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

Peer reviewedPublisher PD

Shelterin-Like Proteins and Yku Inhibit Nucleolytic Processing of Saccharomyces cerevisiae Telomeres

Eukaryotic cells distinguish their chromosome ends from accidental DNA double-strand breaks (DSBs) by packaging them into protective structures called telomeres that prevent DNA repair/recombination activities. Here we investigate the role of key telomeric proteins in protecting budding yeast telomeres from degradation. We show that the Saccharomyces cerevisiae shelterin-like proteins Rif1, Rif2, and Rap1 inhibit nucleolytic processing at both de novo and native telomeres during G1 and G2 cell cycle phases, with Rif2 and Rap1 showing the strongest effects. Also Yku prevents telomere resection in G1, independently of its role in non-homologous end joining. Yku and the shelterin-like proteins have additive effects in inhibiting DNA degradation at G1 de novo telomeres, where Yku plays the major role in preventing initiation, whereas Rif1, Rif2, and Rap1 act primarily by limiting extensive resection. In fact, exonucleolytic degradation of a de novo telomere is more efficient in yku70Δ than in rif2Δ G1 cells, but generation of ssDNA in Yku-lacking cells is limited to DNA regions close to the telomere tip. This limited processing is due to the inhibitory action of Rap1, Rif1, and Rif2, as their inactivation allows extensive telomere resection not only in wild-type but also in yku70Δ G1 cells. Finally, Rap1 and Rif2 prevent telomere degradation by inhibiting MRX access to telomeres, which are also protected from the Exo1 nuclease by Yku. Thus, chromosome end degradation is controlled by telomeric proteins that specifically inhibit the action of different nucleases

Localization of telomeres and telomere-associated proteins in telomerase-negative Saccharomyces cerevisiae

Cells lacking telomerase cannot maintain their telomeres and undergo a telomere erosion phase leading to senescence and crisis in which most cells become nonviable. On rare occasions survivors emerge from these cultures that maintain their telomeres in alternative ways. The movement of five marked telomeres in Saccharomyces cerevisiae was followed in wild-type cells and through erosion, senescence/crisis and eventual survival in telomerase-negative (est2::HYG) yeast cells. It was found that during erosion, movements of telomeres in est2::HYG cells were indistinguishable from wild-type telomere movements. At senescence/crisis, however, most cells were in G2 arrest and the nucleus and telomeres traversed back and forth across the bud neck, presumably until cell death. Type I survivors, using subtelomeric Y′ amplification for telomere maintenance, continued to show this aberrant telomere movement. However, Type II survivors, maintaining telomeres by a sudden elongation of the telomere repeats, became indistinguishable from wild-type cells, consistent with growth properties of the two types of survivors. When telomere-associated proteins Sir2p, Sir3p and Rap1p were tagged, the same general trend was seen—Type I survivors retained the senescence/crisis state of protein localization, while Type II survivors were restored to wild type

Conservation genetics of the annual hemiparasitic plant Melampyrum sylvaticum (Orobanchaceae) in the UK and Scandinavia

Melampyrum sylvaticum is an endangered annual hemiparasitic plant that is found in only 19 small and isolated populations in the United Kingdom (UK). To evaluate the genetic consequences of this patchy distribution we compared levels of diversity, inbreeding and differentiation from ten populations from the UK with eight relatively large populations from Sweden and Norway where the species is more continuously distributed. We demonstrate that in both the UK and Scandinavia, the species is highly inbreeding (global F IS = 0.899). Levels of population differentiation were high (F’ST = 0.892) and significantly higher amongst UK populations (F’ST = 0.949) than Scandinavian populations (F’ST = 0.762; P < 0.01). The isolated populations in the UK have, on average, lower genetic diversity (allelic richness, proportion of loci that are polymorphic, gene diversity) than Scandinavian populations, and this diversity difference is associated with the smaller census size and population area of UK populations. From a conservation perspective, the naturally inbreeding nature of the species may buffer the species against immediate effects of inbreeding depression, but the markedly lower levels of genetic diversity in UK populations may represent a genetic constraint to evolutionary change. In addition, the high levels of population differentiation suggest that gene flow among populations will not be effective at replenishing lost variation. We thus recommend supporting in situ conservation management with ex situ populations and human-mediated seed dispersal among selected populations in the UK

