TERRA Promotes Telomere Shortening through Exonuclease 1–Mediated Resection of Chromosome Ends

The long noncoding telomeric repeat containing RNA (TERRA) is expressed at chromosome ends. TERRA upregulation upon experimental manipulation or in ICF (immunodeficiency, centromeric instability, facial anomalies) patients correlates with short telomeres. To study the mechanism of telomere length control by TERRA in Saccharomyces cerevisiae, we mapped the transcriptional start site of TERRA at telomere 1L and inserted a doxycycline regulatable promoter upstream. Induction of TERRA transcription led to telomere shortening of 1L but not of other chromosome ends. TERRA interacts with the Exo1-inhibiting Ku70/80 complex, and deletion of EXO1 but not MRE11 fully suppressed the TERRA–mediated short telomere phenotype in presence and absence of telomerase. Thus TERRA transcription facilitates the 5′-3′ nuclease activity of Exo1 at chromosome ends, providing a means to regulate the telomere shortening rate. Thereby, telomere transcription can regulate cellular lifespan through modulation of chromosome end processing activities

DNA damage signalling prevents deleterious telomere addition at DNA breaks

The response to DNA damage involves regulation of multiple essential processes to maximize the accuracy of DNA damage repair and cell survival 1. Telomerase has the potential to interfere with repair by inappropriately adding telomeres to DNA breaks. It was unknown whether cells modulate telomerase in response to DNA damage, to increase the accuracy of repair. Here we report that telomerase action is regulated as a part of the cellular response to a DNA double-strand break (DSB). Using yeast, we show that the major ATR/Mec1 DNA damage signalling pathway regulates telomerase action at DSBs. Upon DNA damage, MEC1-RAD53-DUN1-dependent phosphorylation of the telomerase inhibitor Pif1 occurs. Utilizing a separation of function PIF1 mutation, we show that this phosphorylation is required for the Pif1-mediated telomerase inhibition that takes place specifically at DNA breaks, but not telomeres. Hence DNA damage signalling down-modulates telomerase action at a DNA break via Pif1 phosphorylation, thus preventing aberrant healing of broken DNA ends by telomerase. These findings uncover a novel regulatory mechanism that coordinates competing DNA end-processing activities and thereby promotes DNA repair accuracy and genome integrity

Investigating the role of the Est3 protein in yeast telomere replication

The Est3 subunit of yeast telomerase, which adopts a predicted OB-fold, is essential for telomere replication. To assess the possible contributions that Est3 might make to enzyme catalysis, we compared telomerase activity from wild type and est3-Δ strains of Saccharomyces castellii, which revealed that loss of the Est3 subunit results in a 2- to 3-fold decline in nucleotide addition. This effect was not primer-specific, based on assessment of a panel of primers that spanned the template of the S. castellii telomerase RNA. Furthermore, using nuclear magnetic resonance chemical shift perturbation, no chemical shift change was observed at any site in the protein upon addition of single-stranded DNA, arguing against a role for Est3 in recognition of telomeric substrates by telomerase. Addition of exogenous Est3 protein, including mutant Est3 proteins that are severely impaired for telomere replication in vivo, fully restored activity in est3-Δ telomerase reactions. Thus, Est3 performs an in vivo regulatory function in telomere replication, which is distinct from any potential contribution that Est3 might make to telomerase activity

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

Novel Phosphorylation Sites in the S. cerevisiae Cdc13 Protein Reveal New Targets for Telomere Length Regulation

