The G-quadruplex-stabilising agent RHPS4 induces telomeric dysfunction and enhances radiosensitivity in glioblastoma cells

G-quadruplex (G4) interacting agents are a class of ligands that can bind to and stabilise secondary structures located in genomic G-rich regions such as telomeres. Stabilisation of G4 leads to telomere architecture disruption with a consequent detrimental effect on cell proliferation, which makes these agents good candidates for chemotherapeutic purposes. RHPS4 is one of the most effective and well-studied G4 ligands with a very high specificity for telomeric G4.In this work, we tested the in vitro efficacy of RHPS4 in astrocytoma cell lines, and we evaluated whether RHPS4 can act as a radiosensitising agent by destabilising telomeres.In the first part of the study, the response to RHPS4 was investigated in four human astrocytoma cell lines (U251MG, U87MG, T67 and T70) and in two normal primary fibroblast strains (AG01522 and MRC5). Cell growth reduction, histone H2AX phosphorylation and telomere-induced dysfunctional foci (TIF) formation were markedly higher in astrocytoma cells than in normal fibroblasts, despite the absence of telomere shortening. In the second part of the study, the combined effect of submicromolar concentrations of RHPS4 and X-rays was assessed in the U251MG glioblastoma radioresistant cell line. Long-term growth curves, cell cycle analysis and cell survival experiments, clearly showed the synergistic effect of the combined treatment. Interestingly the effect was greater in cells bearing a higher number of dysfunctional telomeres. DNA double-strand breaks rejoining after irradiation revealed delayed repair kinetics in cells pre-treated with the drug and a synergistic increase in chromosome-type exchanges and telomeric fusions.These findings provide the first evidence that exposure to RHPS4 radiosensitizes astrocytoma cells, suggesting the potential for future therapeutic applications.     

p16INK4a as a Second Effector of the Telomere Damage Pathway

Telomere damage resulting from telomere shortening can potentially suppress tumorigenesis by permanently arresting or eliminating incipient cancer cells. Dysfunctional telomeres activate the canonical DNA damage signaling pathway, resulting in a p53-mediated G1/S arrest and senescence or apoptosis. Experimental induction of telomere damage through inhibition of the telomeric protein TRF2 recapitulates aspects of telomere attrition, including a p53-mediated cell cycle arrest. Using this system, we have shown that telomere damage can also elicit a G1/S arrest through the RB-regulator p16INK4a, especially in cells lacking p53 function. Here we discuss the significance of p16INK4a as a second effector of the telomere damage response.     

A basal level of DNA damage and telomere deprotection increases the sensitivity of cancer cells to G-quadruplex interactive compounds

Here, with the aim of obtaining insight into the intriguing selectivity of G-quadruplex (G4) ligands toward cancer compared to normal cells, a genetically controlled system of progressive transformation in human BJ fibroblasts was analyzed. Among the different comparative evaluations, we found a progressive increase of DNA damage response (DDR) markers throughout the genome from normal toward immortalized and transformed cells. More interestingly, sensitivity to G4 ligands strongly correlated with the presence of a basal level of DNA damage, including at the telomeres, where the chromosome ends were exposed to the DDR without concurrent induction of DNA repair activity, as revealed by the lack of 53BP1 recruitment and telomere aberrations. The link between telomere uncapping and the response to G4 stabilization was directly assessed by showing that a partial TRF2 depletion, causing a basal level of telomere localized DDR, rendered telomerized fibroblasts prone to G4-induced telomere damage and anti-proliferative defects. Taken together these data strongly indicate that the presence of a basal level of telomere-associated DDR is a determinant of susceptibility to G4 stabilization.     

