDNA binding protein RPA, and the telomere-specific ssDNA binding protein Cdc13, often colocalize in dna2∆ cells. This suggests that telomeric defects often occur in dna2∆ cells. There are several plausible explanations for why the most critical function of Dna2 is at telomeres. Telomeres modulate the DNA damage response at chromosome ends, inhibiting resection, ligation, and cell-cycle arrest. We suggest that Dna2 nuclease activity contributes to modulating the DNA damage response at telomeres by removing telomeric C-rich ssDNA and thus preventing checkpoint activation.

Nutrient fluctuations in the cellular environment promote changes in cell metabolism and growth to adapt cell proliferation accordingly. The target of rapamycin (TOR) signalling network plays a key role in the coordination of growth and cell proliferation with the nutrient environment and, importantly, nutrient limitation reduces TOR complex 1 (TORC1) signalling. We have performed global quantitative fitness profiling of the collection of Schizosaccharomyces pombe strains from which non-essential genes have been deleted. We identified genes that regulate fitness when cells are grown in a nutrient-rich environment compared with minimal environments, with varying nitrogen sources including ammonium, glutamate and proline. In addition, we have performed the first global screen for genes that regulate fitness when both TORC1 and TORC2 signalling is reduced by Torin1. Analysis of genes whose deletions altered fitness when nutrients were limited, or when TOR signalling was compromised, identified a large number of genes that regulate transmembrane transport, transcription and chromatin organization/regulation and vesicle-mediated transport. The ability to tolerate reduced TOR signalling placed demands upon a large number of biological processes including autophagy, mRNA metabolic processing and nucleocytoplasmic transport. Importantly, novel biological processes and all processes known to be regulated by TOR were identified in our screens. In addition, deletion of 62 genes conserved in humans gave rise to strong sensitivity or resistance to Torin1, and 29 of these 62 genes have novel links to TOR signalling. The identification of chromatin and transcriptional regulation, nutritional uptake and transport pathways in this powerful genetic model now paves the way for a molecular understanding of how cells adapt to the chronic and acute fluctuations in nutrient supply that all eukaryotes experience at some stage, and which is a key feature of cancer cells within solid tumours.

In mammalian cell culture, the Golgi apparatus fragment upon DNA damage. GOLPH3, a Golgi component, is a phosphorylation target of DNA-PK after DNA damage and contributes to Golgi fragmentation. The function of the yeast (Saccharomyces cerevisiae) ortholog of GOLPH3, Vps74, in the DNA damage response has been little studied, although genome-wide screens suggested a role at telomeres. In this study we investigated the role of Vps74 at telomeres and in the DNA damage response. We show that Vps74 decreases the fitness of telomere defective cdc13-1 cells and contributes to the fitness of yku70Δ cells. Importantly, loss of Vps74 in yku70Δ cells exacerbates the temperature dependent growth defects of these cells in a Chk1 and Mec1-dependent manner. Furthermore, Exo1 reduces the fitness of vps74Δ yku70Δ cells suggesting that ssDNA contributes to the fitness defects of vps74Δ yku70Δ cells. Systematic genetic interaction analysis of vps74Δ, yku70Δ and yku70Δ vps74Δ cells suggests that vps74Δ causes a milder but similar defect to that seen in yku70Δ cells. vps74Δ cells have slightly shorter telomeres and loss of VPS74 in yku70Δ or mre11Δ cells further shortens the telomeres of these cells. Interestingly, loss of Vps74 leads to increased levels of Stn1, a partner of Cdc13 in the CST telomere capping complex. Overexpression of Stn1 was previously shown to cause telomere shortening, suppression of cdc13-1 and enhancement of yku70Δ growth defects, suggesting that increased levels of Stn1 may be the route by which Vps74 affects telomere function. These results establish Vps74 as a novel regulator of telomere biology.

