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Telomeres are nucleoprotein structures at the ends of linear chromosomes composed of 5′-TTAGGG-3′ tandem repeat arrays, bound by histone proteins and the telomere-specific binding protein complex shelterin. In somatic cells, telomeres undergo gradual attrition with each cell division, accompanied by a loss of proliferative capacity and eventual cell death. Cancer cells are required to activate a telomere maintenance mechanism in order to circumnavigate this telomere attrition, and thereby gain unlimited proliferative capacity. This can be accomplished through the activation of the ribonucleoprotein telomerase, or through the activation of a homologous recombination (HR) mediated mechanism known as Alternative Lengthening of Telomeres (ALT). Telomere specific proteins, along with telomeric nucleosomes, cap chromosome ends to prevent them from being recognised as DNA double strand breaks. Cancers that use ALT display elevated levels of telomere-specific DNA damage response (DDR) and aberrant telomeric chromatin, implicating a role for telomere chromatin in maintaining structural integrity. Tandem repeat sequences are characterised as being highly recombinogenic. In order to prevent aberrant telomere recombination events, telomeres are maintained in a heterochromatin-like state, being enriched in the H3K9me3 and H4K20me3 heterochromatin marks. The post-translational methylation of H3K9 is achieved by the histone methyl transferase SUV39 family of proteins, which function to increase chromatin compaction. Alteration of these heterochromatin marks in mice results in the induction of ALT phenotypes. As ALT telomeres rely on a HR mechanism of telomere extension, aberrations in telomeric heterochromatin are believed to mediate HR, and therefore facilitate the growth of ALT cancers. The aim of this thesis was to explore the role of SUV39 proteins, and their heterochromatic mark H3K9me3, in maintaining telomere structural integrity. By modulating the expression of SUV39 proteins in a panel of human cancer cell lines, we were able to investigate whether telomeric chromatin state affected telomere recombination, telomere protection and telomere length maintenance. Through SUV39 depletion studies, we demonstrated that loss of H3K9me3 at telomeres results in exacerbated telomere dysfunction, detected by the association of DDR proteins at telomeres. This result revealed for the first time that heterochromatin state is important to the maintenance of telomere structure. One caveat was that this increase was only observed in cells with an already high basal level of DNA damage, implying that loss of H3K9me3 was only able to perturb telomeres with underlying structural defects. This result was independent of the employed TMM of the cell. Through SUV39 overexpression studies, we demonstrated that increased compaction resulted in a decrease in DDR at telomeres only in ALT cells, suggesting that the structural defects that underlie ALT telomeres can be supressed through increased heterochromatin. Not all ALT cell lines were able to be protected from DDR, suggesting that factors other than heterochromatin mediate the extent of telomere dysfunction at these cancer cell lines. Through depletion of the telomere capping protein, telomere repeat binding protein 2 (TRF2), we revealed that SUV39 overexpression was able to confer a protective effect in certain ALT cells. This reduction in DDR, however, could not be maintained following further telomere deprotection by the depletion of both telomere repeat binding protein 1 (TRF1) and TRF2. These results suggest that while telomeric heterochromatin may have a role in telomere DDR suppression, it is not able to confer a protective role upon exhaustive telomere insult. Overall in this thesis we demonstrated that both the depletion and overexpression of SUV39 proteins did not alter telomere length in cells which utilise either ALT or telomerase-mediated telomere extension. These findings are in direct contrast to heterochromatin-based telomere length studies carried out in both mouse and swine cell lines. Furthermore, we were able to show through chromatin immunoprecipitation studies that the SUV39 family of proteins directly associate with telomeric chromatin, albeit at a very low abundance. This result provides evidence demonstrating that there is a direct interaction between telomeres and SUV39 in human cells, but its association does not regulate telomere length. |
