Telomeres are protective structures that preserve genome integrity and stability by preventing chromosome end degradation and misrecognition as DNA damage sites. Dysregulation of telomere structure or length maintenance can have drastic consequences on overall telomere stability, often leading to premature aging diseases or the development of cancer. Though the structural and functional components of telomeres, including the telomere-binding protein complex shelterin, have been well-established, the role of chromatin in maintaining telomere stability remains elusive. Here we investigate the potential role of constitutive heterochromatin using a protein-tethering approach to enrich features of heterochromatin specifically at telomeres. Our results show that telomeric enrichment of the heterochromatin-associated trimethylation of histone 3 on Lysine 9, H3K9me3, does not result in substantial telomere deprotection. Conversely, enrichment of heterochromatin protein 1 (HP1α), of a different heterochromatin feature, produces significant DNA damage and telomere dysfunction. Using fluorescent microscopy techniques, we measured telomere length, replication stress, and levels of telomeric DNA damage after heterochromatin enrichment at telomeres. We demonstrate that cells with telomeric enrichment of HP1α exhibit increased DNA damage at telomeres, diminished telomere length, as well as bridging of telomeres between multiple nuclei, indicative of telomere entanglements. These phenotypes suggest that heterochromatin is detrimental, rather than protective, to normal telomere function. Interestingly, a subset of cancer cells, , which maintain their telomeres through a mechanism called Alternative Lengthening of Telomeres (ALT), rely on HP1a for viability and are known to have high levels of heterochromatin, replication stress, recombination, and DNA damage at their telomeres. The observed telomere dysfunction in non-ALT cells upon HP1a enrichment at telomeres may provide insight into the role of HP1α in the ALT pathway and may thus inform the future development of anti-cancer drug therapies.
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