Rif1 Supports the Function of the CST Complex in Yeast Telomere Capping

Telomere integrity in budding yeast depends on the CST (Cdc13-Stn1-Ten1) and shelterin-like (Rap1-Rif1-Rif2) complexes, which are thought to act independently from each other. Here we show that a specific functional interaction indeed exists among components of the two complexes. In particular, unlike RIF2 deletion, the lack of Rif1 is lethal for stn1ΔC cells and causes a dramatic reduction in viability of cdc13-1 and cdc13-5 mutants. This synthetic interaction between Rif1 and the CST complex occurs independently of rif1Δ-induced alterations in telomere length. Both cdc13-1 rif1Δ and cdc13-5 rif1Δ cells display very high amounts of telomeric single-stranded DNA and DNA damage checkpoint activation, indicating that severe defects in telomere integrity cause their loss of viability. In agreement with this hypothesis, both DNA damage checkpoint activation and lethality in cdc13 rif1Δ cells are partially counteracted by the lack of the Exo1 nuclease, which is involved in telomeric single-stranded DNA generation. The functional interaction between Rif1 and the CST complex is specific, because RIF1 deletion does not enhance checkpoint activation in case of CST-independent telomere capping deficiencies, such as those caused by the absence of Yku or telomerase. Thus, these data highlight a novel role for Rif1 in assisting the essential telomere protection function of the CST complex

Genome-wide patterns of promoter sharing and co-expression in bovine skeletal muscle

Background: Gene regulation by transcription factors (TF) is species, tissue and time specific. To better understand how the genetic code controls gene expression in bovine muscle we associated gene expression data from developing Longissimus thoracis et lumborum skeletal muscle with bovine promoter sequence information.Results: We created a highly conserved genome-wide promoter landscape comprising 87,408 interactions relating 333 TFs with their 9,242 predicted target genes (TGs). We discovered that the complete set of predicted TGs share an average of 2.75 predicted TF binding sites (TFBSs) and that the average co-expression between a TF and its predicted TGs is higher than the average co-expression between the same TF and all genes. Conversely, pairs of TFs sharing predicted TGs showed a co-expression correlation higher that pairs of TFs not sharing TGs. Finally, we exploited the co-occurrence of predicted TFBS in the context of muscle-derived functionally-coherent modules including cell cycle, mitochondria, immune system, fat metabolism, muscle/glycolysis, and ribosome. Our findings enabled us to reverse engineer a regulatory network of core processes, and correctly identified the involvement of E2F1, GATA2 and NFKB1 in the regulation of cell cycle, fat, and muscle/glycolysis, respectively.Conclusion: The pivotal implication of our research is two-fold: (1) there exists a robust genome-wide expression signal between TFs and their predicted TGs in cattle muscle consistent with the extent of promoter sharing; and (2) this signal can be exploited to recover the cellular mechanisms underpinning transcription regulation of muscle structure and development in bovine. Our study represents the first genome-wide report linking tissue specific co-expression to co-regulation in a non-model vertebrate

Do herbivorous minnows have “plug-flow reactor” guts? Evidence from digestive enzyme activities, gastrointestinal fermentation, and luminal nutrient concentrations

Few investigations have empirically analyzed fish gut function in the context of chemical reactor models. In this study, digestive enzyme activities, levels of gastrointestinal fermentation products [short chain fatty acids (SCFA)], luminal nutrient concentrations, and the mass of gut contents were measured along the digestive tract in herbivorous and carnivorous minnows to ascertain whether their guts function as “plug-flow reactors” (PFRs). Four of the species, Campostoma anomalum, C. ornatum, C. oligolepis, and C. pauciradii, are members of a monophyletic herbivorous clade, whereas the fifth species, Nocomis micropogon, is a carnivore from an adjacent carnivorous clade. In the context of a PFR model, the activities of amylase, trypsin and lipase, and the concentrations of glucose, protein, and lipid were predicted to decrease moving from the proximal to the distal intestine. I found support for this as these enzyme activities and nutrient concentrations generally decreased moving distally along the intestine of the four Campostoma species. Furthermore, gut content mass and the low SCFA concentrations did not change (increase or decrease) along the gut of any species. Combined with a previous investigation suggesting that species of Campostoma have rapid gut throughput rates, the data presented here generally support Campostoma as having guts that function as PFRs. The carnivorous N. micropogon showed some differences in the measured parameters, which were interpreted in the contexts of intake and retention time to suggest that PFR function breaks down in this carnivorous species