TopoIIα prevents telomere fragility and formation of ultra thin DNA bridges during mitosis through TRF1-dependent binding to telomeres

Telomeres are repetitive nucleoprotein structures at the ends of chromosomes. Like most genomic regions consisting of repetitive DNA, telomeres are fragile sites prone to replication fork stalling and generation of chromosomal instability. In particular, abrogation of the TRF1 telomere binding protein leads to stalled replication forks and aberrant telomere structures known as “multitelomeric signals”. Here, we report that TRF1 deficiency also leads to the formation of “ultra-fine bridges” (UFB) during mitosis, and to an increased time to complete mitosis mediated by the spindle assembly checkpoint proteins (SAC). We find that topoisomerase IIα (TopoIIα), an enzyme essential for resolution of DNA replication intermediates, binds telomeres in a TRF1-mediated manner. Indeed, similar to TRF1 abrogation, TopoIIα downregulation leads to telomere fragility and UFB, suggesting that these phenotypes are due to decreased TopoIIα at telomeres. We find that SAC proteins bind telomeres in vivo, and that this is disrupted upon TRF1 deletion. These findings suggest that TRF1 links TopoIIα and SAC proteins in a pathway that ensures correct telomere replication and mitotic segregation, unveiling how TRF1 protects from telomere fragility and mitotic defects.     

Timeless tunes: Replicating happy endings

Comment on: Leman AR, et al. Cell Cycle 2012; 11:2337-47.DNA replication is at the heart of the inheritance of genetic material. A single replication fork can progress through hundreds of kilobases of DNA, melting parental double-stranded DNA and leaving newly synthesized strands in its wake. A beautiful illustration showing how the replication machinery accomplishes this complex task is one of the triumphs of molecular biology. However, it is known that DNA replication is not always as processive as the textbooks suggest. Specifically, the rate of fork progression varies depending on the regions being replicated, and the replication fork even stalls in some circumstances, during replication of heterochromatin or damaged DNA, for example. A stalled replication fork has two fates. It may restart DNA replication, or it may collapse after prolonged stalling. A collapsed replication fork is particularly dangerous for the genome, because the DNA intermediate left by the collapsed fork may form a double-stranded break, a highly mutagenic lesion that can undergo illegitimate recombination. To circumvent replication fork collapse, cells are equipped with specialized proteins that stabilize the stalled replication fork. Timeless and Tipin are highly conserved in eukaryotes. from yeast to humans, and form a complex to protect stalled replication forks.In a paper published in Cell Cycle, Noguchi and his group investigated how Timeless plays a role in telomere replication in human cells.¹ Telomeres consist of tandem arrays of short repetitive DNA (TTAGGG/CCCTAA in mammals) at the ends of chromosomes and numerous associated proteins. Telomeres are essential for the stable maintenance of genomic DNA, because they protect the DNA termini from undergoing accidental recombination and exonuclease attack. Dysfunctional telomeres lead to genetic instability that eventually results in senescence and cancer development. Because of the heterochromatic nature of telomeres, it has been recognized that telomere DNA is one of the genomic regions that impede replication fork progression. Indeed, in vitro DNA replication experiments using SV40 DNA, and cell extracts demonstrated that telomere DNA is replicated less efficiently and incurs more fork stalling than non-telomeric DNA.² Moreover, overexpression of telomere-DNA binding protein TRF1 in HeLa cells led to an accumulation of replicating telomeres, consistent with a slower replication rate of telomeres under those circumstance. Furthermore, experiments using TRF1-deleted murine cells showed that TRF1 is essential for efficient telomere DNA replication.³ Collectively, these results confirm that the telomere is a difficult-to-replicate region.There is an apparent contradiction between two earlier studies, however, with TRF1 described as an anti-replication protein in one report² and a pro-replication protein in the other.³ One potential explanation for the inconsistency might be that TRF1 requires other protein(s) to perform its pro-replication function, and the second factor was missing in the TRF1-overexpression experiments. Noguchi and his colleagues investigated this possibility by testing whether Timeless is required for proficient telomere DNA replication.¹ They found that Timeless-knockdown cells displayed telomere length shortening and an increased frequency of dysfunctional telomeres. In vitro replication assays of SV40 DNA revealed that Timeless-depleted extracts supported non-telomere replication proficiently, while telomere replication was inefficient. They then demonstrated that addition of recombinant TRF1 to the replication system slowed telomere replication. Importantly, Timeless depletion and TRF1 addition did not produce additive effects on telomere replication, suggesting that Timeless and TRF1 function in the same pathway. These results suggest a model as described in Figure 1. A replication fork frequently stalls at telomeres because of the molecularly crowded nature of telomeric chromatin. Timeless presumably encounters TRF1 at telomeres and protects the stalled fork from undergoing collapse. In the absence of Timeless, the stalled forks easily collapse, leading to an abrupt shortening of telomeres. Several questions remain to be answered. Given that Timeless moves along the genomic DNA as a component of the replication machinery,⁴ it will be particularly interesting to see how Timeless (or the replication machinery) interacts with telomeric chromatin. In such studies, a dynamic transaction between the regional chromatin at telomeres and the replication machinery may be revealed.Open in a separate windowFigure 1. Hard life at telomeres. (A) Mammalian telomeres consist of repetitive DNA that potentially forms higher-ordered structures [G-quartet(G4)-DNA] and numerous proteins, including telomere DNA-binding protein TRF1. (B) Replication fork is frequently stalled at telomeres. Overexpressed TRF1 slows down fork progression at the telomere, while endogenous TRF1 together with Timeless protein facilitates it. Timeless protects the stalled replication fork from collapse. (C) Telomeres are unique in that the most distal replication fork is not coupled with another fork progressing inversely. (D) Prolonged fork stalling may lead to the formation of a DNA double-strand break. Because of the lack of another fork compensating the telomere replication (C), the break immediately results in the abrupt single-step shortening of telomere DNAs.     

DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion

Telomere attrition and other forms of telomere damage can activate the ATM kinase pathway. What generates the DNA damage signal at mammalian chromosome ends or at other double-strand breaks is not known. Telomere dysfunction is often accompanied by disappearance of the 3' telomeric overhang, raising the possibility that DNA degradation could generate the structure that signals. Here we address these issues by studying telomere structure after conditional deletion of mouse TRF2, the protective factor at telomeres. Upon removal of TRF2 from TRF2(F/-) p53-/- mouse embryo fibroblasts, a telomere damage response is observed at most chromosome ends. As expected, the telomeres lose the 3' overhang and are processed by the non-homologous end-joining pathway. Non-homologous end joining of telomeres was abrogated in DNA ligase IV-deficient (Lig4-/-) cells. Unexpectedly, the telomeres of TRF2-/- Lig4-/- p53-/- cells persisted in a free state without undergoing detectable DNA degradation. Notably, the telomeres retained their 3' overhangs, but they were recognized as sites of DNA damage, accumulating the DNA damage response factors 53BP1 and gamma-H2AX, and activating the ATM kinase. Thus, activation of the ATM kinase pathway at chromosome ends does not require overhang degradation or other overt DNA processing.     

The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins

Most cancer cells activate telomerase to elongate telomeres and achieve unlimited replicative potential. Some cancer cells cannot activate telomerase and use telomere homologous recombination (HR) to elongate telomeres, a mechanism termed alternative lengthening of telomeres (ALT). A hallmark of ALT cells is the recruitment of telomeres to PML bodies (termed APBs). Here, we show that the SMC5/6 complex localizes to APBs in ALT cells and is required for targeting telomeres to APBs. The MMS21 SUMO ligase of the SMC5/6 complex SUMOylates multiple telomere-binding proteins, including TRF1 and TRF2. Inhibition of TRF1 or TRF2 SUMOylation prevents APB formation. Depletion of SMC5/6 subunits by RNA interference inhibits telomere HR, causing telomere shortening and senescence in ALT cells. Thus, the SMC5/6 complex facilitates telomere HR and elongation in ALT cells by promoting APB formation through SUMOylation of telomere-binding proteins.     