Functional telomeres are critically important to eukaryotic genetic stability. Scores of proteins and pathways are known to affect telomere function. Here, we report a series of related genome-wide genetic interaction screens performed on budding yeast cells with acute or chronic telomere defects. Genetic interactions were examined in cells defective in Cdc13 and Stn1, affecting two components of CST, a single stranded DNA (ssDNA) binding complex that binds telomeric DNA. For comparison, genetic interactions were also examined in cells with defects in Rfa3, affecting the major ssDNA binding protein, RPA, which has overlapping functions with CST at telomeres. In more complex experiments, genetic interactions were measured in cells lacking EXO1 or RAD9, affecting different aspects of the DNA damage response, and containing a cdc13-1 induced telomere defect. Comparing fitness profiles across these data sets helps build a picture of the specific responses to different types of dysfunctional telomeres. The experiments show that each context reveals different genetic interactions, consistent with the idea that each genetic defect causes distinct molecular defects. To help others engage with the large volumes of data, the data are made available via two interactive web-based tools: Profilyzer and DIXY. One particularly striking genetic interaction observed was that the chk1∆ mutation improved fitness of cdc13-1 exo1∆ cells more than other checkpoint mutations (ddc1∆, rad9∆, rad17∆, and rad24∆), whereas, in cdc13-1 cells, the effects of all checkpoint mutations were similar. We show that this can be explained by Chk1 stimulating resection-a new function for Chk1 in the eukaryotic DNA damage response network.

Translation Machinery Association 20 (Tma20) and Tma22 are two conserved proteins which form a complex involved in translation initiation. The human homolog of TMA20 is an oncogene, originally identified in a T-cell lymphoma and has been shown to induce cell proliferation. Tma20 and Tma22 homologs in Drosophila have been shown to promote translation of mRNAs which contain uORFs by promoting translation re-initiation. uORFs serve as major regulators of gene expression, repressing translation. In yeast Tma20 and Tma22 have recently been shown to have a role in telomere biology by decreasing fitness of cells with uncapped telomeres. To understand how Tma20 and Tma22 affect growth of cells with uncapped telomeres we have investigated translation re-initiation in yeast using new luciferase reporter constructs. Using transcript leader sequences from 22 genes we showed that, as expected, the majority of uORFs repressed expression but found no evidence to suggest that Tma20 or Tma22 promote translation re-initiation in 21 of these genes. However our data suggests that Tma20 and Tma22 inhibit translation re-initiation of the remaining gene, BRE4. Our data suggests that the function of Tma20 and Tma22 is not conserved from Drosophila to yeast. Our finding that Tma20 and Tma22 inhibit translation re-initiation on BRE4 is in agreement with studies in mammalian cells which have shown that Tma20 and Tma22 promote the dissociation of 40S ribosomal subunit from mRNA following translation termination. Our data suggests that perhaps the human homologs of Tma20 and Tma22 may not promote translation re-initiation as has previously been assumed. Citation Format: Victoria Torrance, David Lydall. Evidence that Tma20 and Tma22 do not promote translation re-initiation in yeast. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B14.

The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here, we report a genome-wide genetic interaction screen in Saccharomyces cerevisiae using the bir1-17 mutant, identifying through quantitative fitness analysis deletion mutations that act as enhancers and suppressors. Gene knockouts affecting the Ctf19 kinetochore complex were identified as the strongest enhancers of bir1-17, while mutations affecting the large ribosomal subunit or the mRNA nonsense-mediated decay pathway caused strong phenotypic suppression. Thus, cells lacking a functional Ctf19 complex become highly dependent on Bir1 function and vice versa. The negative genetic interaction profiles of bir1-17 and the cohesin mutant mcd1-1 showed considerable overlap, underlining the strong functional connection between sister chromatid cohesion and chromosome biorientation. Loss of some Ctf19 components, such as Iml3 or Chl4, impacted differentially on bir1-17 compared with mutations affecting other CPC components: despite the synthetic lethality shown by either iml3∆ or chl4∆ in combination with bir1-17, neither gene knockout showed any genetic interaction with either ipl1-321 or sli15-3. Our data therefore imply a specific functional connection between the Ctf19 complex and Bir1 that is not shared with Ipl1.

We provide a detailed protocol for robot-assisted, genome-wide measurement of fitness in the model yeast Saccharomyces cerevisiae using Quantitative Fitness Analysis (QFA). We first describe how we construct thousands of double or triple mutant yeast strains in parallel using Synthetic Genetic Array (SGA) procedures. Strains are inoculated onto solid agar surfaces by liquid spotting followed by repeated photography of agar plates. Growth curves are constructed and the fitness of each strain is estimated. Robot-assisted QFA, can be used to identify genetic interactions and chemical sensitivity/resistance in genome-wide experiments, but QFA can also be used in smaller scale, manual workflows.

