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Final Report Summary - TELOSCREEN (How spontaneous telomeric fusions occur: unravelling new pathways required for end protection)

Maintaining chromosome ends (known as telomeres) at constant length is a critical process for the cell. If telomeres become critically short, the DNA damage machinery is activated resulting in cell cycle arrest and end-joining DNA repair. In contrast, if they become too long, they become unstable and recombinogenic, leading to sudden telomere loss events. Telomerase, a highly evolutionary conserved reverse transcriptase, is responsible for adding specific repetitive sequences to chromosome ends, thus compensating the cell’s inability to fully replicate the tips of chromosomes. However, how telomere elongation is regulated to avoid constant elongation and therefore unstable telomeres after telomerase activation remains poorly understood.
Making use of the S. pombe as a model organism and carrying out two independent genetic screens, we identified the phosphatase Ssu72 as a regulator of telomerase in fission yeast and higher eukaryotes. First, we used a state-of-the-art plasmid assay developed by the Ferreira’s laboratory combined with transposon-based genetic screen in order to unravel new proteins required for telomere homeostasis. In this screen found Ssu72 as a promoter of telomere fusions in a wild-type unperturbed system. Second, making use of the S. pombe whole genome gene deletion library, we identified the phosphatase Ssu72 as a regulator of telomere length.
Ssu72 is a highly conserved phosphatase previously identified in as an RNA polymerase II C-terminal domain phosphatase from yeast to human. Other roles and targets have been described for this phosphatase, but none of the previous studies described telomere homeostasis defects. Thus we decided to study the role of this phosphatase in telomere regulation.
Both ssu72∆ and ssu72-C13S (phosphatase-dead mutant) exhibit 3-5x longer telomeres than WT cells. Telomere length in ssu72Δ mutants is both trt1+ and rad3+-dependent, consistent with a role as a negative regulator of telomerase. Interestingly, ssu72Δ mutants showed defects in Stn1 recruitment, part of the (C)ST ssDNA-binding complex that promotes lagging strand synthesis and telomerase inhibition. Therefore, we hypothesize that ssu72Δ mutants have longer telomeres due to a defect in lagging strand fill-in reaction. ssu72Δ mutants exhibit longer G-rich overhangs and their telomere defects are genetically epistatic both with stn1 and polα. Consistently, we show that Polα overexpression rescues the telomere length defect of ssu72Δ. ssu72∆ mutants also undergo checkpoint activation and DNA damage responses emanating from telomeres, as measured by live imaging foci co-localization. Overall, our data delineates an unexpected role for Ssu72 in controlling lagging-strand synthesis, and therefore, reducing telomere ssDNA and inhibiting telomerase recruitment.
In agreement with the conserved role of Ssu72 throughout evolution, we observed that human cells down-regulated for SSU72 expression undergo telomere elongation. Similar to fission yeast, depletion of SSU72 in different cancer cell lines also shows checkpoint activation and Telomere induce foci (TIF). Thus, we foresee that these results will make key contributions to our understanding of telomere regulation. Studying how telomere dysfunction arises and identifying new pathways required for telomere length regulation provide insights on how a normal cell can become tumorigenic, thus greatly impacting on our knowledge of cancer development mechanisms.

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