TRF2-independent chromosome end protection during pluripotency.

Mammalian telomeres protect chromosome ends from aberrant DNA repair1. TRF2, a component of the telomere-specific shelterin protein complex, facilitates end protection through sequestration of the terminal telomere repeat sequence within a lariat T-loop structure2,3. Deleting TRF2 (also known as TERF2) in somatic cells abolishes T-loop formation, which coincides with telomere deprotection, chromosome end-to-end fusions and inviability3-9. Here we establish that, by contrast, TRF2 is largely dispensable for telomere protection in mouse pluripotent embryonic stem (ES) and epiblast stem cells. ES cell telomeres devoid of TRF2 instead activate an attenuated telomeric DNA damage response that lacks accompanying telomere fusions, and propagate for multiple generations. The induction of telomere dysfunction in ES cells, consistent with somatic deletion of Trf2 (also known as Terf2), occurs only following the removal of the entire shelterin complex. Consistent with TRF2 being largely dispensable for telomere protection specifically during early embryonic development, cells exiting pluripotency rapidly switch to TRF2-dependent end protection. In addition, Trf2-null embryos arrest before implantation, with evidence of strong DNA damage response signalling and apoptosis specifically in the non-pluripotent compartment. Finally, we show that ES cells form T-loops independently of TRF2, which reveals why TRF2 is dispensable for end protection during pluripotency. Collectively, these data establish that telomere protection is solved by distinct mechanisms in pluripotent and somatic tissues.

TRF2-mediated telomere protection is dispensable in pluripotent stem cells.

In mammals, telomere protection is mediated by the essential protein TRF2, which binds chromosome ends and ensures genome integrity1,2. TRF2 depletion results in end-to-end chromosome fusions in all cell types that have been tested so far. Here we find that TRF2 is dispensable for the proliferation and survival of mouse embryonic stem (ES) cells. Trf2-/- (also known as Terf2) ES cells do not exhibit telomere fusions and can be expanded indefinitely. In response to the deletion of TRF2, ES cells exhibit a muted DNA damage response that is characterized by the recruitment of γH2AX-but not 53BP1-to telomeres. To define the mechanisms that control this unique DNA damage response in ES cells, we performed a CRISPR-Cas9-knockout screen. We found a strong dependency of TRF2-null ES cells on the telomere-associated protein POT1B and on the chromatin remodelling factor BRD2. Co-depletion of POT1B or BRD2 with TRF2 restores a canonical DNA damage response at telomeres, resulting in frequent telomere fusions. We found that TRF2 depletion in ES cells activates a totipotent-like two-cell-stage transcriptional program that includes high levels of ZSCAN4. We show that the upregulation of ZSCAN4 contributes to telomere protection in the absence of TRF2. Together, our results uncover a unique response to telomere deprotection during early development.

FOXK1 Participates in DNA Damage Response by Controlling 53BP1 Function.

53BP1 plays a central role in dictating DNA repair choice between non-homologous end joining (NHEJ) and homologous recombination (HR), which is important for the sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPis) of BRCA1-deficient cancers. In this study, we show that FOXK1 associates with 53BP1 and regulates 53BP1-dependent functions. FOXK1-53BP1 interaction is significantly enhanced upon DNA damage during the S phase in an ATM/CHK2-dependent manner, which reduces the association of 53BP1 with its downstream factors RIF1 and PTIP. Depletion of FOXK1 impairs DNA repair and induces compromised cell survival upon DNA damage. Overexpression of FOXK1 diminishes 53BP1 foci formation, which leads to resistance to PARPis and elevation of HR in BRCA1-deficient cells and decreased telomere fusion in TRF2-depleted cells. Collectively, our findings demonstrate that FOXK1 negatively regulates 53BP1 function by inhibiting 53BP1 localization to sites of DNA damage, which alters the DSB-induced protein complexes centering on 53BP1 and thus influences DNA repair choice.

Persistent telomere cohesion protects aged cells from premature senescence.

