Our long-term goal is to understand the fundamental mechanisms used by human cells to maintain genome integrity and how failures in these processes contribute to human disease. Currently we are using our expertise in the field of telomere biology to study specifically how changes in telomere structure relate to cell growth arrest in healthy cells and conversely how changes in telomere structure cooperate with the loss of tumor suppressors to promote genomic instability and cancer. As our research base and technical capacity grows, we anticipate using our ability to modulate telomere biology as a model to study fundamental mechanisms of genome protection in human cells.
A critical leap in the understanding of telomere biology was our discovery that telomere protection and deprotection during the normal course of cellular ageing progresses through three distinct stages. In young cells with elongated telomeres, chromosome ends are arranged into a protective “closed-state” structure (putatively a t-loop) that sequesters the chromosome end away from the DNA damage response machinery. As telomeres shorten, the capacity to form this protective state diminishes, and some chromosome ends become exposed as “intermediate-state” telomeres. This elicits ATM activity and stimulates the DNA damage response. However, retention of the telomere protein TRF2 at intermediate-state telomeres prevents end-to-end fusion of DNA damage response positive chromosome ends. Our data indicate that passage of five intermediate-state telomeres through mitosis into a G1-phase cell is sufficient to induce p53-dependent growth arrest at the first proliferative barrier of replicative senescence. These data also indicate that telomere deprotection in aged cells is an epigenetic response capable of being passed between cell cycles and cellular generations.
When p53 function is lost, cell division may continue at the cost of telomere shortening until insufficient amounts of TRF2 remain bound to the chromosome end to inhibit end-to-end chromosome fusions. This presumably occurs normally due to telomere shortening removing all TRF2 binding sites at a chromosome end but can be reproduced experimentally by disrupting TRF2 function. Once insufficient TRF2 remains bound at a chromosome end, the result is a fusogenic “uncapped-state” telomere that can become covalently ligated to another uncapped-state telomere to form multi-centromeric chromosomes. Subsequent mitosis in cells with multi-centric chromosomes can then drive genomic instability through what is known as a breakage-fusion-bridge cycle. The appearance of uncapped-state telomeres corresponds with onset of the second proliferative barrier termed crisis, during which time most of the cells in the culture will die. However, in a small fraction of cells the genomic instability at crisis is accompanied by the activation of a mechanism to elongate and maintain telomere length. If this happens, the cell escapes crisis by acquiring the ability to proliferate indefinitely and is now rapidly moving towards oncogencic transformation.
Our recent discoveries have centered on understanding the role of intermediate-state telomeres in human telomere biology. Prior to our discovery of intermediate-state telomeres it was thought that chromosome ends adopted either a protected or unprotected state. What we can now surmise is that the unique properties of intermediate-state provide telomeres with their tumor-suppressive and genome protective qualities.
Unlike broken genomic DNA, intermediate-state telomeres do not induce growth arrest at the G2/M boundary. They are instead passed through cell division and arrest growth in G1-phase. This facilitates growth arrest in aged cells with shortened telomeres at the most stable phase of the cell cycle. The critical separation of DNA damage response activation from the process of DNA repair at intermediate-state telomeres also allows a signal initiating cell growth arrest to be activated and maintained without the consequence of multicentromeric chromosomes and genomic instability. Intermediate-state telomeres also induce a differential DNA damage response where the ATM kinase only activates a subset of targets: a situation unique to the telomeres. The specific G1-phase growth arrest following telomere deprotection also explains why loss of p53 is critical for oncogenesis. Because p53 regulates G1-arrest, and deprotected telomeres do not induce G2/M arrest, once p53 is lost in precancerous cells the deprotected telomeres will longer induce cell growth arrest but will instead be passed between cell cycles and onto daughter cells ad infinitum.
Now that we understand the presence of the critical intermediate-state of telomere deprotection we can begin to establish the molecular framework underlying the telomere-dependent phenomena of cellular ageing, tumor suppression and genomic instability
Our current research expands on these discoveries as follows:
Understanding the molecular basis for telomere deprotection in ageing and cancer
Using human cell culture systems we have established the three-state model of telomere protection. We are now focusing on broadening our understanding of telomere biology by establishing a comprehensive model connecting changes in telomere structure to biological outcomes in vivo.
Deciphering the kinomics of the telomere deprotection response
The observation of differential ATM activity induced by intermediate-state telomeres was an exciting and surprising finding. We are now working with the ACRF Centre for Kinomics at CMRI to understand how spontaneous telomere deprotection is signaled in cancer cells, how differential ATM activation in response to intermediate-state telomeres is regulated and which downstream ATM pathways are upregulated in response to intermediate-state telomeres.
Understanding the telomere deprotection response in the context of the cell cycle
Our observations of deprotected telomeres are consistent with epigenetic marks that are capable of being passed between cell cycles and across cell division to regulate growth arrest specifically in the G1-phase of the cell cycle. We are focusing on understanding the epigientic pathways that allow the passage of DNA damage response positive telomeres across mitosis, the effect deprotected telomeres have on cell division, and putative effects telomere deprotection may have on unexpected areas of cell biology.