PROJECT SUMMARY Telomeres are nucleoprotein structures that protect the ends of linear chromosomes and thereby maintain genome stability. Telomeres solve both the end-protection and the end-replication problems: 1) They inhibit DNA damage at chromosome ends, which would otherwise resemble broken DNA, 2) Since chromosome ends shorten during replication, telomeres act as buffer sequences to prevent loss of coding regions, 3) Once telomeres become too short, they can no longer inhibit DNA damage, leading to permanent cell cycle arrest (senescence). This ?mitotic clock? is a critical tumor-suppressive barrier that forces aging cells to stop dividing. To become cancerous, cells must acquire unlimited division potential by activating a telomere maintenance mechanism, either reactivation of telomerase, the enzyme that elongates telomeres during development, or through the alternative lengthening of telomeres (ALT) mechanism, which is based on recombination. Telomeres consist of 5-15kb of (TTAGGG)n repeats organized into tightly packed nucleosomes and bound by the shelterin, a complex of six non-histone proteins. Telomeres are considered as heterochromatin and are enriched in the repressive H3K9me3 ?histone mark?. Intense focus has been placed on trying to decipher the exact chromatin status of telomeres, but the much more important question has been neglected and remains unanswered: What is the role and function of heterochromatin at telomeres? While the roles of shelterin proteins have been extensively studied, the function of heterochromatin at telomeres remains largely unexplored. Using a novel approach to locally and specifically modulate histone methylation at telomeres, we will thoroughly dissect the function of H3K9me3 in telomere protection and maintenance. By fusing histone modifying enzymes to the shelterin protein TRF1, we can locally enrich or deplete H3K9me3 at telomeres. Our preliminary data revealed that loss of H3K9me3 leads to severe replication defects and de-repression of telomere transcription. These data suggest that heterochromatin could play unanticipated roles in the regulation of replicative aging and the onset of senescence. Moreover, while the general consensus is that ALT is associated with less condensed chromatin at telomeres, we found that H3K9me3 is a driver of ALT activity. Using this unique approach to manipulate H3K9 trimethylation at telomeres, we will methodically determine the function of this heterochromatin mark on the protective properties of telomeres (end- protection, end-replication, entry into senescence) as well as on the ALT mechanism of telomere maintenance.

R35GM143108

2021-08-01

2026-05-31