Human telomeres are bound by the telomere repeat binding proteins TRF1 and TRF2. Telomere shortening in human cells leads to a DNA damage response that signals replicative senescence. While insufficient loading of TRF2 at shortened telomeres contributes to the DNA damage response in senescence, the contribution of TRF1 to senescence induction has not been determined. Here we show that counter to TRF2 deficiency-mediated induction of DNA damage, TRF1 deficiency serves a protective role to limit induction of DNA damage induced by subtelomere recombination. Shortened telomeres recruit insufficient TRF1 and as a consequence inadequate tankyrase 1 to resolve sister telomere cohesion. Our findings suggest that the persistent cohesion protects short telomeres from inappropriate recombination. Ultimately, in the final division, telomeres are no longer able to maintain cohesion and subtelomere copying ensues. Thus, the gradual loss of TRF1 and concomitant persistent cohesion that occurs with telomere shortening ensures a measured approach to replicative senescence.

Endothelial progeria induces adipose tissue senescence and impairs insulin sensitivity through senescence associated secretory phenotype.

Vascular senescence is thought to play a crucial role in an ageing-associated decline of organ functions; however, whether vascular senescence is causally implicated in age-related disease remains unclear. Here we show that endothelial cell (EC) senescence induces metabolic disorders through the senescence-associated secretory phenotype. Senescence-messaging secretomes from senescent ECs induced a senescence-like state and reduced insulin receptor substrate-1 in adipocytes, which thereby impaired insulin signaling. We generated EC-specific progeroid mice that overexpressed the dominant negative form of telomeric repeat-binding factor 2 under the control of the Tie2 promoter. EC-specific progeria impaired systemic metabolic health in mice in association with adipose tissue dysfunction even while consuming normal chow. Notably, shared circulation with EC-specific progeroid mice by parabiosis sufficiently transmitted the metabolic disorders into wild-type recipient mice. Our data provides direct evidence that EC senescence impairs systemic metabolic health, and thus establishes EC senescence as a bona fide risk for age-related metabolic disease.

TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment.

The telomeric protein TRF2 is overexpressed in several human malignancies and contributes to tumorigenesis even though the molecular mechanism is not completely understood. By using a high-throughput approach based on the multiplexed Luminex X-MAP technology, we demonstrated that TRF2 dramatically affects VEGF-A level in the secretome of cancer cells, promoting endothelial cell-differentiation and angiogenesis. The pro-angiogenic effect of TRF2 is independent from its role in telomere capping. Instead, TRF2 binding to a distal regulatory element promotes the expression of SULF2, an endoglucosamine-6-sulfatase that impairs the VEGF-A association to the plasma membrane by inducing post-synthetic modification of heparan sulfate proteoglycans (HSPGs). Finally, we addressed the clinical relevance of our findings showing that TRF2/SULF2 expression is a worse prognostic biomarker in colorectal cancer (CRC) patients.

Induced Trf2 deletion leads to aging vascular phenotype in mice associated with arterial telomere uncapping, senescence signaling, and oxidative stress.

Age-related vascular dysfunction in large elastic and resistance arteries is associated with reductions in microvascular perfusion and elevations in blood pressure. Recent evidence indicates that telomere uncapping-induced senescence in vascular cells may be an important source of oxidative stress and vascular dysfunction in aging, but the causal relationship between these processes has yet to be elucidated. To test this important unexplored hypothesis, we measured arterial senescence signaling and oxidative stress, carotid and mesenteric artery endothelium-dependent vasodilatory capacity, markers of mesenteric microvascular perfusion and endothelial glycocalyx deterioration, and blood pressure in a novel mouse model of Cre-inducible whole body Trf2 deletion and telomere uncapping. Trf2 deletion led to a 320% increase in arterial senescence signaling (P < .05). There was a concurrent 29% and 22% reduction in peak endothelium-dependent vasodilation in carotid and mesenteric arteries, respectively, as well as a 63% reduction in mesenteric microvascular endothelial glycocalyx thickness (all P ≤ .01). Mesenteric microvascular perfusion was reduced by 8% and systolic blood pressure was increased by 9% following Trf2 deletion (both P < .05). Trf2 deletion also led to a pro-oxidative arterial phenotype characterized by increased in NADPH oxidase gene expression; a 210% increase in superoxide levels that was partly dependent on NADPH oxidase activity; and an oxidative stress mediated reduction in carotid artery vasodilation (all P ≤ .05). Collectively, our findings demonstrate that induced Trf2 deletion leads to telomere uncapping, increased senescence signaling, and oxidative stress mediated functional impairments in the vasculature similar to those seen in human aging.

The role of telomere binding molecules for normal and abnormal hematopoiesis.

