ALT induction by ASF1 depletion is associated with TERRA accumulation at telomeres and relies on R-loops

The mechanism of Alternative Lengthening of Telomeres (ALT) relies on homologous recombination processes taking place at telomeres within ALT-associated PML bodies, or APBs. ALT cells express high levels of the telomeric RNA TERRA, which sustains telomere recombination through R-loop formation. Yet, the molecular mechanisms of ALT activation remain undefined. Here we report that during ALT establishment in telomerase-positive HeLa cells by depletion of the histone chaperone ASF1, TERRA accumulates at telomeres, while its levels remain unchanged, independently of the overall telomere length of the cells. TERRA-colocalizing telomeres display DNA damage and engage in APB formation, suggesting a direct involvement of telomeric TERRA molecules in ALT activation. Consistent with this scenario, we report that ASF1 depletion leads to accumulation of telomeric R-loops, and expression of the RNase H1 enzyme, degrading R-loops, impairs the establishment of ALT. Yet, R-loop accumulation per se is not sufficient to induce ALT. Our study provides novel insights into the mechanisms involved in ALT activation, revealing the critical role of TERRA and R-loops in this process triggered by ASF1 depletion.