The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation

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Off-Target Effects of Psychoactive Drugs Revealed by Genome-Wide Assays in Yeast

To better understand off-target effects of widely prescribed psychoactive drugs, we performed a comprehensive series of chemogenomic screens using the budding yeast Saccharomyces cerevisiae as a model system. Because the known human targets of these drugs do not exist in yeast, we could employ the yeast gene deletion collections and parallel fitness profiling to explore potential off-target effects in a genome-wide manner. Among 214 tested, documented psychoactive drugs, we identified 81 compounds that inhibited wild-type yeast growth and were thus selected for genome-wide fitness profiling. Many of these drugs had a propensity to affect multiple cellular functions. The sensitivity profiles of half of the analyzed drugs were enriched for core cellular processes such as secretion, protein folding, RNA processing, and chromatin structure. Interestingly, fluoxetine (Prozac) interfered with establishment of cell polarity, cyproheptadine (Periactin) targeted essential genes with chromatin-remodeling roles, while paroxetine (Paxil) interfered with essential RNA metabolism genes, suggesting potential secondary drug targets. We also found that the more recently developed atypical antipsychotic clozapine (Clozaril) had no fewer off-target effects in yeast than the typical antipsychotics haloperidol (Haldol) and pimozide (Orap). Our results suggest that model organism pharmacogenetic studies provide a rational foundation for understanding the off-target effects of clinically important psychoactive agents and suggest a rational means both for devising compound derivatives with fewer side effects and for tailoring drug treatment to individual patient genotypes

Quantitative Fitness Analysis Shows That NMD Proteins and Many Other Protein Complexes Suppress or Enhance Distinct Telomere Cap Defects

To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70Δ). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time. The data are fitted to a logistic population growth model; and growth parameters, such as maximum growth rate and maximum doubling potential, are deduced. QFA reveals that as many as 5% of systematic gene deletions, affecting numerous functional classes, strongly interact with telomere capping defects. We show that, while Cdc13 and Yku70 perform complementary roles in telomere capping, their genetic interaction profiles differ significantly. At least 19 different classes of functionally or physically related proteins can be identified as interacting with cdc13-1, yku70Δ, or both. Each specific genetic interaction informs the roles of individual gene products in telomere biology. One striking example is with genes of the nonsense-mediated RNA decay (NMD) pathway which, when disabled, suppress the conditional cdc13-1 mutation but enhance the null yku70Δ mutation. We show that the suppressing/enhancing role of the NMD pathway at uncapped telomeres is mediated through the levels of Stn1, an essential telomere capping protein, which interacts with Cdc13 and recruitment of telomerase to telomeres. We show that increased Stn1 levels affect growth of cells with telomere capping defects due to cdc13-1 and yku70Δ. QFA is a sensitive, high-throughput method that will also be useful to understand other aspects of microbial cell biology

Genome-Wide Association Study of Relative Telomere Length

Telomere function is essential to maintaining the physical integrity of linear chromosomes and healthy human aging. The probability of forming proper telomere structures depends on the length of the telomeric DNA tract. We attempted to identify common genetic variants associated with log relative telomere length using genome-wide genotyping data on 3,554 individuals from the Nurses' Health Study and the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial that took part in the National Cancer Institute Cancer Genetic Markers of Susceptibility initiative for breast and prostate cancer. After genotyping 64 independent SNPs selected for replication in additional Nurses' Health Study and Women's Genome Health Study participants, we did not identify genome-wide significant loci; however, we replicated the inverse association of log relative telomere length with the minor allele variant [C] of rs16847897 at the TERC locus (per allele β = −0.03, P = 0.003) identified by a previous genome-wide association study. We did not find evidence for an association with variants at the OBFC1 locus or other loci reported to be associated with telomere length. With this sample size we had >80% power to detect β estimates as small as ±0.10 for SNPs with minor allele frequencies of ≥0.15 at genome-wide significance. However, power is greatly reduced for β estimates smaller than ±0.10, such as those for variants at the TERC locus. In general, common genetic variants associated with telomere length homeostasis have been difficult to detect. Potential biological and technical issues are discussed

Vowel reduction in word-final position by early and late Spanish-English bilinguals

Vowel reduction is a prominent feature of American English, as well as other stress-timed languages. As a phonological process, vowel reduction neutralizes multiple vowel quality contrasts in unstressed syllables. For bilinguals whose native language is not characterized by large spectral and durational differences between tonic and atonic vowels, systematically reducing unstressed vowels to the central vowel space can be problematic. Failure to maintain this pattern of stressed-unstressed syllables in American English is one key element that contributes to a ?foreign accent? in second language speakers. Reduced vowels, or ?schwas,? have also been identified as particularly vulnerable to the co-articulatory effects of adjacent consonants. The current study examined the effects of adjacent sounds on the spectral and temporal qualities of schwa in word-final position. Three groups of English-speaking adults were tested: Miami-based monolingual English speakers, early Spanish-English bilinguals, and late Spanish-English bilinguals. Subjects performed a reading task to examine their schwa productions in fluent speech when schwas were preceded by consonants from various points of articulation. Results indicated that monolingual English and late Spanish-English bilingual groups produced targeted vowel qualities for schwa, whereas early Spanish-English bilinguals lacked homogeneity in their vowel productions. This extends prior claims that schwa is targetless for F2 position for native speakers to highly-proficient bilingual speakers. Though spectral qualities lacked homogeneity for early Spanish-English bilinguals, early bilinguals produced schwas with near native-like vowel duration. In contrast, late bilinguals produced schwas with significantly longer durations than English monolinguals or early Spanish-English bilinguals. Our results suggest that the temporal properties of a language are better integrated into second language phonologies than spectral qualities. Finally, we examined the role of nonstructural variables (e.g. linguistic history measures) in predicting native-like vowel duration. These factors included: Age of L2 learning, amount of L1 use, and self-reported bilingual dominance. Our results suggested that different sociolinguistic factors predicted native-like reduced vowel duration than predicted native-like vowel qualities across multiple phonetic environments

