Telomere Biology

Telomeres make up the ends of linear chromosomes and are composed of TG-rich repetitive DNA. They ensure that the chromosome ends are not recognized as DNA damage, which could result in their aberrant repair and lead to the formation of chromosome end-to-end fusions and in turn, genome instability. Telomeres shorten with each round of cell division due to the ‘end replication problem’. The telomerase enzyme is able to counteract loss by adding telomeric repeats, thereby re-extending the chromosome ends. In spite of this, telomeres shorten following each cell division, and with age, due to insufficient telomerase expression in most human somatic cells. When telomeres become critically short, they activate the DNA Damage Response (DDR), which arrests cell growth and eventually leads to cellular senescence/apoptosis. In this respect telomere shortening acts as a tumor suppressor by limiting the replicative lifespan of a cell. In the absence of a proper checkpoint response, critically short telomeres may lead to chromosomal rearrangements that result in cellular transformation and tumorigenesis. Telomere shortening plays a critical role in the increase in chromosomal abnormalities associated with ageing. Therefore it is important to understand how telomere maintenance is upheld to prevent premature cellular senescence and preserve genome integrity, which constitutes the main priority of our research goals. We are focused on multiple aspects of telomere biology that are relatively new to the field and as a result, poorly characterized. Indeed, we have discovered that checkpoint adaptation, chromatin looping and the non-coding telomere repeat containing RNA (TERRA), have significant influences on telomere dynamics and rates of cellular senescence.

Checkpoint Adaptation

Figure. 1. Telomere dysfunction activates the DNA damage checkpoint leading to cell cycle arrest. In the presence of high nutrient levels, cells adapt and proliferate in the presence of damage which leads to cell death and genome instability. Rapamycin (nutrient deprivation) can prevent adaptation and uphold the checkpoint until repair has been completed (Klermund et al., 2014).

Dysfunctional telomeres result in checkpoint activation, which leads to cell cycle arrest at the G2/M transition. Following prolonged telomere dysfunction cells eventually down-regulate the checkpoint response and progress through mitosis even in the presence of damaged telomeres - a phenomenon referred to as "checkpoint adaptation". We observed that the inhibition of the metabolically regulated TORC1 signalling pathway, through the addition of rapamycin, drastically improves the viability of prolonged telomere dysfunction. We have further shown that the addition of rapamycin prevents checkpoint adaptation and that the inhibition of checkpoint adaptation is required for the viability rescue of telomere capping mutants. Whereas cells with telomere dysfunction eventually pass into G1 and resume the cell cycle, TORC1 inhibition holds the cells for a longer period of time in the G2/M block in a Rad53Chk2 dependent manner (Figure 1). 

We believe these results could have important implications for human pathologies associated with chronic telomere dysfunction (e.g. dyskeratosis congenita) and increased genomic instability as well as for cancer therapy. Further studies will focus on characterizing checkpoint adaptation in yeast and human cells, as well as elucidating the role of adaptation during telomere induced cellular senescence. Moreover, we are trying to understand how rapamycin regulates Cdc5 (PLK1) during checkpoint adaptation. Finally, we will investigate how nutrient regulation can potentially, be exploited for therapeutic purposes.

TERRA (a non-coding TElomere Repeat containing RNA)

Although telomeres are heterochromatic they get transcribed, producing a long non-coding RNA, TERRA. There have been many hypotheses regarding TERRA function, however it remains an unsolved mystery and will require continued investigation. Using chromatin immunoprecipitation (ChIP) directed against RNA-DNA hybrids we found that telomeres were enriched, suggesting that TERRA RNA-DNA hybrids (R-loops) exist at chromosome ends. The number of R-loops at telomeres increases when we deplete RNase H activity from the cell. We found that RNase H depleted cells senesce rapidly and have increased rates of telomere loss in the absence of telomerase and homologous recombination (HR). Importantly, in the presence of HR, telomere lengthening occurred with increasing RNA-DNA hybrids and replicative senescence was delayed, consistent with R-loops being hyper-recombinogenic substrates. These results lead us to our current working model: that in telomerase negative cells TERRA containing R-loops at telomeres lead to either telomere lengthening or shortening depending on the HR status of the cell. Further work on TERRA will focus on trying to understand how telomere transcription is regulated during the senescence process both in terms of R-loop and transcription regulation. In addition, in telomerase positive cells we are trying to understand the mechanistic details regarding how TERRA promotes telomerase function. Finally, we are elucidating the role of TERRA in ALT positive human tumor cells, which exclusively use recombination to maintain their telomere length

Telomere Looping

Telomeric chromatin folds back onto itself and forms a lariat structure, which is conserved from yeast to man. It has been proposed that this structure protects the chromosome ends from degradation; much like the plastic cap on a shoelace protects the lace from fraying. We have recently developed a 3C (Chromatin conformation capture) -based approach to detect the loop structure in budding yeast. Using the method we are exploring how telomere looping is regulated in terms of cell cycle, telomere length and genetic context. Furthermore, we are testing possible links between telomere loops and telomere (TERRA) transcription. Our data suggests that the telomere loop requires a minimal telomere length to be maintained. Indeed, telomeres in telomerase negative pre-senescent and senescent cells are looping defective, which may explain why they are more prone to nucleolytic resection and unscheduled DNA repair events.