Chromosome end protection in stem cells and development

Telomeres have evolved to protect linear chromosome ends, preventing them from degradation and activating DNA damage response (DDR) pathways. Essential for "end protection" is the Shelterin subunit, TRF2, which inhibits the DDR and facilitates T-loop formation to sequester the telomere end, thereby hiding it from promiscuous DNA repair. The essentiality of "end protection" is revealed upon removal of TRF2, which triggers the DDR and rapid telomere-telomere fusions mediated by end joining. While dependency of "end protection" on TRF2 has been seen in numerous somatic cell types, we recently discovered that TRF2 is dispensable for "end protection" in stem cells and early development. Trf2-/- stem cells proliferate normally and remain free from telomere fusions, but rapidly switch to become reliant on TRF2 to prevent end-to-end fusions upon differentiation and loss of pluripotency. These findings challenge current dogma and reveal fundamental differences in "end protection" between pluripotent and somatic cell states. In this ERC proposal, we will exploit genetic, proteomic and super-resolution imaging methods to address how end protection is achieved in pluripotent cells, how this differs from somatic cells, and why different cell states have evolved distinct end protection mechanisms? To further our understanding of telomere maintenance mechanisms, we will also exploit our recent advances in biochemistry and single molecule (SM) biophysics to reconstitute telomeres in vitro. Using fluorescently labelled Shelterin, telomerase and telomere-associated factors, we will interrogate the mechanism of 1) T-loop assembly/disassembly, 2) telomerase recruitment to telomeres, and 3) Shelterin-chromatin interactions at telomeres. Our multi-disciplinary approach will establish the mechanistic basis of telomere end protection in pluripotent cells and will provide unprecedented insight into telomere maintenance mechanisms in real-time and at a SM level.