ALT telomeres get together with nuclear receptors.

Nuclear receptors bind chromosome ends in "alternative lengthening of telomeres" (ALT) cancer cells that maintain their ends by homologous recombination instead of telomerase. Marzec et al. now demonstrate that, in ALT cells, nuclear receptors not only trigger distal chromatin associations to mediate telomere-telomere recombination events, but also drive chromosome-internal targeted telomere insertions (TTI).

TFIIH moonlighting at telomeres.

Although telomeres are essential for chromosome stability, they represent fragile structures in our genome. Telomere shortening occurs during aging in cells lacking telomerase due to the end replication problem. In addition, recent work uncovered that the bulk of telomeric DNA poses severe hurdles for the semiconservative DNA replication machinery, requiring the assistance of an increasing number of specialized factors that prevent accidental telomere loss or damage events. In this issue of Genes & Development, Yang and colleagues (pp. 956-969) discover that TFIIH, a basic component of the PolII transcription initiation and nucleotide excision repair machinery, facilitates telomere replication. TFIIH is recruited to telomeres by the shelterin component TRF1, taking on at telomeres a moonlighting function.

RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops.

Telomeres-repeated, noncoding nucleotide motifs and associated proteins that are found at the ends of eukaryotic chromosomes-mediate genome stability and determine cellular lifespan1. Telomeric-repeat-containing RNA (TERRA) is a class of long noncoding RNAs (lncRNAs) that are transcribed from chromosome ends2,3; these RNAs in turn regulate telomeric chromatin structure and telomere maintenance through the telomere-extending enzyme telomerase4-6 and homology-directed DNA repair7,8. The mechanisms by which TERRA is recruited to chromosome ends remain poorly defined. Here we develop a reporter system with which to dissect the underlying mechanisms, and show that the UUAGGG repeats of TERRA are both necessary and sufficient to target TERRA to chromosome ends. TERRA preferentially associates with short telomeres through the formation of telomeric DNA-RNA hybrid (R-loop) structures that can form in trans. Telomere association and R-loop formation trigger telomere fragility and are promoted by the recombinase RAD51 and its interacting partner BRCA2, but counteracted by the RNA-surveillance factors RNaseH1 and TRF1. RAD51 physically interacts with TERRA and catalyses R-loop formation with TERRA in vitro, suggesting a direct involvement of this DNA recombinase in the recruitment of TERRA by strand invasion. Together, our findings reveal a RAD51-dependent pathway that governs TERRA-mediated R-loop formation after transcription, providing a mechanism for the recruitment of lncRNAs to new loci in trans.

PRDX1 and MTH1 cooperate to prevent ROS-mediated inhibition of telomerase.

Telomerase counteracts telomere shortening and cellular senescence in germ, stem, and cancer cells by adding repetitive DNA sequences to the ends of chromosomes. Telomeres are susceptible to damage by reactive oxygen species (ROS), but the consequences of oxidation of telomeres on telomere length and the mechanisms that protect from ROS-mediated telomere damage are not well understood. In particular, 8-oxoguanine nucleotides at 3' ends of telomeric substrates inhibit telomerase in vitro, whereas, at internal positions, they suppress G-quadruplex formation and were therefore proposed to promote telomerase activity. Here, we disrupt the peroxiredoxin 1 (PRDX1) and 7,8-dihydro-8-oxoguanine triphosphatase (MTH1) genes in cancer cells and demonstrate that PRDX1 and MTH1 cooperate to prevent accumulation of oxidized guanine in the genome. Concomitant disruption of PRDX1 and MTH1 leads to ROS concentration-dependent continuous shortening of telomeres, which is due to efficient inhibition of telomere extension by telomerase. Our results identify antioxidant systems that are required to protect telomeres from oxidation and are necessary to allow telomere maintenance by telomerase conferring immortality to cancer cells.

The THO complex counteracts TERRA R-loop-mediated telomere fragility in telomerase+ cells and telomeric recombination in ALT+ cells.

Telomeres are the nucleoprotein structures at the ends of linear chromosomes. Telomeres are transcribed into long non-coding Telomeric Repeat-Containing RNA (TERRA), whose functions rely on its ability to associate with telomeric chromatin. The conserved THO complex (THOC) was previously identified at human telomeres. It links transcription with RNA processing, decreasing the accumulation of co-transcriptional DNA:RNA hybrids throughout the genome. Here, we explore the role of THOC at human telomeres, as a regulator of TERRA localization to chromosome ends. We show that THOC counteracts TERRA association with telomeres via R-loops formed co-transcriptionally and also post-transcriptionally, in trans. We demonstrate that THOC binds nucleoplasmic TERRA, and that RNaseH1 loss, which increases telomeric R-loops, promotes THOC occupancy at telomeres. Additionally, we show that THOC counteracts lagging and mainly leading strand telomere fragility, suggesting that TERRA R-loops can interfere with replication fork progression. Finally, we observed that THOC suppresses telomeric sister-chromatid exchange and C-circle accumulation in ALT cancer cells, which maintain telomeres by recombination. Altogether, our findings reveal crucial roles of THOC in telomeric homeostasis through the co- and post-transcriptional regulation of TERRA R-loops.