During interactions with its mammalian host, the pathogenic yeast Candida albicans is exposed to a range of stresses such as superoxide radicals and cationic fluxes. Unexpectedly, a nonbiased screen of transcription factor deletion mutants revealed that the phosphate-responsive transcription factor Pho4 is vital for the resistance of C. albicans to these diverse stresses. RNA-Seq analysis indicated that Pho4 does not induce stress-protective genes directly. Instead, we show that loss of Pho4 affects metal cation toxicity, accumulation, and bioavailability. We demonstrate that pho4Δ cells are sensitive to metal and nonmetal cations and that Pho4-mediated polyphosphate synthesis mediates manganese resistance. Significantly, we show that Pho4 is important for mediating copper bioavailability to support the activity of the copper/zinc superoxide dismutase Sod1 and that loss of Sod1 activity contributes to the superoxide sensitivity of pho4Δ cells. Consistent with the key role of fungal stress responses in countering host phagocytic defenses, we also report that C. albicans pho4Δ cells are acutely sensitive to macrophage-mediated killing and display attenuated virulence in animal infection models. The novel connections between phosphate metabolism, metal homeostasis, and superoxide stress resistance presented in this study highlight the importance of metabolic adaptation in promoting C. albicans survival in the host.

All organisms live in changeable, stressful environments. It has been reported that exposure to low-dose stresses or poisons can improve fitness. However, examining the effects of chronic low-dose chemical exposure is challenging. To address this issue we used temperature sensitive mutations affecting the yeast cell division cycle to induce low-dose stress for 40 generation times, or more. We examined cdc13-1 mutants, defective in telomere function, and cdc15-2 mutants, defective in mitotic kinase activity. We found that each stress induced similar adaptive responses. Stress-exposed cells became resistant to higher levels of stress but less fit, in comparison with unstressed cells, in conditions of low stress. The costs and benefits of adaptation to chronic stress were reversible. In the cdc13-1 context we tested the effects of Rad9, a central player in the response to telomere defects, Exo1, a nuclease that degrades defective telomeres, and Msn2 and Msn4, 2 transcription factors that contribute to the environmental stress response. We also observed, as expected, that Rad9 and Exo1 modulated the response of cells to stress. In addition we observed that adaptation to stress could still occur in these contexts, with associated costs and benefits. We conclude that functionally redundant cellular networks control the adaptive responses to low dose chronic stress. Our data suggests that if organisms adapt to low dose stress it is helpful if stress continues or increases but harmful should stress levels reduce.

Telomere attrition is linked to cancer, diabetes, cardiovascular disease and aging. This is because telomere losses trigger further genomic modifications, culminating with loss of cell function and malignant transformation. However, factors regulating the transition from cells with short telomeres, to cells with profoundly altered genomes, are little understood. Here, we use budding yeast engineered to lack telomerase and other forms of telomere maintenance, to screen for such factors. We show that initially, different DNA damage checkpoint proteins act together with Exo1 and Mre11 nucleases, to inhibit proliferation of cells undergoing telomere attrition. However, this situation changes when survivors lacking telomeres emerge. Intriguingly, checkpoint pathways become tolerant to loss of telomeres in survivors, yet still alert to new DNA damage. We show that Rif1 is responsible for the checkpoint tolerance and proliferation of these survivors, and that is also important for proliferation of cells with a broken chromosome. In contrast, Exo1 drives extensive genomic modifications in survivors. Thus, the conserved proteins Rif1 and Exo1 are critical for survival and evolution of cells with lost telomeres.

Three major DNA polymerases replicate the linear eukaryotic chromosomes. DNA polymerase α-primase (Pol α) and DNA polymerase δ (Pol δ) replicate the lagging-strand and Pol α and DNA polymerase ε (Pol ε) the leading-strand. To identify factors affecting coordination of DNA replication, we have performed genome-wide quantitative fitness analyses of budding yeast cells containing defective polymerases. We combined temperature-sensitive mutations affecting the three replicative polymerases, Pol α, Pol δ, and Pol ε with genome-wide collections of null and reduced function mutations. We identify large numbers of genetic interactions that inform about the roles that specific genes play to help Pol α, Pol δ, and Pol ε function. Surprisingly, the overlap between the genetic networks affecting the three DNA polymerases does not represent the majority of the genetic interactions identified. Instead our data support a model for division of labor between the different DNA polymerases during DNA replication. For example, our genetic interaction data are consistent with biochemical data showing that Pol ε is more important to the Pre-Loading complex than either Pol α or Pol δ. We also observed distinct patterns of genetic interactions between leading- and lagging-strand DNA polymerases, with particular genes being important for coupling proliferating cell nuclear antigen loading/unloading (Ctf18, Elg1) with nucleosome assembly (chromatin assembly factor 1, histone regulatory HIR complex). Overall our data reveal specialized genetic networks that affect different aspects of leading- and lagging-strand DNA replication. To help others to engage with these data we have generated two novel, interactive visualization tools, DIXY and Profilyzer.

