Telomeres are nucleoprotein structures that protect chromosome ends. Progressive telomeric DNA erosion accompanies cell ageing and eventually compromises chromosome end protection. Cellular age-dependent telomere erosion initiates two separate proliferative boundaries known as senescence and crisis that function as potent tumour suppressor programs. Cancer cells overcome these barriers by activating one of two telomere maintenance mechanisms. Approximately 15% of tumours use the ‘alternative lengthening of telomeres’ (ALT) mechanism to synthesise new telomeric DNA. ALT relies on cell cycle-specific interactions occurring at chromosome ends that have been difficult to discern. We have aimed to elucidate these interactions and determine their effect on ALT activity.
To purify telomere interacting proteins we generated novel proximity biotinylation tools combined with mass spectrometry in ALT-positive U2OS cells. We have enriched and analysed the telomere binding dynamics of 8327 proteins at seven distinct timepoints across the cell cycle (n = 5). This quantitative atlas is the largest dataset of telomere interacting proteins and the only database to include cell cycle specificity. Within these data 59 stringent interactors were identified by permutation-based FDR testing. 24 of these proteins have validated impacts on ALT activity, and the remaining 35 are entirely novel. Using a siRNA panel against these 35 novel targets we assessed the impact of their depletion on two ALT phenotypes. Quantifying the number of ALT-associated PML nuclear bodies by high-throughput microscopy, and extra-chromosomal telomere repeat intensity we have shown that seven of the novel regulators have significant impact on ALT activity.
In conclusion, we have compiled the largest proteomic atlas of the telomere, which has identified seven novel regulators of ALT activity. Our work has expanded the mechanistic insight of ALT activity and may prove to unveil targetable vulnerabilities in ALT-positive cancers.