Both the classical and alternative non-homologous end joining pathways contribute to the fusion of drastically shortened telomeres induced by TRF2 overexpression

The double-stranded telomeric binding protein TRF2 is expressed in many human cancers at elevated levels. Moreover, experimental overexpression of TRF2 in human cells causes replication stalling in telomeric tracts, which leads to drastic telomere shortening and fusion of deprotected chromosome ends. To understand which end joining pathway is involved in mediating these chromosome fusions, we overexpressed TRF2 in human HCT116 cell lines that were deficient for the DNA Ligase 4 (Lig4)-dependent classical non-homologous end joining (C-NHEJ) or the DNA Ligase 3 (Lig3)-dependent alternative non-homologous end joining (A-NHEJ) pathway. Surprisingly, abrogation of either Lig4 or nuclear Lig3 significantly reduced inter-chromosomal fusion of drastically shortened telomeres, suggesting that both the C-NHEJ and A-NHEJ pathways are involved in mediating this type of fusion. Fusion between deprotected sister chromatids, however, only required the Lig3-dependent A-NHEJ pathway. Interestingly, a previous study reported similar end joining pathway requirements for the fusion of critically shortened telomeres during a telomere attrition-based cellular crisis. We speculate that, as in cellular crisis, the same repair pathway(s) may drive clonal and genomic evolution in human cancers containing elevated TRF2 levels.     

The role of the poly(ADP-ribose) polymerase tankyrase1 in telomere length control by the TRF1 component of the shelterin complex

Tankyrase1 is a multifunctional poly(ADP-ribose) polymerase that can localize to telomeres through its interaction with the shelterin component TRF1. Tankyrase1 poly(ADP-ribosyl)ates TRF1 in vitro, and its nuclear overexpression leads to loss of TRF1 and telomere elongation, suggesting that tankyrase1 is a positive regulator of telomere length. In agreement with this proposal, we show that tankyrase1 RNA interference results in telomere shortening proportional to the level of knockdown. Furthermore, we show that a tankyrase1-resistant form of TRF1 enforced normal telomere length control, indicating that tankyrase1 is not required downstream of TRF1 in this pathway. Thus, in human cells, tankyrase1 appears to act upstream of TRF1, promoting telomere elongation through the removal of TRF1. This pathway appears absent from mouse cells. We show that murine TRF1, which lacks the canonical tankyrase1-binding site, is not a substrate for tankyrase1 poly(ADP-ribosyl)sylation in vitro. Furthermore, overexpression of tankyrase1 in mouse nuclei did not remove TRF1 from telomeres and had no detectable effect on other components of mouse shelterin. We propose that the tankyrase1-controlled telomere extension is a human-specific elaboration that allows additional control over telomere length in telomerase positive cells.     

Dynamics of TRF1 organizing a single human telomere

Chromosome stability is primarily determined by telomere length. TRF1 is the core subunit of shelterin that plays a critical role in telomere organization and replication. However, the dynamics of TRF1 in scenarios of telomere-processing activities remain elusive. Using single-molecule magnetic tweezers, we here investigated the dynamics of TRF1 upon organizing a human telomere and the protein-DNA interactions at a moving telomeric fork. We first developed a method to obtain telomeres from human cells for directly measuring the telomere length by single-molecule force spectroscopy. Next, we examined the compaction and decompaction of a telomere by TRF1 dimers. TRF1 dissociates from a compacted telomere with heterogenous loops in ∼20 s. We also found a negative correlation between the number of telomeric loops and loop sizes. We further characterized the dynamics of TRF1 at a telomeric DNA fork. With binding energies of 11 k_BT, TRF1 can modulate the forward and backward steps of DNA fork movements by 2–9 s at a critical force of F_1/2, temporarily maintaining the telomeric fork open. Our results shed light on the mechanisms of how TRF1 organizes human telomeres and facilitates the efficient replication of telomeric DNA. Our work will help future research on the chemical biology of telomeres and shelterin-targeted drug discovery.     

PinX1, a Telomere Repeat-binding Factor 1 (TRF1)-interacting Protein,Maintains Telomere Integrity by Modulating TRF1 Homeostasis,the Process in Which Human Telomerase Reverse Transcriptase (hTERT) Plays Dual Roles

