The Role of End Synapsis Factors During Telomere Dysfunction - Proliferative barriers are key tumor suppressive mechanisms and includes cellular senescence and replicative crisis. Telomere dysfunction (e.g., telomere shortening or telomere deprotection) plays a critical role in such barriers, such that cells with disrupted telomeric ends can induce cellular senescence. If cells bypass senescence, they enter the second proliferative barrier called replicative crisis. Many cells succumb to cell death at this stage, however cells that survive crisis are prone to malignant transformation. Therefore, characterizing the mechanisms behind telomere dysfunction is significant to give insight into the factors that drive cancer initiation and progression. This proposal will focus on understanding aspects of telomere dysfunction and replicative crisis, including the mechanisms behind telomere fusions and how they affect cellular survival during replicative crisis. Aim 1: Define the role of DSB DNA end synapsis factors during telomere fusions. Telomere fusions are a marker of telomere dysfunction and are mediated by the DNA double strand (DSB) repair pathways Canonical Non-homologous end joining (C-NHEJ) or the Alternative End Joining (Alt-EJ). A key aspect of DNA DSB repair is the synapsis of two DNA ends, however the mechanisms of DNA end synapsis for telomere fusions remain poorly understood. Recent work has uncovered a possible crosstalk between C-NHEJ and Alt-EJ for telomere fusions, and I posit that preparation of DNA DSB ends for EJ repair by both pathways involves synapsis via DNA-PKcs, XLF, or 53BP1 to promote telomere fusions. Furthermore, I posit that these synapsis factors may show close interactions with factors in the Alt-EJ pathway within the cell. In performing this research, I will expand my scientific skill set in several ways, including learning microscopy methods to assess EJ outcomes by examining types of telomere fusions. I will also use protein-protein association assays to assess interaction between C-NHEJ and Alt-EJ factors. Lastly, I will also examine how loss of end synapsis affects DNA end resection by assessing DNA-PKcs-bound DNA fragments at DSBs since a key step in Alt-EJ is initiation of end resection. Aim 2: Define the role of telomere fusions for genomic instability during replicative crisis. The goal of this aim is to understand the mechanisms of replicative crisis, as this is a crucial tumor suppressive proliferation barrier. Cell death during crisis has been shown to be mediated by the proinflammatory cGAS/STING pathway and activation of autophagy. Telomere fusions are a marker of cells undergoing crisis and have been shown to activate the cGAS/STING pathway. Therefore, I hypothesize suppression of end synapsis, which causes telomere fusions, can lead to decreased cGAS/STING activation and allow for cells to continue to replicate. To test my hypothesis, I will use a replicative crisis cell line model and suppress telomere fusions by depletion of end synapsis factors. With this model, I will examine telomere dysfunction, inflammatory and autophagy markers, as well as cell survival. Overall, my scientific training at the Salk Institute in the Karlseder laboratory will provide key skills and a strong foundation for my career as an independent researcher.