The Sno Oncogene Antagonizes Wingless Signaling during Wing Development in Drosophila

The Sno oncogene (Snoo or dSno in Drosophila) is a highly conserved protein and a well-established antagonist of Transforming Growth Factor-β signaling in overexpression assays. However, analyses of Sno mutants in flies and mice have proven enigmatic in revealing developmental roles for Sno proteins. Thus, to identify developmental roles for dSno we first reconciled conflicting data on the lethality of dSno mutations. Then we conducted analyses of wing development in dSno loss of function genotypes. These studies revealed ectopic margin bristles and ectopic campaniform sensilla in the anterior compartment of the wing blade suggesting that dSno functions to antagonize Wingless (Wg) signaling. A subsequent series of gain of function analyses yielded the opposite phenotype (loss of bristles and sensilla) and further suggested that dSno antagonizes Wg signal transduction in target cells. To date Sno family proteins have not been reported to influence the Wg pathway during development in any species. Overall our data suggest that dSno functions as a tissue-specific component of the Wg signaling pathway with modest antagonistic activity under normal conditions but capable of blocking significant levels of extraneous Wg, a role that may be conserved in vertebrates

Cancer Risk in Diabetic Patients Treated with Metformin: A Systematic Review and Meta-analysis

BACKGROUND: A growing body of evidence has suggested that metformin potentially reduces the risk of cancer. Our objective was to enhance the precision of estimates of the effect of metformin on the risk of any-site and site-specific cancers in patients with diabetes. METHODS/PRINCIPAL FINDINGS: We performed a search of MEDLINE, EMBASE, ISI Web of Science, Cochrane Library, and ClinicalTrials.gov for pertinent articles published as of October 12, 2011, and included them in a systematic review and meta-analysis. We calculated pooled risk ratios (RRs) for overall cancer mortality and cancer incidence. Of the 21,195 diabetic patients reported in 6 studies (4 cohort studies, 2 RCTs), 991 (4.5%) cases of death from cancer were reported. A total of 11,117 (5.3%) cases of incident cancer at any site were reported among 210,892 patients in 10 studies (2 RCTs, 6 cohort studies, 2 case-control studies). The risks of cancer among metformin users were significantly lower than those among non-metformin users: the pooled RRs (95% confidence interval) were 0.66 (0.49-0.88) for cancer mortality, 0.67 (0.53-0.85) for all-cancer incidence, 0.68 (0.53-0.88) for colorectal cancer (n = 6), 0.20 (0.07-0.59) for hepatocellular cancer (n = 4), 0.67 (0.45-0.99) for lung cancer (n = 3). CONCLUSION/SIGNIFICANCE: The use of metformin in diabetic patients was associated with significantly lower risks of cancer mortality and incidence. However, this analysis is mainly based on observational studies and our findings underscore the more need for long-term RCTs to confirm this potential benefit for individuals with diabetes

Taming the tiger by the tail: modulation of DNA damage responses by telomeres

Telomeres are by definition stable and inert chromosome ends, whereas internal chromosome breaks are potent stimulators of the DNA damage response (DDR). Telomeres do not, as might be expected, exclude DDR proteins from chromosome ends but instead engage with many DDR proteins. However, the most powerful DDRs, those that might induce chromosome fusion or cell-cycle arrest, are inhibited at telomeres. In budding yeast, many DDR proteins that accumulate most rapidly at double strand breaks (DSBs), have important functions in physiological telomere maintenance, whereas DDR proteins that arrive later tend to have less important functions. Considerable diversity in telomere structure has evolved in different organisms and, perhaps reflecting this diversity, different DDR proteins seem to have distinct roles in telomere physiology in different organisms. Drawing principally on studies in simple model organisms such as budding yeast, in which many fundamental aspects of the DDR and telomere biology have been established; current views on how telomeres harness aspects of DDR pathways to maintain telomere stability and permit cell-cycle division are discussed