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.     

DNA damage foci at dysfunctional telomeres

We report cytologic and genetic data indicating that telomere dysfunction induces a DNA damage response in mammalian cells. Dysfunctional, uncapped telomeres, created through inhibition of TRF2, became associated with DNA damage response factors, such as 53BP1, gamma-H2AX, Rad17, ATM, and Mre11. We refer to the domain of telomere-associated DNA damage factors as a Telomere Dysfunction-Induced Focus (TIF). The accumulation of 53BP1 on uncapped telomeres was reduced in the presence of the PI3 kinase inhibitors caffeine and wortmannin, which affect ATM, ATR, and DNA-PK. By contrast, Mre11 TIFs were resistant to caffeine, consistent with previous findings on the Mre11 response to ionizing radiation. A-T cells had a diminished 53BP1 TIF response, indicating that the ATM kinase is a major transducer of this pathway. However, in the absence of ATM, TRF2 inhibition still induced TIFs and senescence, pointing to a second ATM-independent pathway. We conclude that the cellular response to telomere dysfunction is governed by proteins that also control the DNA damage response. TIFs represent a new tool for evaluating telomere status in normal and malignant cells suspected of harboring dysfunctional telomeres. Furthermore, induction of TIFs through TRF2 inhibition provides an opportunity to study the DNA damage response within the context of well-defined, physically marked lesions.     

TRF2 dysfunction elicits DNA damage responses associated with senescence in proliferating neural cells and differentiation of neurons

Telomeres are specialized structures at the ends of chromosomes that consist of tandem repeats of the DNA sequence TTAGGG and several proteins that protect the DNA and regulate the plasticity of the telomeres. The telomere-associated protein TRF2 (telomeric repeat binding factor 2) is critical for the control of telomere structure and function; TRF2 dysfunction results in the exposure of the telomere ends and activation of ATM (ataxia telangiectasin mutated)-mediated DNA damage response. Recent findings suggest that telomere attrition can cause senescence or apoptosis of mitotic cells, but the function of telomeres in differentiated neurons is unknown. Here, we examined the impact of telomere dysfunction via TRF2 inhibition in neurons (primary embryonic hippocampal neurons) and mitotic neural cells (astrocytes and neuroblastoma cells). We demonstrate that telomere dysfunction induced by adenovirus-mediated expression of dominant-negative TRF2 (DN-TRF2) triggers a DNA damage response involving the formation of nuclear foci containing phosphorylated histone H2AX and activated ATM in each cell type. In mitotic neural cells DN-TRF2 induced activation of both p53 and p21 and senescence (as indicated by an up-regulation of beta-galactosidase). In contrast, in neurons DN-TRF2 increased p21, but neither p53 nor beta-galactosidase was induced. In addition, TRF2 inhibition enhanced the morphological, molecular and biophysical differentiation of hippocampal neurons. These findings demonstrate divergent molecular and physiological responses to telomere dysfunction in mitotic neural cells and neurons, indicate a role for TRF2 in regulating neuronal differentiation, and suggest a potential therapeutic application of inhibition of TRF2 function in the treatment of neural tumors.     

No overt nucleosome eviction at deprotected telomeres

Dysfunctional telomeres elicit the canonical DNA damage response, which includes the activation of the ATM or ATR kinase signaling pathways and end processing by nonhomologous end joining (NHEJ) or homologous recombination (HR). The cellular response to DNA double-strand breaks has been proposed to involve chromatin remodeling and nucleosome eviction, but whether dysfunctional telomeres undergo chromatin reorganization is not known. Here, we report on the nucleosomal organization of telomeres that have become deprotected through the deletion of the shelterin components TRF2 or POT1. We found no evidence of changes in the nucleosomal organization of the telomeric chromatin or nucleosome eviction near the telomere terminus. An unaltered chromatin structure was observed at telomeres lacking TRF2, which activate the ATM kinase and are a substrate for NHEJ. Similarly, telomeres lacking POT1a and POT1b, which activate the ATR kinase, showed no overt nucleosome eviction. Finally, telomeres lacking TRF2 and Ku70, which are processed by HR, appeared to maintain their original nucleosomal organization. We conclude that ATM signaling, ATR signaling, NHEJ, and HR at deprotected telomeres can take place in the absence of overt nucleosome eviction.     