TRF1, a telomere-binding protein, is important for telomere protection and homeostasis. PinX1 interacts with TRF1, but the physiological consequences of their interaction in telomere protection are not yet understood. Here we investigated PinX1 function on TRF1 stability in HeLa cells. PinX1 overexpression stabilized TRF1, but PinX1 depletion by siRNA led to TRF1 degradation, TRF1 ubiquitination, and less TRF1 telomere association. The depletion also induced DNA damage responses at telomeres and chromosome instability. These telomere dysfunctional phenotypes were in fact due to TRF1 deficiency. We also report that hTERT, a catalytic component of telomerase, plays dual roles in the TRF1 steady state pathway. PinX1-mediated TRF1 stability was not observed in hTERT-negative immortal cells, but was pronounced when hTERT was ectopically expressed in the cells, suggesting that hTERT may be needed in the PinX1-mediated TRF1 stability pathway. Interestingly, the knockdown of both PinX1 and hTERT in HeLa cells stabilized TRF1, suppressed DNA damage response activation, and restored chromosome stability. In summary, our findings suggested that PinX1 may maintain telomere integrity by regulating TRF1 stability and that hTERT may act as both a positive and a negative regulator of TRF1 homeostasis in a PinX1-dependent manner.     

A mathematical model of cellular apoptosis and senescence through the dynamics of telomere loss

The shortening of telomeric repeats as a cell replicates has long been implicated as a determinant of cell viability. However, recent studies have indicated that it is not telomere length, but rather whether telomeres have bound a telomere-related protein, which in mammals is TTAGGG repeat binding factor-2 (TRF2), that determines whether a cell undergoes apoptosis (programmed cell death), enters senescence (a quiescent, non-replicative state), or continues to proliferate. When bound to a telomere, TRF2 allows a cell to recognize the telomere as the point where a chromosome ends rather than a break in DNA. When telomeres are not bound by TRF2, the cell can either immediately trigger senescence or apoptosis via the DNA damage response pathway, or indirectly trigger it by attempting to repair the chromosome, which results in chromosomal end joining. We model the ability of telomeres to bind TRF2 as a function of telomere length and apply the resulting binding probability to a model of cellular replication that assumes a homogeneous cell population. The model fits data from cultured human fibroblasts and human embryonic kidney cells for two free parameters well. We extract values for the percent of telomere loss at which cell proliferation ceases. We show, in agreement with previous experiments, that overexpression of TRF2 allows a cell to delay the senescence setpoint. We explore the effect of oxidative stress, which increases the rate of telomere loss, on cell viability and show that cells in the presence of oxidative stress have reduced lifespans. We also show that the addition of telomerase, an enzyme that maintains telomere length, is sufficient to result in cell immortality. We conclude that the increasing inability of TRF2 to bind telomeres as they shorten is a quantitatively reasonable model for a cause of either cellular apoptosis or senescence.     

The basic N-terminal domain of TRF2 limits recombination endonuclease action at human telomeres

The stability of mammalian telomeres depends upon TRF2, which prevents inappropriate repair and checkpoint activation. By using a plasmid integration assay in yeasts carrying humanized telomeres, we demonstrated that TRF2 possesses the intrinsic property to both stimulate initial homologous recombination events and to prevent their resolution via its basic N-terminal domain. In human cells, we further showed that this TRF2 domain prevents telomere shortening mediated by the resolvase-associated protein SLX4 as well as GEN1 and MUS81, 2 different types of endonucleases with resolvase activities. We propose that various types of resolvase activities are kept in check by the basic N-terminal domain of TRF2 in order to favor an accurate repair of the stalled forks that occur during telomere replication.     

Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases

Werner syndrome is a human premature aging disorder displaying cellular defects associated with telomere maintenance including genomic instability, premature senescence, and accelerated telomere erosion. The yeast homologue of the Werner protein (WRN), Sgs1, is required for recombination-mediated lengthening of telomeres in telomerase-deficient cells. In human cells, we report that WRN co-localizes and physically interacts with the critical telomere maintenance protein TRF2. This interaction is mediated by the RecQ conserved C-terminal region of WRN. In vitro, TRF2 demonstrates high affinity for WRN and for another RecQ family member, the Bloom syndrome protein (BLM). TRF2 interaction with either WRN or BLM results in a notable stimulation of their helicase activities. Furthermore, the WRN and BLM helicases, partnered with replication protein A, actively unwind long telomeric duplex regions that are pre-bound by TRF2. These results suggest that TRF2 functions with WRN, and possibly BLM, in a common pathway at telomeric ends.     