Expression variability of co-regulated genes differentiates Saccharomyces cerevisiae strains

Background: Saccharomyces cerevisiae (Baker’s yeast) is found in diverse ecological niches and is characterized by high adaptive potential under challenging environments. In spite of recent advances on the study of yeast genome diversity, little is known about the underlying gene expression plasticity. In order to shed new light onto this biological question, we have compared transcriptome profiles of five environmental isolates, clinical and laboratorial strains at different time points of fermentation in synthetic must medium, during exponential and stationary growth phases. Results: Our data unveiled diversity in both intensity and timing of gene expression. Genes involved in glucose metabolism and in the stress response elicited during fermentation were among the most variable. This gene expression diversity increased at the onset of stationary phase (diauxic shift). Environmental isolates showed lower average transcript abundance of genes involved in the stress response, assimilation of nitrogen and vitamins, and sulphur metabolism, than other strains. Nitrogen metabolism genes showed significant variation in expression among the environmental isolates. Conclusions: Wild type yeast strains respond differentially to the stress imposed by nutrient depletion, ethanol accumulation and cell density increase, during fermentation of glucose in synthetic must medium. Our results support previous data showing that gene expression variability is a source of phenotypic diversity among closely related organisms.Fundação para a Ciência e TecnologiaThe authors wish to thank Adega Cooperativa da Bairrada, Cantanhede, Portugal, for providing the commercial strains

Natural Single-Nucleosome Epi-Polymorphisms in Yeast

Epigenomes commonly refer to the sequence of presence/absence of specific epigenetic marks along eukaryotic chromatin. Complete histone-borne epigenomes have now been described at single-nucleosome resolution from various organisms, tissues, developmental stages, or diseases, yet their intra-species natural variation has never been investigated. We describe here that the epigenomic sequence of histone H3 acetylation at Lysine 14 (H3K14ac) differs greatly between two unrelated strains of the yeast Saccharomyces cerevisiae. Using single-nucleosome chromatin immunoprecipitation and mapping, we interrogated 58,694 nucleosomes and found that 5,442 of them differed in their level of H3K14 acetylation, at a false discovery rate (FDR) of 0.0001. These Single Nucleosome Epi-Polymorphisms (SNEPs) were enriched at regulatory sites and conserved non-coding DNA sequences. Surprisingly, higher acetylation in one strain did not imply higher expression of the relevant gene. However, SNEPs were enriched in genes of high transcriptional variability and one SNEP was associated with the strength of gene activation upon stimulation. Our observations suggest a high level of inter-individual epigenomic variation in natural populations, with essential questions on the origin of this diversity and its relevance to gene x environment interactions

Natural Polymorphism in BUL2 Links Cellular Amino Acid Availability with Chronological Aging and Telomere Maintenance in Yeast

Aging and longevity are considered to be highly complex genetic traits. In order to gain insight into aging as a polygenic trait, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard strain RM11 and a laboratory strain S288c, to identify quantitative trait loci that control chronological lifespan. Among the major loci that regulate chronological lifespan in this cross, one genetic linkage was found to be congruent with a previously mapped locus that controls telomere length variation. We found that a single nucleotide polymorphism in BUL2, encoding a component of an ubiquitin ligase complex involved in trafficking of amino acid permeases, controls chronological lifespan and telomere length as well as amino acid uptake. Cellular amino acid availability changes conferred by the BUL2 polymorphism alter telomere length by modulating activity of a transcription factor Gln3. Among the GLN3 transcriptional targets relevant to this phenotype, we identified Wtm1, whose upregulation promotes nuclear retention of ribonucleotide reductase (RNR) components and inhibits the assembly of the RNR enzyme complex during S-phase. Inhibition of RNR is one of the mechanisms by which Gln3 modulates telomere length. Identification of a polymorphism in BUL2 in this outbred yeast population revealed a link among cellular amino acid availability, chronological lifespan, and telomere length control

Depleting Components of the THO Complex Causes Increased Telomere Length by Reducing the Expression of the Telomere-Associated Protein Rif1p

Telomere length is regulated mostly by proteins directly associated with telomeres. However, genome-wide analysis of Saccharomyces cerevisiae mutants has revealed that deletion of Hpr1p, a component of the THO complex, also affects telomere length. The THO complex comprises four protein subunits, namely, Tho2p, Hpr1p, Mft1p, and Thp2p. These subunits interplay between transcription elongation and co-transcriptional assembly of export-competent mRNPs. Here we found that the deletion of tho2 or hpr1 caused telomere lengthening by ∼50–100 bps, whereas that of mft1 or thp2 did not affect telomere length. Since the THO complex functions in transcription elongation, we analyzed the expression of telomere-associated proteins in mutants depleted of complex components. We found that both the mRNA and protein levels of RIF1 were decreased in tho2 and hpr1 cells. RIF1 encodes a 1917-amino acid polypeptide that is involved in regulating telomere length and the formation of telomeric heterochromatin. Hpr1p and Tho2p appeared to affect telomeres through Rif1p, as increased Rif1p levels suppressed the telomere lengthening in tho2 and hpr1 cells. Moreover, yeast cells carrying rif1 tho2 or rif1 hpr1 double mutations showed telomere lengths and telomere silencing effects similar to those observed in the rif1 mutant. Thus, we conclude that mutations of components of the THO complex affect telomere functions by reducing the expression of a telomere-associated protein, Rif1p