53BP1 deficiency combined with telomere dysfunction activates ATR-dependent DNA damage response

TRF1 protects mammalian telomeres from fusion and fragility. Depletion of TRF1 leads to telomere fusions as well as accumulation of γ-H2AX foci and activation of both the ataxia telangiectasia mutated (ATM)- and the ataxia telangiectasia and Rad3 related (ATR)-mediated deoxyribonucleic acid (DNA) damage response (DDR) pathways. 53BP1, which is also present at dysfunctional telomeres, is a target of ATM that accumulates at DNA double-strand breaks and favors nonhomologous end-joining (NHEJ) repair over ATM-dependent resection and homology-directed repair (homologous recombination [HR]). To address the role of 53BP1 at dysfunctional telomeres, we generated mice lacking TRF1 and 53BP1. 53BP1 deficiency significantly rescued telomere fusions in mouse embryonic fibroblasts (MEFs) lacking TRF1, but they showed evidence of a switch from the NHEJ- to HR-mediated repair of uncapped telomeres. Concomitantly, double-mutant MEFs showed evidence of hyperactivation of the ATR-dependent DDR. In intact mice, combined 53BP1/TRF1 deficiency in stratified epithelia resulted in earlier onset of DNA damage and increased CHK1 phosphorylation during embryonic development, leading to aggravation of skin phenotypes.     

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.     

The radiomimetic enediyne C-1027 induces unusual DNA damage responses to double-strand breaks

Cells lacking the protein kinase ataxia telangiectasia mutated (ATM) have defective responses to DNA double-strand breaks (DSBs), including an inability to activate damage response proteins such as p53. However, we previously showed that cells lacking ATM robustly activate p53 in response to DNA strand breaks induced by the radiomimetic enediyne C-1027. To gain insight into the nature of C-1027-induced ATM-independent damage responses to DNA DSBs, we further examined the molecular mechanisms underlying the cellular response to this unique radiomimetic agent. Like ionizing radiation (IR) and other radiomimetics, breaks induced by C-1027 efficiently activate ATM by phosphorylation at Ser1981, yet unlike other radiomimetics and IR, DNA breaks induced by C-1027 result in normal phosphorylation of p53 and the cell cycle checkpoint kinases (Chk1 and Chk2) in the absence of ATM. In the presence of ATM, but under ATM and Rad3-related kinase (ATR) deficient conditions, C-1027 treatment resulted in a decrease in the level of Chk1 phosphorylation but not in the level of p53 and Chk2 phosphorylation. Only when cells were deficient in both ATM and ATR was there a reduction in the level of phosphorylation of each of these DNA damage response proteins. This reduction was also accompanied by an increased level of cell death in comparison to that of wild-type cells or cells lacking either ATM or ATR. Our findings demonstrate a unique cellular response to C-1027-induced DNA DSBs in that DNA damage response proteins are unaffected by the absence of ATM, as long as ATR is present.     

Stabilization of quadruplex DNA perturbs telomere replication leading to the activation of an ATR-dependent ATM signaling pathway

Functional telomeres are required to maintain the replicative ability of cancer cells and represent putative targets for G-quadruplex (G4) ligands. Here, we show that the pentacyclic acridinium salt RHPS4, one of the most effective and selective G4 ligands, triggers damages in cells traversing S phase by interfering with telomere replication. Indeed, we found that RHPS4 markedly reduced BrdU incorporation at telomeres and altered the dynamic association of the telomeric proteins TRF1, TRF2 and POT1, leading to chromosome aberrations such as telomere fusions and telomere doublets. Analysis of the molecular damage pathway revealed that RHPS4 induced an ATR-dependent ATM signaling that plays a functional role in the cellular response to RHPS4 treatment. We propose that RHPS4, by stabilizing G4 DNA at telomeres, impairs fork progression and/or telomere processing resulting in telomere dysfunction and activation of a replication stress response pathway. The detailed understanding of the molecular mode of action of this class of compounds makes them attractive tools to understand telomere biology and provides the basis for a rational use of G4 ligands for the therapy of cancer.     

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 critical role for Pin2/TRF1 in ATM-dependent regulation. Inhibition of Pin2/TRF1 function complements telomere shortening, radiosensitivity, and the G(2)/M checkpoint defect of ataxia-telangiectasia cells.