Telomere-bound TRF1 and TRF2 stall the replication fork at telomeric repeats

Vertebrate telomeres consist of tandem repeats of T₂AG₃ and associated proteins including the telomeric DNA-binding proteins, TRF1 and TRF2. It has been proposed that telomeres assume two interswitchable states, the open state that is accessible to various trans-acting factors and the closed state that excludes those factors. TRF1 and TRF2 are believed to promote the formation of the closed state. However, little is known about how those two states influence DNA replication. We analyzed the effects of TRF1 and TRF2 on telomeric replication both in vitro and in vivo. By exploiting the in vitro replication system of linear SV40 DNA, we found that telomeric repeats are a poor replication template. Moreover, the addition of recombinant TRF1 and TRF2 significantly stalled the replication fork progression at telomeric repeats. When TRF1 was overexpressed in HeLa cells, cells with 4N DNA content were accumulated. Furthermore, cytological analyses revealed that the replication focus overlapped with telomere signals at a significantly higher frequency in TRF1-overexpressing cells than in control cells. The results suggest that TRF1 and TRF2 exert inhibitory effects on replication fork progression.     

DNA ligase IV-dependent NHEJ of deprotected mammalian telomeres in G1 and G2

BACKGROUND: Telomeres are required to prevent end-to-end chromosome fusions. End-to-end fusions of metaphase chromosomes are observed in mammalian cells with dysfunctional telomeres due to diminished function of telomere-associated proteins and in cells experiencing extensive attrition of telomeric DNA. However, the molecular nature of these fusions and the mechanism by which they occur have not been elucidated. RESULTS: We document that telomere fusions resulting from inhibition of the telomere-protective factor TRF2 are generated by DNA ligase IV-dependent nonhomologous end joining (NHEJ). NHEJ gives rise to covalent ligation of the C strand of one telomere to the G strand of another. Breakage of the resulting dicentric chromosomes results in nonreciprocal translocations, a hallmark of human cancer. Telomere NHEJ took place before and after DNA replication, and both sister telomeres participated in the reaction. Telomere fusions were accompanied by active degradation of the 3' telomeric overhangs. CONCLUSIONS: The main threat to dysfunctional mammalian telomeres is degradation of the 3' overhang and subsequent telomere end-joining by DNA ligase IV. The involvement of NHEJ in telomere fusions is paradoxical since the NHEJ factors Ku70/80 and DNA-PKcs are present at telomeres and protect chromosome ends from fusion.     

TRF2-mediated ORC recruitment underlies telomere stability upon DNA replication stress

Telomeres are intrinsically difficult-to-replicate region of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2–ORC interaction for telomere maintenance remains largely elusive. Here, we employed a TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that inhibited the TRF2–ORC interaction in human cells. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244–511), which competitively inhibited the TRF2–ORC interaction, increased telomeric DNA damage under replication stress conditions. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.     

TRF1 Ensures the Centromeric Function of Aurora-B and Proper Chromosome Segregation

A cancer is a robustly evolving cell population originating from a normal diploid cell. Improper chromosome segregation causes aneuploidy, a driving force of cancer development and malignant progression. Telomeric repeat binding factor 1 (TRF1) has been established as a telomeric protein that negatively regulates telomere elongation by telomerase and promotes efficient DNA replication at telomeres. Intriguingly, overexpression of a mitotic kinase, Aurora-A, compromises efficient microtubule-kinetochore attachment in a TRF1-dependent manner. However, the precise role of TRF1 in mitosis remains elusive. Here we demonstrate that TRF1 is required for the centromeric function of Aurora-B, which ensures proper chromosome segregation. TRF1 depletion abolishes centromeric recruitment of Aurora-B and loosens sister centromere cohesion, resulting in the induction of merotelic kinetochore attachments, lagging chromosomes, and micronuclei. Accordingly, an absence of TRF1 in human and mouse diploid cells induces aneuploidy. These phenomena seem to be telomere independent, because a telomere-unbound TRF1 mutant can suppress the TRF1 knockdown phenotype. These observations indicate that TRF1 regulates the rigidity of the microtubule-kinetochore attachment, contributing to proper chromosome segregation and the maintenance of genomic integrity.