In order to maintain the homeostasis of the hematopoietic system, hematopoietic stem cells (HSCs) need to be maintained while slowly dividing over their lifetime. However, repeated cell divisions lead to the gradual accumulation of DNA damage and ultimately impair HSC function. Since telomeres are particularly fragile when subjected to replication stress, cells have several defense machinery to protect telomeres. Moreover, HSCs must protect their genome against possible DNA damage, while maintaining telomere length. A group of proteins called the shelterin complex are deeply involved in this two-way role, and it is highly resistant to the replication stress to which HSCs are subjected. Most shelterin-deficient experimental models suffer acute cytotoxicity and severe phenotypes, as each shelterin component is essential for telomere protection. The Tin2 point mutant mice show a dyskeratosis congenita (DC) like phenotype, and the Tpp1 deletion impairs the hematopoietic system. POT1/Pot1a is highly expressed in HSCs and contributes to the maintenance of the HSC pool during in vitro culture. Here, we discuss the role of shelterin molecules in HSC regulation and review current understanding of how these are regulated in the maintenance of the HSC pool and the development of hematological disorders.

Cell death during crisis is mediated by mitotic telomere deprotection.

Tumour formation is blocked by two barriers: replicative senescence and crisis. Senescence is triggered by short telomeres and is bypassed by disruption of tumour-suppressive pathways. After senescence bypass, cells undergo crisis, during which almost all of the cells in the population die. Cells that escape crisis harbour unstable genomes and other parameters of transformation. The mechanism of cell death during crisis remains unexplained. Here we show that human cells in crisis undergo spontaneous mitotic arrest, resulting in death during mitosis or in the following cell cycle. This phenotype is induced by loss of p53 function, and is suppressed by telomerase overexpression. Telomere fusions triggered mitotic arrest in p53-compromised non-crisis cells, indicating that such fusions are the underlying cause of cell death. Exacerbation of mitotic telomere deprotection by partial TRF2 (also known as TERF2) knockdown increased the ratio of cells that died during mitotic arrest and sensitized cancer cells to mitotic poisons. We propose a crisis pathway wherein chromosome fusions induce mitotic arrest, resulting in mitotic telomere deprotection and cell death, thereby eliminating precancerous cells from the population.

Knockdown of telomeric repeat binding factor 2 enhances tumor radiosensitivity regardless of telomerase status.

PURPOSE: To investigate the effects of TRF2 depletion on radiosensitivity in both the telomerase-positive cell lines (A549) and alternative lengthening of telomere (ALT) cell lines (U2OS). METHODS: X-ray irradiation was used to establish two radioresistant cancer models (A549R and U2OSR) from A549 and U2OS. Colony formation assay was applied to examine the radiosensitivity of radioresistant A549R and U2OSR cells and TRF2 low-expression cells. Real-time PCR and TeloTAGGG Telomerase PCR ELISA Kit were performed to examine telomere length and telomerase activity separately. γ-H2AX was detected by immunofluorescence to assess the radiation-induced DSBs. RESULTS: Radioresistant cancer models were established, in which TRF2 was significantly over-expressed. Low expression of TRF2 protein could enhance the radiosensitivity and induce telomere length of A549 and U2OS cell shortening. In A549 cells with TRF2 down-regulated, the telomerase activity was inhibited, too. TRF2 deficiency increases γ-H2AX foci and fails to protect telomere from radiation. CONCLUSION: The data suggest that TRF2 is a radioresistant protein in A549 and U2OS cells, and could potentially be a target for radiosensitization of both telomerase-positive and ALT cells in radiotherapy.

Role of 53BP1 oligomerization in regulating double-strand break repair.

Tumor suppressor p53-binding protein 1 (53BP1) regulates the repair of dysfunctional telomeres lacking the shelterin protein TRF2 by promoting their mobility, their nonhomologous end-joining (NHEJ), and, as we show here, by blocking 5' resection by CtIP. We report that these functions of 53BP1 required its N-terminal ATM/ATR target sites and its association with H4K20diMe, but not the BRCT domain, the GAR domain, or the binding of 53BP1 to dynein. A mutant lacking the oligomerization domain (53BP1(oligo)) was only modestly impaired in promoting NHEJ of dysfunctional telomeres and showed no defect with regard to the repression of CtIP. This 53BP1(oligo) allele was previously found to be unable to support class switch recombination or to promote radial chromosome formation in PARP1 inhibitor-treated Brca1-deficient cells. The data therefore support two conclusions. First, the requirements for 53BP1 in mediating NHEJ at dysfunctional telomeres and in class switch recombination are not identical. Second, 53BP1-dependent repression of CtIP at double-strand breaks (DSBs) is unlikely to be sufficient for the generation of radial chromosomes in PARP1 inhibitor-treated Brca1-deficient cells.

