The termini of eukaryotic chromosomes are potentially dangerous sites, as their resemblance to damage-induced DNA breaks makes them vulnerable to degradation and end-joining pathways that provoke cancer. Telomeres protect chromosome ends from these hazards, and solve the 'DNA end replication problem'- the inability of DNA polymerases to fully duplicate the ends of linear molecules - by engaging telomerase, a reverse transcriptase that replenishes telomere repeats lost during replication. We study the spectrum and mechanisms of telomere function. Human stem cells express telomerase, insufficient levels of which cause diseases reflecting stem cell failure. In contrast, the lack of telomerase expression in most somatic cells limits cellular lifespan and contributes to human aging. To become immortal, cancer cells must avert critical telomere shortening by activating telomerase or an alternative telomere maintenance pathway. Hence, while loss of telomere function promotes early tumorigenesis, the genomic instability that stems from telomere loss would eventually halt cellular growth, making telomeres intriguing universal targets for anti-cancer therapy. Fission yeast telomeres are remarkably similar to those of human but present substantial experimental benefits, like precise genetic manipulability. The components of human 'shelterin'are also found in fission yeast and we are building an integrated picture of how these proteins interact to protect chromosome ends. We have also identified unforeseen additional functions of telomeres that are likely to be widely conserved. Advances over the past year include: Replication fork obstruction through telomeres as a key challenge, and a trigger for telomerase: Having shown previously that 'naked'telomeric repeats impede replication fork (RF) progression while telomeres bound by the telomere binding protein Taz1 experience smooth RF progression, we found that stalled telomeric RFs trigger chromosome breakage and entanglement. We now find that the conserved Rif1 protein plays a crucial role in controlling the final resolution of such entanglements at mitosis, thus uncovering an unanticipated facet (and time of action) of Rif1's activities and illuminating a final, regulated step of chromosome segregation. Homeostatic mechanisms ensure that telomere lengths are maintained within a set range, preventing individual telomeres from sporadically triggering cell death, but the bases for these homeostatic mechanisms are largely mysterious. We found that Taz1 enforces both temporal and spatial control of formation of the telomerase substrate (a 3'overhang) and telomerase activity. Along with our studies of semi-conservative telomere replication, these results suggest that stalled telomeric RFs confer the preferential recruitment of telomerase to short telomeres. Survival without telomeres Cells can occasionally survive the absence of telomerase, by maintaining telomeres via recombination or by circularizing their chromosomes. We had identified a third mode of telomerase-minus survival in which linear chromosomes are maintained using a strategy we dubbed 'HAATI'(heterochromatin amplification-mediated and telomerase independent). In HAATI cells, telomere repeats are absent but tracts of 'generic'heterochromatin jump to each chromosome end. This heterochromatin, along with a non-telomeric terminal 3'-overhang, recruits Pot1, which is essential for HAATI chromosome linearity. This discovery revealed an alternative mode by which cancer cells might survive without telomerase activation. We have now found that HAATI formation is limited only by the chromosome rearrangements that place generic heterochromatin at HAATI chromosome ends, and that this 'jumping'of DNA sequences is controlled by the RNAi pathway. We also have uncovered a role for specific subnuclear domain positioning in HAATI maintenance. Surprising functions for telomeres during meiosis The spindles that form at mitosis and meiosis are often thought of as semi-autonomous architectural structures that control the movement of chromosomes. Our recent findings overturned this notion by revealing that telomeres, which gather together near the centrosome in early stages of meiosis to form the highly conserved 'bouquet', control both the formation of meiotic spindles and the attachment of chromosomes, via their centromeres, to those spindles. These observations raise exciting and novel questions about mechanisms by which chromosomes control cell cycle progression during both meiosis and mitosis. We now find that centromeres and telomeres share the ability to control spindle formation. Most excitingly, we find that the domain surrounding the telomere bouquet constitutes a nuclear microenvironment conducive to centromere assembly. Moreover, we find that centromeres are prone to dismantling during meiosis, making this telomeric environment crucial for centromere assembly and proper meiotic chromosome segregation. These observations have potential implications not only for cancers that activate meiotic chromosome maintenance pathways, but also for our understanding of fertility.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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National Cancer Institute Division of Basic Sciences
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Klutstein, Michael; Cooper, Julia Promisel (2014) The Chromosomal Courtship Dance-homolog pairing in early meiosis. Curr Opin Cell Biol 26:123-31
Tomita, Kazunori; Bez, Cecile; Fennell, Alex et al. (2013) A single internal telomere tract ensures meiotic spindle formation. EMBO Rep 14:252-60