HIRA protects ALT telomeres from aberrant R-loop accumulation

The Alternative Lengthening of Telomeres (ALT) pathway is a homology-directed repair (HDR) mechanism of telomere extension that enables the proliferation of several aggressive cancers. Recurrent mutations inactivate the ATRX (alpha-thalassemia/mental retardation, X-linked)–DAXX (death domain associated protein) chromatin remodeling complex, which coordinates histone H3.3 deposition at telomeres. Recently, we showed that HIRA, the remaining H3.3 chaperone, adapts to compensate for ATRX loss by maintaining telomeric chromatin to sustain ALT cancer cell survival. However, the specific mechanisms underlying HIRA's ability to rescue ALT telomeres from the repercussions of ATRX loss remain unclear. Here, we demonstrate that the HIRA-UBN1/2 complex mediates new H3.3 deposition, limiting the accumulation of unchromatinized, single-stranded DNA (ssDNA) presence at telomeres following ALT-associated HDR. HIRA adapts to maintain H3.3 levels but cannot entirely prevent R-loops, leading to collisions between RNA polymerase II and the ALT replisome and unwarranted replication stress. Furthermore, we show that Chk1-mediated H3.3 S31 phosphorylation is crucial for suppressing aberrant ssDNA accumulation and maintaining the integrity of telomeric chromatin. These studies suggest that the deposition of new histone H3.3 maintains productive ALT, and HIRA ensures ALT cell survival by limiting TERRA R-loop formation. This implicates HIRA in TERRA R-loop homeostasis, which we propose is essential in ensuring ALT cancer cell survival following ATRX loss.