Multiple roles for MRE11 at uncapped telomeres.

Progressive telomere attrition or uncapping of the shelterin complex elicits a DNA damage response as a result of a cell's inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. Telomere deprotection activates both ataxia telangiectasia mutated (ATM) and telangiectasia and Rad3-related (ATR) kinase-dependent DNA damage response pathways, and promotes efficient non-homologous end-joining (NHEJ) of dysfunctional telomeres. The mammalian MRE11-RAD50-NBS1 (MRN; NBS1 is also known as NBN) complex interacts with ATM to sense chromosomal double-strand breaks and coordinate global DNA damage responses. Although the MRN complex accumulates at dysfunctional telomeres, it is not known whether mammalian MRN promotes repair at these sites. Here we address this question by using mouse alleles that either inactivate the entire MRN complex or eliminate only the nuclease activities of MRE11 (ref. 8). We show that cells lacking MRN do not activate ATM when telomeric repeat binding factor 2 (TRF2) is removed from telomeres, and ligase 4 (LIG4)-dependent chromosome end-to-end fusions are markedly reduced. Residual chromatid fusions involve only telomeres generated by leading strand synthesis. Notably, although cells deficient for MRE11 nuclease activity efficiently activate ATM and recruit 53BP1 (also known as TP53BP1) to deprotected telomeres, the 3' telomeric overhang persists to prevent NHEJ-mediated chromosomal fusions. Removal of shelterin proteins that protect the 3' overhang in the setting of MRE11 nuclease deficiency restores LIG4-dependent chromosome fusions. Our data indicate a critical role for the MRN complex in sensing dysfunctional telomeres, and show that in the absence of TRF2, MRE11 nuclease activity removes the 3' telomeric overhang to promote chromosome fusions. MRE11 can also protect newly replicated leading strand telomeres from NHEJ by promoting 5' strand resection to generate POT1a-TPP1-bound 3' overhangs.

53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility.

Double-strand breaks activate the ataxia telangiectasia mutated (ATM) kinase, which promotes the accumulation of DNA damage factors in the chromatin surrounding the break. The functional significance of the resulting DNA damage foci is poorly understood. Here we show that 53BP1 (also known as TRP53BP1), a component of DNA damage foci, changes the dynamic behaviour of chromatin to promote DNA repair. We used conditional deletion of the shelterin component TRF2 (also known as TERF2) from mouse cells (TRF2(fl/-)) to deprotect telomeres, which, like double-strand breaks, activate the ATM kinase, accumulate 53BP1 and are processed by non-homologous end joining (NHEJ). Deletion of TRF2 from 53BP1-deficient cells established that NHEJ of dysfunctional telomeres is strongly dependent on the binding of 53BP1 to damaged chromosome ends. To address the mechanism by which 53BP1 promotes NHEJ, we used time-lapse microscopy to measure telomere dynamics before and after their deprotection. Imaging showed that deprotected telomeres are more mobile and sample larger territories within the nucleus. This change in chromatin dynamics was dependent on 53BP1 and ATM but did not require a functional NHEJ pathway. We propose that the binding of 53BP1 near DNA breaks changes the dynamic behaviour of the local chromatin, thereby facilitating NHEJ repair reactions that involve distant sites, including joining of dysfunctional telomeres and AID (also known as AICDA)-induced breaks in immunoglobulin class-switch recombination.

WRN controls formation of extrachromosomal telomeric circles and is required for TRF2DeltaB-mediated telomere shortening.

Telomere dysfunction has been proposed to contribute to the pathogenesis of Werner syndrome (WS), a premature-aging disorder. The WS protein WRN binds TRF2, a telomere-specific factor that protects chromosome ends. TRF2 possesses an amino-terminal domain that plays an essential role in preventing telomere shortening, as expression of TRF2(DeltaB), which lacks this domain, leads to the formation of telomeric circles, telomere shortening, and cell senescence. Our data show that the TRF2(DeltaB)-induced telomeric-loop homologous-recombination pathway requires WRN helicase. In addition, we show that WRN represses the formation of spontaneous telomeric circles, as demonstrated by the increased levels of telomeric circles observed in telomerase-positive WS fibroblasts. The mechanism of circle formation in WS cells does not involve XRCC3 function. Circle formation in WS cells is reduced by reconstitution with wild-type WRN but not mutant forms lacking either exonuclease or helicase activity, demonstrating that both enzymatic activities of WRN are required to suppress telomeric-circle formation in normal cells expressing telomerase reverse transcriptase. Thus, WRN has a key protective function at telomeres which influences telomere topology and inhibits accelerated attrition of telomeres.

