Local heterochromatin enrichment promotes telomere clustering and PML nuclear body assembly at telomeres

• Telomeric heterochromatin promotes telomere clustering and APB formation in ALT-positive cells • HP1α tethering to TRF1 is sufficient to induce PML nuclear body formation at telomeres • ATRX and DAXX inhibit heterochromatin-driven telomere nuclear body assembly Telomeric heterochromatin promotes telomere clustering and APB formation in ALT-positive cells HP1α tethering to TRF1 is sufficient to induce PML nuclear body formation at telomeres ATRX and DAXX inhibit heterochromatin-driven telomere nuclear body assembly The alternative lengthening of telomeres (ALT) pathway is a recombination-based telomere maintenance mechanism used by a subset of human cancers and is characterized by telomere clustering within telomere-associated promyelocytic leukemia (PML) nuclear bodies (APBs). Although ALT telomeres exhibit reduced nucleosome density, they are paradoxically enriched for heterochromatin-associated factors, raising questions about how chromatin state contributes to ALT. Here, we use a targeted system to locally modulate heterochromatin features at telomeres. We show that telomeric heterochromatin promotes telomere clustering and multiple hallmarks of APB-associated telomere processing in ALT-positive (ALT+) cells. Remarkably, molecular tethering of HP1α at telomeres is sufficient to nucleate PML nuclear bodies in non-ALT cells and, in specific contexts, induce biomarkers of ALT-like recombination. We further demonstrate that heterochromatin-driven PML-telomere colocalization is inhibited by α-thalassemia/mental retardation, X-linked and death domain-associated protein (ATRX/DAXX), factors frequently mutated in ALT+ tumors. Together, these findings establish telomeric heterochromatin as a driver of telomere clustering and PML nuclear body assembly, shaping ALT-associated subnuclear compartmentalization.