HIRA protects telomeres against R-loop-induced instability in ALT cancer cells

Inactivating mutations in chromatin modifiers, like the α-thalassemia/mental retardation, X-linked (ATRX)-death domain-associated protein (DAXX) chromatin remodeling/histone H3.3 deposition complex, drive the cancer-specific alternative lengthening of telomeres (ALT) pathway. Prior studies revealed that HIRA, another histone H3.3 chaperone, compensates for ATRX-DAXX loss at telomeres to sustain ALT cancer cell survival. How HIRA rescues telomeres from the consequences of ATRX-DAXX deficiency remains unclear. Here, using an assay for transposase-accessible chromatin using sequencing (ATAC-seq) and cleavage under targets and release using nuclease (CUT&RUN), we establish that HIRA-mediated deposition of new H3.3 maintains telomeric chromatin accessibility to prevent the detrimental accumulation of nucleosome-free single-stranded DNA (ssDNA) in ATRX-DAXX-deficient ALT cells. We show that the HIRA-UBN1/UBN2 complex deposits new H3.3 to prevent TERRA R-loop buildup and transcription-replication conflicts (TRCs) at telomeres. Furthermore, HIRA-mediated H3.3 incorporation into telomeric chromatin links productive ALT to the phosphorylation of serine 31, an H3.3-specific amino acid, by Chk1. Therefore, we identify a critical role for HIRA-mediated H3.3 deposition that ensures the survival of ATRX-DAXX-deficient ALT cancer cells. [Display omitted] •HIRA establishes greater telomeric chromatin accessibility after ATRX-DAXX loss•Deposition of new H3.3 by HIRA-UBN restricts telomeric ssDNA and TERRA R-loops•Unresolved TERRA R-loops block new H3.3 deposition by HIRA-UBN•CHK1 phosphorylation of H3.3 is critical to prevent ssDNA and TERRA R-loop buildup Lynskey et al. report links between HIRA-mediated histone H3.3 deposition and R-loop homeostasis at ALT telomeres. In most ALT cancer cells, after ATRX-DAXX is inactivated, HIRA regulates telomeric chromatin assembly. HIRA limits ssDNA accumulation and mitigates out-of-control R-loop formation and transcription-replication conflicts at telomeres, threatening cancer cell survival.