Cells derived from patients with the human genetic disorder ataxia-telangiectasia (A-T) display many abnormalities, including telomere shortening, premature senescence, and defects in the activation of S phase and G(2)/M checkpoints in response to double-strand DNA breaks induced by ionizing radiation. We have previously demonstrated that one of the ATM substrates is Pin2/TRF1, a telomeric protein that binds the potent telomerase inhibitor PinX1, negatively regulates telomere elongation, and specifically affects mitotic progression. Following DNA damage, ATM phosphorylates Pin2/TRF1 and suppresses its ability to induce abortive mitosis and apoptosis (Kishi, S., Zhou, X. Z., Nakamura, N., Ziv, Y., Khoo, C., Hill, D. E., Shiloh, Y., and Lu, K. P. (2001) J. Biol. Chem. 276, 29282-29291). However, the functional importance of Pin2/TRF1 in mediating ATM-dependent regulation remains to be established. To address this question, we directly inhibited the function of endogenous Pin2/TRF1 in A-T cells by stable expression of two different dominant-negative Pin2/TRF1 mutants and then examined their effects on telomere length and DNA damage response. Both the Pin2/TRF1 mutants increased telomere length in A-T cells, as shown in other cells. Surprisingly, both the Pin2/TRF1 mutants reduced radiosensitivity and complemented the G(2)/M checkpoint defect without inhibiting Cdc2 activity in A-T cells. In contrast, neither of the Pin2/TRF1 mutants corrected the S phase checkpoint defect in the same cells. These results indicate that inhibition of Pin2/TRF1 in A-T cells is able to bypass the requirement for ATM in specifically restoring telomere shortening, the G(2)/M checkpoint defect, and radiosensitivity and demonstrate a critical role for Pin2/TRF1 in the ATM-dependent regulation of telomeres and DNA damage response.     

ATM and Chk2-dependent phosphorylation of MDMX contribute to p53 activation after DNA damage

The p53 tumor suppressor is activated after DNA damage to maintain genomic stability and prevent transformation. Rapid activation of p53 by ionizing radiation is dependent on signaling by the ATM kinase. MDM2 and MDMX are important p53 regulators and logical targets for stress signals. We found that DNA damage induces ATM-dependent phosphorylation and degradation of MDMX. Phosphorylated MDMX is selectively bound and degraded by MDM2 preceding p53 accumulation and activation. Reduction of MDMX level by RNAi enhances p53 response to DNA damage. Loss of ATM prevents MDMX degradation and p53 stabilization after DNA damage. Phosphorylation of MDMX on S342, S367, and S403 were detected by mass spectrometric analysis, with the first two sites confirmed by phosphopeptide-specific antibodies. Mutation of MDMX on S342, S367, and S403 each confers partial resistance to MDM2-mediated ubiquitination and degradation. Phosphorylation of S342 and S367 in vivo require the Chk2 kinase. Chk2 also stimulates MDMX ubiquitination and degradation by MDM2. Therefore, the E3 ligase activity of MDM2 is redirected to MDMX after DNA damage and contributes to p53 activation.     

Differential roles of ATM- and Chk2-mediated phosphorylations of Hdmx in response to DNA damage

The p53 tumor suppressor plays a major role in maintaining genomic stability. Its activation and stabilization in response to double strand breaks (DSBs) in DNA are regulated primarily by the ATM protein kinase. ATM mediates several posttranslational modifications on p53 itself, as well as phosphorylation of p53's essential inhibitors, Hdm2 and Hdmx. Recently we showed that ATM- and Hdm2-dependent ubiquitination and subsequent degradation of Hdmx following DSB induction are mediated by phosphorylation of Hdmx on S403, S367, and S342, with S403 being targeted directly by ATM. Here we show that S367 phosphorylation is mediated by the Chk2 protein kinase, a downstream kinase of ATM. This phosphorylation, which is important for subsequent Hdmx ubiquitination and degradation, creates a binding site for 14-3-3 proteins which controls nuclear accumulation of Hdmx following DSBs. Phosphorylation of S342 also contributed to optimal 14-3-3 interaction and nuclear accumulation of Hdmx, but phosphorylation of S403 did not. Our data indicate that binding of a 14-3-3 dimer and subsequent nuclear accumulation are essential steps toward degradation of p53's inhibitor, Hdmx, in response to DNA damage. These results demonstrate a sophisticated control by ATM of a target protein, Hdmx, which itself is one of several ATM targets in the ATM-p53 axis of the DNA damage response.     

S1219 residue of 53BP1 is phosphorylated by ATM kinase upon DNA damage and required for proper execution of DNA damage response

53BP1 is phosphorylated by the protein kinase ATM upon DNA damage. Even though several ATM phosphorylation sites in 53BP1 have been reported, those sites have little functional implications in the DNA damage response. Here, we show that ATM phosphorylates the S1219 residue of 53BP1 in vitro and that the residue is phosphorylated in cells exposed to ionizing radiation (IR). Transfection with siRNA targeting ATM abolished IR-induced phosphorylation at this residue, supporting the theory that this process is mediated by the kinase. To determine the functional relevance of this phosphorylation event, a U2OS cell line expressing S1219A mutant 53BP1 was established. IR-induced foci formation of MDC1 and γH2AX, DNA damage signaling molecules, was reduced in this cell line, implying that S1219 phosphorylation is required for recruitment of these molecules to DNA damage sites. Furthermore, overexpression of the mutant protein impeded IR-induced G2 arrest. In conclusion, we have shown that S1219 phosphorylation by ATM is required for proper execution of DNA damage response.     

