Telomere length heterogeneity in ALT cells is maintained by PML-dependent localization of the BTR complex to telomeres.

Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by using the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed alternative lengthening of telomeres (ALT). Cells that use ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML bodies (APBs). APBs localize to and cluster telomeric ends together with telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can occur. However, the necessity of APBs and their function in the ALT pathway has remained unclear. Here, we used CRISPR/Cas9 to delete PML and APB components from ALT-positive cells to cleanly define the function of APBs in ALT. We found that PML is required for the ALT mechanism, and that this necessity stems from APBs' role in localizing the BLM-TOP3A-RMI (BTR) complex to ALT telomere ends. Strikingly, recruitment of the BTR complex to telomeres in a PML-independent manner bypasses the need for PML in the ALT pathway, suggesting that BTR localization to telomeres is sufficient to sustain ALT activity.

Caught with One's Zinc Fingers in the Genome Integrity Cookie Jar.

Zinc finger (ZnF) domains are present in at least 5% of human proteins. First characterized as binding to DNA, ZnFs display extraordinary binding plasticity and can bind to RNA, lipids, proteins, and protein post-translational modifications (PTMs). The diverse binding properties of ZnFs have made their functional characterization challenging. While once confined to large and poorly characterized protein families, proteomic, cellular, and molecular studies have begun to shed light on their involvement as protectors of the genome. We focus here on the emergent roles of ZnF domain-containing proteins in promoting genome integrity, including their involvement in telomere maintenance and DNA repair. These findings have highlighted the need for further characterization of ZnF proteins, which can reveal the functions of this large gene class in normal cell function and human diseases, including those involving genome instability such as aging and cancer.

Telomere Replication Stress Induced by POT1 Inactivation Accelerates Tumorigenesis.

Genome sequencing studies have revealed a number of cancer-associated mutations in the telomere-binding factor POT1. Here, we show that when combined with p53 deficiency, depletion of murine POT1a in common lymphoid progenitor cells fosters genetic instability, accelerates the onset, and increases the severity of T cell lymphomas. In parallel, we examined human and mouse cells carrying POT1 mutations found in cutaneous T cell lymphoma (CTCL) patients. Inhibition of POT1 activates ATR-dependent DNA damage signaling and induces telomere fragility, replication fork stalling, and telomere elongation. Our data suggest that these phenotypes are linked to impaired CST (CTC1-STN1-TEN1) function at telomeres. Lastly, we show that proliferation of cancer cells lacking POT1 is enabled by the attenuation of the ATR kinase pathway. These results uncover a role for defective telomere replication during tumorigenesis.

DUB-resistant ubiquitin to survey ubiquitination switches in mammalian cells.

The ubiquitin-modification status of proteins in cells is highly dynamic and maintained by specific ligation machineries (E3 ligases) that tag proteins with ubiquitin or by deubiquitinating enzymes (DUBs) that remove the ubiquitin tag. The development of tools that offset this balance is critical in characterizing signaling pathways that utilize such ubiquitination switches. Herein, we generated a DUB-resistant ubiquitin mutant that is recalcitrant to cleavage by various families of DUBs both in vitro and in mammalian cells. As a proof-of-principle experiment, ectopic expression of the uncleavable ubiquitin stabilized monoubiquitinated PCNA in the absence of DNA damage and also revealed a defect in the clearance of the DNA damage response at unprotected telomeres. Importantly, a proteomic survey using the uncleavable ubiquitin identified ubiquitinated substrates, validating the DUB-resistant ubiquitin expression system as a valuable tool for interrogating cell signaling pathways.

A two-step mechanism for TRF2-mediated chromosome-end protection.

Mammalian telomeres repress DNA-damage activation at natural chromosome ends by recruiting specific inhibitors of the DNA-damage machinery that form a protective complex termed shelterin. Within this complex, TRF2 (also known as TERF2) has a crucial role in end protection through the suppression of ATM activation and the formation of end-to-end chromosome fusions. Here we address the molecular properties of TRF2 that are both necessary and sufficient to protect chromosome ends in mouse embryonic fibroblasts. Our data support a two-step mechanism for TRF2-mediated end protection. First, the dimerization domain of TRF2 is required to inhibit ATM activation, the key initial step involved in the activation of a DNA-damage response (DDR). Next, TRF2 independently suppresses the propagation of DNA-damage signalling downstream of ATM activation. This novel modulation of the DDR at telomeres occurs at the level of the E3 ubiquitin ligase RNF168 (ref. 3). Inhibition of RNF168 at telomeres involves the deubiquitinating enzyme BRCC3 and the ubiquitin ligase UBR5, and is sufficient to suppress chromosome end-to-end fusions. This two-step mechanism for TRF2-mediated end protection helps to explain the apparent paradox of frequent localization of DDR proteins at functional telomeres without concurrent induction of detrimental DNA-repair activities.

Persistent telomere damage induces bypass of mitosis and tetraploidy.

Tetraploidization has been proposed as an intermediate step toward aneuploidy in human cancer but a general mechanism for the induction of tetraploidy during tumorigenesis is lacking. We report that tetraploidization occurs in p53-deficient cells experiencing a prolonged DNA damage signal due to persistent telomere dysfunction. Live-cell imaging revealed that these cells have an extended G2 due to ATM/ATR- and Chk1/Chk2-mediated inhibition of Cdk1/CyclinB and eventually bypass mitosis. Despite their lack of mitosis, the cells showed APC/Cdh1-dependent degradation of the replication inhibitor geminin, followed by accumulation of Cdt1, which is required for origin licensing. Cells then entered a second S phase resulting in whole-genome reduplication and tetraploidy. Upon restoration of telomere protection, these tetraploid cells resumed cell division cycles and proliferated. These observations suggest a general mechanism for the induction of tetraploidization in the early stages of tumorigenesis when telomere dysfunction can result from excessive telomere shortening.

Give me a break: how telomeres suppress the DNA damage response.

Linear organization of the genome requires mechanisms to protect and replicate chromosome ends. To this end eukaryotic cells evolved telomeres, specialized nucleoproteic complexes, and telomerase, the enzyme that maintains the telomeric DNA. Telomeres allow cells to distinguish chromosome ends from sites of DNA damage. In mammalian cells this is accomplished by a protein complex, termed shelterin, that binds to telomeric DNA and is able to shield chromosome ends from the DNA damage machinery. In recent years, we have seen major advances in our understanding of how this protein complex works due to the generation of mouse models carrying mutations of individual shelterin components. This review will focus on our current understanding of how the shelterin complex is able to suppress the DNA damage response pathways, and on the cellular and organismal outcomes of telomere dysfunction.

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.