The telomeric DNA damage response occurs in the absence of chromatin decompaction.

Telomeres are specialized nucleoprotein structures that protect chromosome ends from DNA damage response (DDR) and DNA rearrangements. The telomeric shelterin protein TRF2 suppresses the DDR, and this function has been attributed to its abilities to trigger t-loop formation or prevent massive decompaction and loss of density of telomeric chromatin. Here, we applied stochastic optical reconstruction microscopy (STORM) to measure the sizes and shapes of functional human telomeres of different lengths and dysfunctional telomeres that elicit a DDR. Telomeres have an ovoid appearance with considerable plasticity in shape. Examination of many telomeres demonstrated that depletion of TRF2, TRF1, or both affected the sizes of only a small subset of telomeres. Costaining of telomeres with DDR markers further revealed that the majority of DDR signaling telomeres retained a normal size. Thus, DDR signaling at telomeres does not require decompaction. We propose that telomeres are monitored by the DDR machinery in the absence of telomere expansion and that the DDR is triggered by changes at the molecular level in structure and protein composition.

Human shelterin protein POT1 prevents severe telomere instability induced by homology-directed DNA repair.

The evolutionarily conserved POT1 protein binds single-stranded G-rich telomeric DNA and has been implicated in contributing to telomeric DNA maintenance and the suppression of DNA damage checkpoint signaling. Here, we explore human POT1 function through genetics and proteomics, discovering that a complete absence of POT1 leads to severe telomere maintenance defects that had not been anticipated from previous depletion studies in human cells. Conditional deletion of POT1 in HEK293E cells gives rise to rapid telomere elongation and length heterogeneity, branched telomeric DNA structures, telomeric R-loops, and telomere fragility. We determine the telomeric proteome upon POT1-loss, implementing an improved telomeric chromatin isolation protocol. We identify a large set of proteins involved in nucleic acid metabolism that engage with telomeres upon POT1-loss. Inactivation of the homology-directed repair machinery suppresses POT1-loss-mediated telomeric DNA defects. Our results unravel as major function of human POT1 the suppression of telomere instability induced by homology-directed repair.

SMCHD1 promotes ATM-dependent DNA damage signaling and repair of uncapped telomeres.

Structural maintenance of chromosomes flexible hinge domain-containing protein 1 (SMCHD1) has been implicated in X-chromosome inactivation, imprinting, and DNA damage repair, and mutations in SMCHD1 can cause facioscapulohumeral muscular dystrophy. More recently, SMCHD1 has also been identified as a component of telomeric chromatin. Here, we report that SMCHD1 is required for DNA damage signaling and non-homologous end joining (NHEJ) at unprotected telomeres. Co-depletion of SMCHD1 and the shelterin subunit TRF2 reduced telomeric 3'-overhang removal in time-course experiments, as well as the number of chromosome end fusions. SMCHD1-deficient cells displayed reduced ATM S1981 phosphorylation and diminished formation of γH2AX foci and of 53BP1-containing telomere dysfunction-induced foci (TIFs), indicating defects in DNA damage checkpoint signaling. Removal of TPP1 and subsequent activation of ATR signaling rescued telomere fusion events in TRF2-depleted SMCHD1 knockout cells. Together, these data indicate that SMCHD1 depletion reduces telomere fusions in TRF2-depleted cells due to defects in ATM-dependent checkpoint signaling and that SMCHD1 mediates DNA damage response activation upstream of ATM phosphorylation at uncapped telomeres.

Challenging endings: How telomeres prevent fragility.

It has become apparent that difficulties to replicate telomeres concern not only the very ends of eukaryotic chromosomes. The challenges already start when the replication fork enters the telomeric repeats. The obstacles encountered consist mainly of noncanonical nucleic acid structures that interfere with replication if not resolved. Replication stress at telomeres promotes the formation of so-called fragile telomeres displaying an abnormal appearance in metaphase chromosomes though their exact molecular nature remains to be elucidated. A substantial number of factors is required to counteract fragility. In this review we promote the hypothesis that telomere fragility is not caused directly by an initial insult during replication but it results as a secondary consequence of DNA repair of damaged replication forks by the homologous DNA recombination machinery. Incomplete DNA synthesis at repair sites or partial chromatin condensation may become apparent as telomere fragility. Fragility and DNA repair during telomere replication emerges as a common phenomenon which exacerbates in multiple disease conditions.

