ATRX loss induces telomere dysfunction and necessitates induction of alternative lengthening of telomeres during human cell immortalization

Loss of the histone H3.3-specific chaperone component ATRXor its partner DAXX frequently occurs in human cancers that employ alternative lengthening of telomeres(ALT) for chromosomal end protection, yet the underlying mechanism remains unclear. Here, we report that ATRX/DAXX does not serve as an immediate repressive switch for ALT. Instead, ATRXor DAXX depletion gradually induces telomereDNA replication dysfunction that activates not only homology-directed DNA repair responses but also cell cycle checkpointcontrol. Mechanistically, we demonstrate that this process is contingent on ATRX/DAXX histone chaperone function, independently of telomerelength. Combined ATAC-seqand telomere chromatin immunoprecipitationstudies reveal that ATRXloss provokes progressive telomeredecondensation that culminates in the inception of persistent telomerereplication dysfunction. We further show that endogenous telomerase activity cannot overcome telomeredysfunction induced by ATRXloss, leaving telomererepair-based ALT as the only viable mechanism for telomeremaintenance during immortalization. Together, these findings implicate ALT activation as an adaptive responseto ATRX/DAXX loss-induced telomerereplication dysfunction.