Nuclear-receptor-mediated telomere insertion leads to genome instability in ALT cancers.

The breakage-fusion-bridge cycle is a classical mechanism of telomere-driven genome instability in which dysfunctional telomeres are fused to other chromosomal extremities, creating dicentric chromosomes that eventually break at mitosis. Here, we uncover a distinct pathway of telomere-driven genome instability, specifically occurring in cells that maintain telomeres with the alternative lengthening of telomeres mechanism. We show that, in these cells, telomeric DNA is added to multiple discrete sites throughout the genome, corresponding to regions regulated by NR2C/F transcription factors. These proteins drive local telomere DNA addition by recruiting telomeric chromatin. This mechanism, which we name targeted telomere insertion (TTI), generates potential common fragile sites that destabilize the genome. We propose that TTI driven by NR2C/F proteins contributes to the formation of complex karyotypes in ALT tumors.

TRF2-independent chromosome end protection during pluripotency.

Mammalian telomeres protect chromosome ends from aberrant DNA repair1. TRF2, a component of the telomere-specific shelterin protein complex, facilitates end protection through sequestration of the terminal telomere repeat sequence within a lariat T-loop structure2,3. Deleting TRF2 (also known as TERF2) in somatic cells abolishes T-loop formation, which coincides with telomere deprotection, chromosome end-to-end fusions and inviability3-9. Here we establish that, by contrast, TRF2 is largely dispensable for telomere protection in mouse pluripotent embryonic stem (ES) and epiblast stem cells. ES cell telomeres devoid of TRF2 instead activate an attenuated telomeric DNA damage response that lacks accompanying telomere fusions, and propagate for multiple generations. The induction of telomere dysfunction in ES cells, consistent with somatic deletion of Trf2 (also known as Terf2), occurs only following the removal of the entire shelterin complex. Consistent with TRF2 being largely dispensable for telomere protection specifically during early embryonic development, cells exiting pluripotency rapidly switch to TRF2-dependent end protection. In addition, Trf2-null embryos arrest before implantation, with evidence of strong DNA damage response signalling and apoptosis specifically in the non-pluripotent compartment. Finally, we show that ES cells form T-loops independently of TRF2, which reveals why TRF2 is dispensable for end protection during pluripotency. Collectively, these data establish that telomere protection is solved by distinct mechanisms in pluripotent and somatic tissues.

SLX4IP Antagonizes Promiscuous BLM Activity during ALT Maintenance.

Cancer cells acquire unlimited proliferative capacity by either re-expressing telomerase or inducing alternative lengthening of telomeres (ALT), which relies on telomere recombination. Here, we show that ALT recombination requires coordinate regulation of the SMX and BTR complexes to ensure the appropriate balance of resolution and dissolution activities at recombining telomeres. Critical to this control is SLX4IP, which accumulates at ALT telomeres and interacts with SLX4, XPF, and BLM. Loss of SLX4IP increases ALT-related phenotypes, which is incompatible with cell growth following concomitant loss of SLX4. Inactivation of BLM is sufficient to rescue telomere aggregation and the synthetic growth defect in this context, suggesting that SLX4IP favors SMX-dependent resolution by antagonizing promiscuous BLM activity during ALT recombination. Finally, we show that SLX4IP is inactivated in a subset of ALT-positive osteosarcomas. Collectively, our findings uncover an SLX4IP-dependent regulatory mechanism critical for telomere maintenance in ALT cancer cells.

Oncogenic herpesvirus KSHV triggers hallmarks of alternative lengthening of telomeres.

To achieve replicative immortality, cancer cells must activate telomere maintenance mechanisms to prevent telomere shortening. ~85% of cancers circumvent telomeric attrition by re-expressing telomerase, while the remaining ~15% of cancers induce alternative lengthening of telomeres (ALT), which relies on break-induced replication (BIR) and telomere recombination. Although ALT tumours were first reported over 20 years ago, the mechanism of ALT induction remains unclear and no study to date has described a cell-based model that permits the induction of ALT. Here, we demonstrate that infection with Kaposi's sarcoma herpesvirus (KSHV) induces sustained acquisition of ALT-like features in previously non-ALT cell lines. KSHV-infected cells acquire hallmarks of ALT activity that are also observed in KSHV-associated tumour biopsies. Down-regulating BIR impairs KSHV latency, suggesting that KSHV co-opts ALT for viral functionality. This study uncovers KSHV infection as a means to study telomere maintenance by ALT and reveals features of ALT in KSHV-associated tumours.

RTEL1 Regulates G4/R-Loops to Avert Replication-Transcription Collisions.

Regulator of telomere length 1 (RTEL1) is an essential helicase that maintains telomere integrity and facilitates DNA replication. The source of replication stress in Rtel1-deficient cells remains unclear. Here, we report that loss of RTEL1 confers extensive transcriptional changes independent of its roles at telomeres. The majority of affected genes in Rtel1-/- cells possess G-quadruplex (G4)-DNA-forming sequences in their promoters and are similarly altered at a transcriptional level in wild-type cells treated with the G4-DNA stabilizer TMPyP4 (5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine). Failure to resolve G4-DNAs formed in the displaced strand of RNA-DNA hybrids in Rtel1-/- cells is suggested by increased R-loops and elevated transcription-replication collisions (TRCs). Moreover, removal of R-loops by RNaseH1 overexpression suppresses TRCs and alleviates the global replication defects observed in Rtel1-/- and Rtel1PIP_box knockin cells and in wild-type cells treated with TMPyP4. We propose that RTEL1 unwinds G4-DNA/R-loops to avert TRCs, which is important to prevent global deregulation in both transcription and DNA replication.

Molecular basis of telomere dysfunction in human genetic diseases.

Mutations in genes encoding proteins required for telomere structure, replication, repair and length maintenance are associated with several debilitating human genetic disorders. These complex telomere biology disorders (TBDs) give rise to critically short telomeres that affect the homeostasis of multiple organs. Furthermore, genome instability is often a hallmark of telomere syndromes, which are associated with increased cancer risk. Here, we summarize the molecular causes and cellular consequences of disease-causing mutations associated with telomere dysfunction.