During the last reporting period we were able to show that telomerase aberrantly accumulates at telomeres in the absence of RTEL1 and eliminating telomerase or blocking its recruitment to telomeres is sufficient to rescue telomere dysfunction in Rtel1 null cells. We presented evidence that the abnormal association of telomerase with telomeres in these cells corresponds to its binding to single-ended DSBs generated at reversed replication forks that form as a consequence of persistent t-loops or unresolved telomeric G4-DNA structures. Consistent with this conclusion, blocking fork reversal is sufficient to rescue telomere dysfunction in Rtel1-/- cells, whereas inhibiting the restart of reversed replication forks mimics the toxic effects of telomerase. These data revealled an unappreciated source of critically short telomeres that results from the aberrant binding and stabilization of reversed replication forks by telomerase (Margalef et al, Cell 2018). We also identified a CDK phosphorylation site in TRF2 (Ser365), whose dephosphorylation in S-phase by the PP6C/R3 phosphatase provides a narrow window during which the helicase RTEL1 is able to transiently unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 on Ser365 outside of S-phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate ATM activation, but also counteracts replication conflicts at DNA secondary structures arising within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response (Sarek et al., Nature 2019). AIM2: identifying novel telomere maintenance mechanisms: Our recent work has challenged the current dogma of chromosome end protection through the unexpected discovery that TRF2 is entirely dispensable for end protection in embryonic stem cells (ESCs) and the pluripotent compartment of early mouse development. Importantly, we confirmed that TRF2 is required for end protection in somatic cells, but found that ESCs without TRF2 are viable, proliferate normally and their telomeres remain free from fusions. Similarly, Trf2 deficiency mouse embryos survive until the late blastula transition at a time when lineage specification begins. Indeed, we showed that upon differentiation and loss of pluripotency, cells rapidly switch to become reliant on TRF2 to prevent fusions and maintain viability. We further demonstrated that DNA repair by NHEJ is functional in ESCs and telomeres in pluripotent cells form T-loops both in the presence and absence of TRF2, suggesting the existence of an alternative mechanism of T-loop formation/stabilization in ESCs. Since in somatic cells TRF2 and T-loops are inextricably linked, it has never previously been possible to conclusively demonstrate that T-loops themselves can maintain end protection in the absence of TRF2. Our findings provide the first conclusive evidence that looped telomeres without TRF2 protect chromosomes ends. Furthermore, the retention of telomere protection in the presence of T-loops, but absence of TRF2, confirms a long-suspected view that T-loops are a key mediator of telomere protection irrespectively of how they form. Phil’s work predicts the presence of a developmental switch upon exit from pluripotency that transitions T-loops from forming independently of TRF2 to being reliant on TRF2 for their formation/stabilization (Ruis et al., Nature, In press; Ruis & Boulton, Genes & Dev, In Press). How this developmental switch is controlled, how T-loops form and are stabilized without TRF2, whether this alternative mechanism occurs in other contexts, and why it has evolved are open questions we are currently investigating. Using PICh methods we have identified three new telomere associated proteins, which we are characterising using established methods in the lab. Finally, we have recently discovered that infection with Kaposi’s sarcoma herpesvirus (KSHV) induces sustained acquisition of ALT telomere maintenance in previously non-ALT/telomerase positive cell lines. KSHV-infected cells acquire an ALT-associated telomeric proteome and molecular hallmarks of ALT activity that are also observed in KSHV-associated patient tumour biopsies. We have further shown that down-regulating BIR impairs KSHV latency, suggesting that KSHV induces and then co-opts ALT as part of its own life cycle. This study uncovers KSHV infection as a means to induce telomere maintenance by ALT and reveals features of ALT in KSHV-associated cancer (Lippert et al., Nature Comms, In revision).
