The ends of eukaryotic linear chromosomes are potentially dangerous sites, as their resemblance to damage-induced DNA double strand breaks (DSBs) makes them vulnerable to degradation and end-joining pathways. If left unchecked at chromosome ends, these pathways cause chromosome shortening and rearrangement, which in turn provoke cancer. Telomeres protect chromosome ends from these dangerous events. We study the components of telomeres, the spectrum and mechanisms of telomere function, and the events that follow telomere loss.
Telomeres also solve the 'DNA end replication problem' - the inability of DNA polymerases to fully replicate the ends of linear DNA molecules. Telomeres solve this problem by engaging telomerase, a specialised reverse transcriptase whose internal RNA subunit templates synthesis of telomere repeats. The field of telomere biology was recognised this year by the Nobel Prize in Physiology or Medicine, which was awarded to Elizabeth Blackburn, Carol Greider and Jack Szostak for their discovery of telomeres and telomerase.
In humans, telomerase is expressed in stem cells and germ cells but not most somatic cells. However, tumor cells must activate telomerase or an alternative telomere maintenance pathway. While loss of telomere function promotes early tumorigenesis, the genomic instability that stems from telomere loss is incompatible with longterm cellular survival. Therefore, regeneration of telomeres is critical for the eventual 'immortalisation' of cancer cells and suggests intriguing universal targets for anti-cancer therapy.
Fission yeast telomeres are remarkably similar to human telomeres but present substantial experimental benefits, like easy and precise genetic manipulation. Taz1, the only known ortholog of human TRF1 and TRF2, binds double strand telomeric DNA, regulates numerous telomere functions and has been extensively studied in our lab; other 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. Taz1 prevents DSB repair reactions from acting inappropriately on chromosome ends - loss of Taz1 leads to lethal chromosome end-fusions during G1 when nonhomologous end-joining (NHEJ) activities are elevated but not during G2 when NHEJ levels are low. Hence, telomere dysfunction yields strikingly different outcomes during G1 versus G2, conferring an advantage to studying telomeres in fission yeast, whose mainly G2 cell cycle allows cells lacking Taz1 to grow despite their dysfunctional telomeres.
Cell cycle-dependent control of telomere accessibility and telomerase
Our recent studies shed light on how the telomere complex changes through the cell cycle and regulates telomerase. Taz1 and Rap1 are both required to prevent excessive telomeric elongation, but we find that they play very different roles in controlling telomerase, as Taz1 is required to restrain telomerase activity to Sphase while Rap1 is dispensable for this timing. Furthermore, the unique role of Taz1 in allowing smooth replication fork passage through telomeres confers telomeric breakage in taz1Δ¿cells, in turn enhancing telomerase recruitment. However, telomerase activity is only possible when Pot1 is phosphorylated by a cell cycle controlled kinase. These observations provide a foothold for deciphering the molecular underpinnings of cell cycle control of telomere accessibility to both telomerase and DNA damage response factors.
Figure 1. Model for alternative mode of survival with linear chromosomes that we have observed in the absence of telomeres and telomerase. Blocks of heterochromatin undergo constant recombination, resulting in changeable chromosome ends harbouring non-telomeric heterochromatic DNA repeats. This heterochromatin recruits end protection factors that prevent massive terminal degradation.
Figure 2. The centrosome fails to separate properly upon division in the absence of the meiotic telomere bouquet. This results in the inability of the chromosomes to segregate (see unseparated chromatin mass in the `without bouquet¿ photograph). We are investigating how the telomeres control this process.
Challenges associated with stalled replication forks at telomeres
While telomerase synthesizes the most terminal telomere repeats, the majority of telomere repeats are synthesized by semi-conservative DNA replication. We have shown that stalled replication forks accumulate at telomeres lacking Taz1, in turn leading not only to telomeric breakage but also to entangled chromosome ends that fail to segregate properly. Intriguingly, the activity of the RecQ helicase Rqh1 (ortholog of human Werners Syndrome helicase, whose mutation causes the eponymous premature aging disease) prevents resumption of fork movement through telomeres, triggering telomere loss and entanglement. Furthermore, SUMO modification of Rqh1 is required for its detrimental activity at telomeres. We have also found a non-canonical function for the essential decatenation enzyme Topoisomerase II (Top2) in detangling telomeres, heralding an unforeseen role of Top2 in promoting genome stability.
A newly recognized way to survive without telomeres
When telomeres are lost in telomerase-minus cells, some cells acquire the ability to maintain telomeres via recombination. In addition, fission yeast can survive telomere loss by chromosome circularization. These 'circular strains' are viable but exhibit several conspicuous defects, like slow growth and hypersensitivity to agents that induce DSBs. We have identified a DSB-resistant subclass of telomerase minus cells that survive using a third strategy that we have dubbed `HAATI¿, in which telomere sequences are absent but large blocks of heterochromatin are amplified. This survival mode resembles that found in the fruit fly Drosophila, and may illuminate the universal 'stripped-down' requirements for chromosome end maintenance.
Telomere function during meiosis
When cells progress to the meiotic cell cycle, telomere function changes dramatically. Telomere clustering during early stages of meiosis, or 'bouquet formation', is observed throughout the Eukaryota. Our earlier studies showed that formation of this telomere bouquet depends on Taz1 and is required for successful meiosis. In the absence of the telomere bouquet (e.g. in taz1Δ¿cells), the centrosome fails to divide properly and form a spindle at meiosis I and often dissociates from the nucleus. Thus, the highly conserved bouquet plays an unanticipated role in controlling spindle formation. Our data suggest that an artificial 'tether' between non-telomeric heterochromatin and the meiotic centrosome can confer proper meiotic spindle formation in the absence of a true 'telomere bouquet', and raise the possibility that the bouquet arrangement also affects centromere localization and kinetochore function. We are currently investigating potential mechanisms by which the gathered telomeres control these diverse meiotic events, as well as the relevance of these observations to mitosis.
For a list of refereed research papers, see Publications (in navigation on left).