The S. cerevisiae Cdc13 is a multifunctional protein with key roles in regulation of telomerase, telomere end protection, and conventional telomere replication, all of which are cell cycle-regulated processes. Given that phosphorylation is a key mechanism for regulating protein function, we identified sites of phosphorylation using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). We also determined phosphorylation abundance on both wild type (WT) and a telomerase deficient form of Cdc13, encoded by the cdc13-2 allele, in both G1 phase cells, when telomerase is not active, and G2/M phase cells, when it is. We identified 21 sites of in vivo phosphorylation, of which only five had been reported previously. In contrast, phosphorylation of two in vitro targets of the ATM-like Tel1 kinase, S249 and S255, was not detected. This result helps resolve conflicting data on the importance of phosphorylation of these residues in telomerase recruitment. multiple residues showed differences in their cell cycle pattern of modification. For example, phosphorylation of S314 was significantly higher in the G2/M compared to the G1 phase and in WT versus mutant Cdc13, and a S314D mutation negatively affected telomere length. Our findings provide new targets in a key telomerase regulatory protein for modulation of telomere dynamics. [Image: see text

The Individual Blood Cell Telomere Attrition Rate Is Telomere Length Dependent

Age-associated telomere shortening is a well documented feature of peripheral blood cells in human population studies, but it is not known to what extent these data can be transferred to the individual level. Telomere length (TL) in two blood samples taken at ∼10 years interval from 959 individuals was investigated using real-time PCR. TL was also measured in 13 families from a multigenerational cohort. As expected, we found an age-related decline in TL over time (r = –0.164, P<0.001, n = 959). However, approximately one-third of the individuals exhibited a stable or increased TL over a decade. The individual telomere attrition rate was inversely correlated with initial TL at a highly significant level (r = –0.752, P<0.001), indicating that the attrition rate was most pronounced in individuals with long telomeres at baseline. In accordance, the age-associated telomere attrition rate was more prominent in families with members displaying longer telomeres at a young age (r = –0.691, P<0.001). Abnormal blood TL has been reported at diagnosis of various malignancies, but in the present study there was no association between individual telomere attrition rate or prediagnostic TL and later tumor development. The collected data strongly suggest a TL maintenance mechanism acting in vivo, providing protection of short telomeres as previously demonstrated in vitro. Our findings might challenge the hypothesis that individual TL can predict possible life span or later tumor development

Sub-Telomeric core X and Y' Elements in S.cerevisiae Suppress Extreme Variations in Gene Silencing

Telomere Position Effect (TPE) is governed by strong repression signals emitted by telomeres via the Sir2/3/4 Histone Deacetylase complex. These signals are then relayed by weak proto-silencers residing in the subtelomeric core X and Y' elements. Subtelomeres also contain Sub-Telomeric Anti-silencing Regions (STARs). In this study we have prepared telomeres built of different combinations of core X, Y' and STARs and have analyzed them in strains lacking Histone-Acetyltransferase genes as well as in cdc6-1 and Δrif1 strains. We show that core X and Y' dramatically reduce both positive and negative variations in TPE, that are caused by these mutations. We also show that the deletion of Histone-Acetyltransferase genes reduce the silencing activity of an ACS proto-silencer, but also reduce the anti-silencing activity of a STAR. We postulate that core X and Y' act as epigenetic “cushioning” cis-elements

Integrated Genome-Scale Prediction of Detrimental Mutations in Transcription Networks

A central challenge in genetics is to understand when and why mutations alter the phenotype of an organism. The consequences of gene inhibition have been systematically studied and can be predicted reasonably well across a genome. However, many sequence variants important for disease and evolution may alter gene regulation rather than gene function. The consequences of altering a regulatory interaction (or “edge”) rather than a gene (or “node”) in a network have not been as extensively studied. Here we use an integrative analysis and evolutionary conservation to identify features that predict when the loss of a regulatory interaction is detrimental in the extensively mapped transcription network of budding yeast. Properties such as the strength of an interaction, location and context in a promoter, regulator and target gene importance, and the potential for compensation (redundancy) associate to some extent with interaction importance. Combined, however, these features predict quite well whether the loss of a regulatory interaction is detrimental across many promoters and for many different transcription factors. Thus, despite the potential for regulatory diversity, common principles can be used to understand and predict when changes in regulation are most harmful to an organism

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