The human telomeric proteome during telomere replication.

Telomere shortening can cause detrimental diseases and contribute to aging. It occurs due to the end replication problem in cells lacking telomerase. Furthermore, recent studies revealed that telomere shortening can be attributed to difficulties of the semi-conservative DNA replication machinery to replicate the bulk of telomeric DNA repeats. To investigate telomere replication in a comprehensive manner, we develop QTIP-iPOND - Quantitative Telomeric chromatin Isolation Protocol followed by isolation of Proteins On Nascent DNA - which enables purification of proteins that associate with telomeres specifically during replication. In addition to the core replisome, we identify a large number of proteins that specifically associate with telomere replication forks. Depletion of several of these proteins induces telomere fragility validating their importance for telomere replication. We also find that at telomere replication forks the single strand telomere binding protein POT1 is depleted, whereas histone H1 is enriched. Our work reveals the dynamic changes of the telomeric proteome during replication, providing a valuable resource of telomere replication proteins. To our knowledge, this is the first study that examines the replisome at a specific region of the genome.

Expression and differential regulation of human TERRA at several chromosome ends.

The telomeric long noncoding RNA TERRA has been implicated in regulating telomere maintenance by telomerase and homologous recombination, and in influencing telomeric protein composition during the cell cycle and the telomeric DNA damage response. TERRA transcription starts at subtelomeric regions resembling the CpG islands of eukaryotic genes extending toward chromosome ends. TERRA contains chromosome-specific subtelomeric sequences at its 5' end and long tracts of UUAGGG-repeats toward the 3' end. Conflicting studies have been published as to whether TERRA is expressed from one or several chromosome ends. Here, we quantify TERRA species by RT-qPCR in normal and several cancerous human cell lines. By using chromosome-specific subtelomeric DNA primers, we demonstrate that TERRA is expressed from a large number of telomeres. Deficiency in DNA methyltransferases leads to TERRA up-regulation only at the subset of chromosome ends that contain CpG-island sequences, revealing differential regulation of TERRA promoters by DNA methylation. However, independently of the differences in TERRA expression, short telomeres were uniformly present in a DNA methyltransferase deficient cell line, indicating that telomere length was not dictated by TERRA expression in cis Bioinformatic analyses indicated the presence of a large number of putative transcription factors binding sites at TERRA promoters, and we identified a subset of them that repress TERRA expression. Altogether, our study confirms that TERRA corresponds to a large gene family transcribed from multiple chromosome ends where we identified two types of TERRA promoters, only one of which is regulated by DNA methylation.

Molecular basis of telomere syndrome caused by CTC1 mutations.

Mutations in CTC1 lead to the telomere syndromes Coats Plus and dyskeratosis congenita (DC), but the molecular mechanisms involved remain unknown. CTC1 forms with STN1 and TEN1 a trimeric complex termed CST, which binds ssDNA, promotes telomere DNA synthesis, and inhibits telomerase-mediated telomere elongation. Here we identify CTC1 disease mutations that disrupt CST complex formation, the physical interaction with DNA polymerase α-primase (polα-primase), telomeric ssDNA binding in vitro, accumulation in the nucleus, and/or telomere association in vivo. While having diverse molecular defects, CTC1 mutations commonly lead to the accumulation of internal single-stranded gaps of telomeric DNA, suggesting telomere DNA replication defects as a primary cause of the disease. Strikingly, mutations in CTC1 may also unleash telomerase repression and telomere length control. Hence, the telomere defect initiated by CTC1 mutations is distinct from the telomerase insufficiencies seen in classical forms of telomere syndromes, which cause short telomeres due to reduced maintenance of distal telomeric ends by telomerase. Our analysis provides molecular evidence that CST collaborates with DNA polα-primase to promote faithful telomere DNA replication.

A Role for Human DNA Polymerase λ in Alternative Lengthening of Telomeres.

Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.

AUF1/HnRNP D RNA binding protein functions in telomere maintenance.

In this issue of Molecular Cell, Pont et al. (2012) show that AUF1/hnRNP D promotes TERT transcription, which is required for telomere maintenance in mice.

Impact of oxidative stress on telomere biology.

Telomere integrity is essential for genome stability and it regulates cell proliferation and tissue renewal. Several lines of evidence indicate that telomeres are particularly sensitive to oxidative damage. Moreover, recent studies demonstrate striking inhibitory effects of oxidative damage on telomerase activity. On the other hand, several mechanisms have been uncovered that either counteract oxidative damage at telomeres or remove the modified lesions. Here, we review the current understanding of oxidative damage and protection of telomeric DNA. We discuss how oxidative telomeric lesions impact on telomerase, the regenerative capacity of stem cells and cancer. Finally, we propose how through a better understanding of the involved pathways it may become possible to target telomerase in cancer cells in future cancer therapies.

