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Telomere metabolism in Genome Stability and Disease

Periodic Reporting for period 2 - TelMetab (Telomere metabolism in Genome Stability and Disease)

Reporting period: 2019-03-01 to 2020-08-31

Our genetic information encoded in DNA is packaged into linear chromosomes in cells. The work proposed in this application focuses on telomeres a highly specialised and essential structure that protects DNA at the ends of chromosomes. Defects in maintaining telomeres results in several debilitating human diseases including Dyskeratosis Congenita and Hoyeraal-Hreidarrson Syndrome. Telomeres also progressively shorten with each cell division, eventually stopping the cell from growing. A specially adapted enzyme called telomerase is able to extend and maintain telomeres in stem cells, which is very important for tissue repair and renewal. In normal cells, telomerase expression is lost as cells commit to a specific fate. However, re-expression of telomerase is a major route to cancer as this allows the cells to divide indefinitely. Hence, telomeres must be subject to exquisite regulation to maintain their normal function. My laboratory has a long-standing interest in telomeres and our efforts in this important area of research has led to the discovery of new genes that function in telomere maintenance and are linked to human disease. Recently, we my laboratory has developed sensitive methods that allow us to determine the protein composition of telomeres under different conditions. This proposal is divided into two complementary objective that will 1) determine in detail how RTEL1 functions in telomere maintenance and how this is compromised by RTEL1 mutations that cause Hoyeraal-Hreidarrson Syndrome, and 2) we will identify new telomere binding proteins and will determine how they function in telomere maintenance and human disease. Our proposal will lead to a greater understanding of the causes/consequences of telomere dysfunction, the factors that mitigate these effects to maintain telomere homeostasis and how these processes are compromised in human diseases.
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).
The discovery that TRF2 a key protein required for telomere end protection in differentiated cells is dispensable in stem cells, is beyond state of the art and entirely unexpected. There is no doubt that this discovery will have a major impact on our understanding of telomere end protection. We are also very encouraged by the preliminary data with the three newly identified telomere binding proteins and anticipate completing the CRISPR screen to identify new proteins important for ALT telomere maintenance. Finally, our discovery that KSHV infection can induces the sustained acquisition of ALT in culture is a major break-through, which will allow us to investigate the mechanisms responsible for the induction and maintenance of ALT - an important telomere maintenance process in 10-15% of all cancers.
The illustration depicts the goal of the TelMetab proposal. Our approach is to define the proteomic