DNA-end resection, the generation of single-stranded DNA at DNA double strand break (DSB) ends, is critical for controlling the many cellular responses to breaks. Here we show that the conserved DNA damage checkpoint sliding clamp (the 9-1-1 complex) plays two opposing roles coordinating DSB resection in budding yeast. We show that the major effect of 9-1-1 is to inhibit resection by promoting the recruitment of Rad953BP1 near DSBs. However, 9-1-1 also stimulates resection by Exo1- and Dna2-Sgs1-dependent nuclease/helicase activities, and this can be observed in the absence of Rad953BP1. Our new data resolve the controversy in the literature about the effect of the 9-1-1 complex on DSB resection. Interestingly, the inhibitory role of 9-1-1 on resection is not observed near uncapped telomeres because less Rad953BP1 is recruited near uncapped telomeres. Thus, 9-1-1 both stimulates and inhibits resection and the effects of 9-1-1 are modulated by different regions of the genome. Our experiments illustrate the central role of the 9-1-1 checkpoint sliding clamp in the DNA damage response network that coordinates the response to broken DNA ends. Our results have implications in all eukaryotic cells.

Quantitative fitness analysis (QFA) is a high throughput experimental and computational methodology for measuring the growth of microbial populations. QFA screens can be used to compare the health of cell populations with and without a mutation in a query gene to infer genetic interaction strengths genomewide, examining thousands of separate genotypes. We introduce Bayesian hierarchical models of population growth rates and genetic interactions that better reflect QFA experimental design than current approaches. Our new approach models population dynamics and genetic interaction simultaneously, thereby avoiding passing information between models via a univariate fitness summary. Matching experimental structure more closely, Bayesian hierarchical approaches use data more efficiently and find new evidence for genes which interact with yeast telomeres within a published data set.

The telomere is present at the ends of all eukaryotic chromosomes and usually consists of repetitive TG-rich DNA that terminates in a single-stranded 3' TG extension and a 5' CA-rich recessed strand. A biochemical assay that allows the in vitro observation of exonuclease-catalyzed degradation (resection) of telomeres has been developed. The approach uses an oligodeoxynucleotide that folds to a stem-loop with a TG-rich double-stranded region and a 3' single-stranded extension, typical of telomeres. Cdc13, the major component of the telomere-specific CST complex, strongly protects the recessed strand from the 5'→3' exonuclease activity of the model exonuclease from bacteriophage λ. The isolated DNA binding domain of Cdc13 is less effective at shielding telomeres. Protection is specific, not being observed in control DNA lacking the specific TG-rich telomere sequence. RPA, the eukaryotic single-stranded DNA binding protein, also inhibits telomere resection. However, this protein is non-specific, equally hindering the degradation of non-telomere controls.

Synthetic genetic array (SGA) has been successfully used to identify genetic interactions in S. cerevisiae and S. pombe. In S. pombe, SGA methods use either cycloheximide (C) or heat shock (HS) to select double mutants before measuring colony size as a surrogate for fitness. Quantitative Fitness Analysis (QFA) is a different method for determining fitness of microbial strains. In QFA, liquid cultures are spotted onto solid agar and growth curves determined for each spot by photography and model fitting. Here, we compared the two S. pombe SGA methods and found that the HS method was more reproducible for us. We also developed a QFA procedure for S. pombe. We used QFA to identify genetic interactions affecting two temperature sensitive, telomere associated query mutations (taz1Δ and pot1-1). We identify exo1∆ and other gene deletions as suppressors or enhancers of S. pombe telomere defects. Our study identifies known and novel gene deletions affecting the fitness of strains with telomere defects. The interactions we identify may be relevant in human cells.