Lack of TRF2 in ALT cells causes PML-dependent p53 activation and loss of telomeric DNA.

Alternative lengthening of telomere (ALT) tumors maintain telomeres by a telomerase-independent mechanism and are characterized by a nuclear structure called the ALT-associated PML body (APB). TRF2 is a component of a telomeric DNA/protein complex called shelterin. However, TRF2 function in ALT cells remains elusive. In telomerase-positive tumor cells, TRF2 inactivation results in telomere de-protection, activation of ATM, and consequent induction of p53-dependent apoptosis. We show that in ALT cells this sequence of events is different. First, TRF2 inactivation/silencing does not induce cell death in p53-proficient ALT cells, but rather triggers cellular senescence. Second, ATM is constitutively activated in ALT cells and colocalizes with TRF2 into APBs. However, it is only following TRF2 silencing that the ATM target p53 is activated. In this context, PML is indispensable for p53-dependent p21 induction. Finally, we find a substantial loss of telomeric DNA upon stable TRF2 knockdown in ALT cells. Overall, we provide insight into the functional consequences of shelterin alterations in ALT cells.

Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1.

When telomeres are rendered dysfunctional through replicative attrition of the telomeric DNA or by inhibition of shelterin, cells show the hallmarks of ataxia telangiectasia mutated (ATM) kinase signalling. In addition, dysfunctional telomeres might induce an ATM-independent pathway, such as ataxia telangiectasia and Rad3-related (ATR) kinase signalling, as indicated by the phosphorylation of the ATR target CHK1 in senescent cells and the response of ATM-deficient cells to telomere dysfunction. However, because telomere attrition is accompanied by secondary DNA damage, it has remained unclear whether there is an ATM-independent pathway for the detection of damaged telomeres. Here we show that damaged mammalian telomeres can activate both ATM and ATR and address the mechanism by which the shelterin complex represses these two important DNA damage signalling pathways. We analysed the telomere damage response on depletion of either or both of the shelterin proteins telomeric repeat binding factor 2 (TRF2) and protection of telomeres 1 (POT1) from cells lacking ATM and/or ATR kinase signalling. The data indicate that TRF2 and POT1 act independently to repress these two DNA damage response pathways. TRF2 represses ATM, whereas POT1 prevents activation of ATR. Unexpectedly, we found that either ATM or ATR signalling is required for efficient non-homologous end-joining of dysfunctional telomeres. The results reveal how mammalian telomeres use multiple mechanisms to avoid DNA damage surveillance and provide an explanation for the induction of replicative senescence and genome instability by shortened telomeres.

MDC1 accelerates nonhomologous end-joining of dysfunctional telomeres.

Here we document the role of MDC1 (mediator of DNA damage checkpoint 1) in the detection and repair of human and mouse telomeres rendered dysfunctional through inhibition of TRF2. Consistent with its role in promoting DNA damage foci, MDC1 knockdown affected the formation of telomere dysfunction-induced foci (TIFs), diminishing the accumulation of phosphorylated ATM, 53BP1, Nbs1, and to a lesser extent, gamma-H2AX. In addition to this effect on TIFs, the rate of nonhomologous end-joining (NHEJ) of dysfunctional telomeres was significantly decreased when MDC1 itself or its recruitment to chromatin was inhibited. MDC1 appeared to promote a step in the NHEJ pathway after the removal of the 3' telomeric overhang. The acceleration of NHEJ was unlikely to be due to increased presence of 53BP1 and Mre11 in TIFs, since knockdown of neither factor affected telomere fusions. Furthermore, relevant cell cycle effectors (Chk2, p53, and p21) of the ATM kinase pathway were unaffected and there was no change in the rate of cell cycle progression. We propose that the binding of MDC1 to gamma-H2AX directly affects NHEJ in a manner that is independent of the ATM-dependent cell cycle arrest pathway.