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. ALT+ cancers cells are characterized by clustering of telomeres into ALT-associated PML bodies (APBs) that serve as platforms for telomere recombination. ALT+ telomeres also display singular chromatin, with a lower nucleosome density and loss of the ATRX/DAXX pathway that normally deposits H3.3 at telomeres. We found, however, that H3K9 trimethylation at telomeres promotes formation of APBs, as well as subsequent ALT activity. We propose here to determine which chromatin regulators as well as DNA damage and repair genes modulate APB formation in ALT+ cells.