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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - TelMetab (Telomere metabolism in Genome Stability and Disease)

Teaser

Summary

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.

Work performed

We have made significant developments in the two main aims of the project. AIM1: Function of RTEL1 at telomeres: 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 In revision). AIM2: identifying novel telomere maintenance mechanisms: we have discovered that TRF2 a key protein required for telomere end protection in differentiated cells is dispensable in stem cells. This is a highly unexpected finding, which we are currently investigating. Using PICh methods we have identified three new telomere associated proteins, which are characterising using established methods in the lab. We have also generated a comprehensive network of proteins that are specifically enriched at ALT telomeres and have produced a bespoke CRISPR library to interrogate the function of these proteins in the ALT process. Finally, we have defined how SLX4IP functions at ALT telomeres. We have shown that SLX4IP accumulates specifically at ALT telomeres and interacts with SLX4, XPF and BLM. Loss of SLX4IP results in a hyper-ALT phenotype that is incompatible with cell viability following concomitant loss of SLX4. Inactivation of BLM is sufficient to rescue toxic telomere aggregation and synthetic lethality in this context, suggesting that SLX4IP favours SMX-dependent resolution by antagonising promiscuous BLM activity during ALT recombination. Finally, we show that SLX4IP is inactivated in a subset of ALT-positive osteosarcomas. Collectively, our findings uncover an SLX4IP-dependent regulatory mechanism critical for telomere maintenance in ALT cancer cells (Panier et al., Mol Cell In revision.

Final results

Given the progress we have made since starting the grant (outline above) we anticipate making transformative discoveries over the remaining 4 years. 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 in the coming months.