ATM activation is accompanied with earlier stages of prostate tumorigenesis

The ATM (ataxia telangiectasia mutated) kinase plays an essential role in maintaining genome integrity by coordinating cell cycle arrest, apoptosis, and DNA damage repair. Phosphorylation of ATM at serine 1981 (ATMpSer1981) by DNA damage activates ATM, which subsequently phosphorylates H2AX Ser139 (gammaH2AX), Chk2 Thr68 (Chk2pThr68), and p53 Ser15 (p53pSer15). To determine the role of the ATM pathway in prostate cancer tumorigenesis, we have analyzed 35 primary prostate cancer specimens for ATMpSer1981 (ATM activation), Chk2pThr68, gammaH2AX, and p53pSer15 by immunohistochemistry (IHC) in normal glands, prostatic intraepithelial neoplasias (PINs), and carcinomas. Increases in the intensities of ATMpSer1981, Chk2pThr68, and gammaH2AX and in the percentage of cells that are positive for ATMpSer1981, Chk2pThr68, or gammaH2AX were observed in PINs (p<0.001) compared to normal prostatic glands and carcinoma. However, this pattern of immunostaining was not seen for p53pSer15. Thus, ATM and Chk2 are specifically activated in PINs. As PINs are generally regarded as precursors of prostatic carcinoma, our results suggest that ATM and Chk2 activation at earlier stages of prostate tumorigenesis suppresses tumor progression, with attenuation of ATM activation leading to cancer progression.     

Involvement of novel autophosphorylation sites in ATM activation

ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.     

Aven-dependent activation of ATM following DNA damage

BACKGROUND: In response to DNA damage, cells undergo either cell-cycle arrest or apoptosis, depending on the extent of damage and the cell's capacity for DNA repair. Cell-cycle arrest induced by double-stranded DNA breaks depends on activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell-cycle effectors such as Chk2 and p53 to inhibit cell-cycle progression. ATM is recruited to double-stranded DNA breaks by a complex of sensor proteins, including Mre11/Rad50/Nbs1, resulting in autophosphorylation, monomerization, and activation of ATM kinase. RESULTS: In characterizing Aven protein, a previously reported apoptotic inhibitor, we have found that Aven can function as an ATM activator to inhibit G2/M progression. Aven bound to ATM and Aven overexpressed in cycling Xenopus egg extracts prevented mitotic entry and induced phosphorylation of ATM and its substrates. Immunodepletion of endogenous Aven allowed mitotic entry even in the presence of damaged DNA, and RNAi-mediated knockdown of Aven in human cells prevented autophosphorylation of ATM at an activating site (S1981) in response to DNA damage. Interestingly, Aven is also a substrate of the ATM kinase. Mutation of ATM-mediated phosphorylation sites on Aven reduced its ability to activate ATM, suggesting that Aven activation of ATM after DNA damage is enhanced by ATM-mediated Aven phosphorylation. CONCLUSIONS: These results identify Aven as a new ATM activator and describe a positive feedback loop operating between Aven and ATM. In aggregate, these findings place Aven, a known apoptotic inhibitor, as a critical transducer of the DNA-damage signal.     

PARP1 Is a TRF2-associated poly(ADP-ribose)polymerase and protects eroded telomeres

Poly(ADP-ribose)polymerase 1 (PARP1) is well characterized for its role in base excision repair (BER), where it is activated by and binds to DNA breaks and catalyzes the poly(ADP-ribosyl)ation of several substrates involved in DNA damage repair. Here we demonstrate that PARP1 associates with telomere repeat binding factor 2 (TRF2) and is capable of poly(ADP-ribosyl)ation of TRF2, which affects binding of TRF2 to telomeric DNA. Immunostaining of interphase cells or metaphase spreads shows that PARP1 is detected sporadically at normal telomeres, but it appears preferentially at eroded telomeres caused by telomerase deficiency or damaged telomeres induced by DNA-damaging reagents. Although PARP1 is dispensable in the capping of normal telomeres, Parp1 deficiency leads to an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA in primary murine cells after induction of DNA damage. Our results suggest that upon DNA damage, PARP1 is recruited to damaged telomeres, where it can help protect telomeres against chromosome end-to-end fusions and genomic instability.