A practical qPCR approach to detect TERRA, the elusive telomeric repeat-containing RNA.

Telomeres, the heterochromatic structures that protect the ends of the chromosomes, are transcribed into a class of long non-coding RNAs, telomeric repeat-containing RNAs (TERRA), whose transcriptional regulation and functions are not well understood. The identification of TERRA adds a novel level of structural and functional complexity at telomeres, opening up a new field of research. TERRA molecules are expressed at several chromosome ends with transcription starting from the subtelomeric DNA proceeding into the telomeric tracts. TERRA is heterogeneous in length and its expression is regulated during the cell cycle and upon telomere damage. Little is known about the mechanisms of regulation at the level of transcription and post transcription by RNA stability. Furthermore, it remains to be determined to what extent the regulation at different chromosome ends may differ. We present an overview on the methodology of how RT-qPCR and primer pairs that are specific for different subtelomeric sequences can be used to detect and quantify TERRA expressed from different chromosome ends.

Quantitative telomeric chromatin isolation protocol for human cells.

The ends of eukaryotic chromosomes, known as telomeres, consist of repetitive DNA sequences, multiple proteins and noncoding RNAs. Telomeres are dynamic structures that play crucial roles as guardians of genome stability and tumor suppressors. Defects in telomere length or protein composition can accelerate aging and are seen in telomere syndromes, which affect various proliferative tissues such as the bone marrow or the lungs. One of the biggest challenges in the telomere field is to identify the molecular changes at telomeres that occur during normal development, in cancer and in telomere syndromes. To tackle this problem, our laboratory has established a quantitative telomeric chromatin isolation protocol (QTIP) for human cells, in which chromatin is cross-linked, immunopurified and analyzed by mass spectrometry. QTIP involves stable isotope labeling by amino acids in cell culture (SILAC) to compare and identify quantitative differences in telomere protein composition of cells from various states.

The human CST complex is a terminator of telomerase activity.

The lengths of human telomeres, which protect chromosome ends from degradation and end fusions, are crucial determinants of cell lifespan. During embryogenesis and in cancer, the telomerase enzyme counteracts telomeric DNA shortening. As shown in cancer cells, human telomerase binds the shelterin component TPP1 at telomeres during the S phase of the cell cycle, and adds ~60 nucleotides in a single round of extension, after which telomerase is turned off by unknown mechanisms. Here we show that the human CST (CTC1, STN1 and TEN1) complex, previously implicated in telomere protection and DNA metabolism, inhibits telomerase activity through primer sequestration and physical interaction with the protection of telomeres 1 (POT1)­TPP1 telomerase processivity factor. CST competes with POT1­TPP1 for telomeric DNA, and CST­telomeric-DNA binding increases during late S/G2 phase only on telomerase action, coinciding with telomerase shut-off. Depletion of CST allows excessive telomerase activity, promoting telomere elongation. We propose that through binding of the telomerase-extended telomere, CST limits telomerase action at individual telomeres to approximately one binding and extension event per cell cycle. Our findings define the sequence of events that occur to first enable and then terminate telomerase-mediated telomere elongation.

The THO complex component Thp2 counteracts telomeric R-loops and telomere shortening.

Telomere maintenance by the conventional DNA replication machinery and telomerase is assisted by specialized DNA helicases, nucleases and telomere binding proteins. Here, we identify the THO components at telomeres and define critical roles of this complex in telomere stability. Deletion of the THO-subunit THP2 leads to telomere shortening. We discover that telomeres contain RNA:DNA hybrid structures or R-loops which involve the long-noncoding RNA TERRA and which accumulate in thp2-Δ cells. Telomere length is not restored by R-loop removal upon RNase H overexpression, but by deletion of Exonuclease 1 (Exo1). Replication stress further enhances the short telomere phenotype of THP2 mutants. Similar events occur upon induced transcription of TERRA and genetic analysis links Thp2 to TERRA function. Altogether, our data indicate that THO, through the interplay with TERRA, regulates chromosome end processing activities and prevents interference with semiconservative DNA replication of telomeric DNA.

Related mechanisms for end processing at telomeres and DNA double-strand breaks.

In a recent issue of Molecular Cell, Bonetti et al. (2009) identify in the yeast Saccharomyces cerevisiae that the molecular activities that generate 3' overhangs at telomeric DNA ends are the same as those that resect DNA at double-strand breaks.