Telomere length heterogeneity in ALT cells is maintained by PML-dependent localization of the BTR complex to telomeres

Abstract

Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by using the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed alternative lengthening of telomeres (ALT). Cells that use ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML bodies (APBs). APBs localize to and cluster telomeric ends together with telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can occur. However, the necessity of APBs and their function in the ALT pathway has remained unclear. Here, we used CRISPR/Cas9 to delete PML and APB components from ALT-positive cells to cleanly define the function of APBs in ALT. We found that PML is required for the ALT mechanism, and that this necessity stems from APBs' role in localizing the BLM-TOP3A-RMI (BTR) complex to ALT telomere ends. Strikingly, recruitment of the BTR complex to telomeres in a PML-independent manner bypasses the need for PML in the ALT pathway, suggesting that BTR localization to telomeres is sufficient to sustain ALT activity.

Tousled-Like Kinases Suppress Innate Immune Signaling Triggered by Alternative Lengthening of Telomeres

“…BLM depletion strongly attenuated C-circles and reduced STING and RSAD2 induction in HeLa LT cells following TLK depletion ( Figures 6 A, 6B, and S6 A). We next examined U-2-OS cells lacking PML, a key component of APBs that is required for efficient C-circle production ( Figure 6 C) ( Loe et al., 2020 ). Deletion of PML also strongly attenuated RSAD2 induction following TLK1/2 depletion and reduced extranuclear telomeric FISH signal, indicating that ECTR production contributes to innate immune activation in TLK-depleted cells ( Figures 6 D, S6 B, and S6C).…”

Nuclear body phase separation drives telomere clustering in ALT cancer cells

“…It is reported that BLM helicase was recruited to synthetic condensates formed by polySUMO and polySIM only when the condensate was SUMO rich ( Min et al , 2019 ). A recent study showed that the presence of PML protein is required for the recruitment of BTR complex to telomeres for ALT telomere maintenance ( Loe et al , 2020 ). It is possible that the chemistry of APB condensates is actively regulated during the course of telomere elongation to selectively recruit different factors based on direct SUMO–SIM interactions or indirect interactions with existing APB components.…”

“…PML nuclear bodies are dynamic structures in the nucleus that transiently sequester up to 100 different proteins that are implicated in many cellular functions including tumor suppression, DNA replication, gene transcription, DNA repair, viral pathogenicity, cellular senescence, and apoptosis ( Lallemand-Breitenbach and de The, 2010 ). Inhibiting APB formation by knocking down PML protein, an essential component of PML nuclear bodies, leads to telomere shortening ( Draskovic et al , 2009 ; Osterwald et al , 2015 ; Loe et al , 2020 ), indicating that APBs contribute to ALT telomere maintenance. In addition to typical PML nuclear body components, APBs contain proteins involved in homologous recombination such as replication protein A (RPA), Rad51, and breast cancer susceptibility protein 1 (BRCA1) ( Nabetani and Ishikawa, 2011 ), which suggests that APBs promote telomere synthesis.…”

PML nuclear bodies and chromatin dynamics: catch me if you can!

“…Overexpression of the helicase BLM triggered ALT-like phenotypes in presence of these reconstituted APB-like condensates suggesting that concentration of specific DNA repair factors, together with the clustering of telomeres to provide repair templates, are required for induction of the ALT phenotype ( 33 ). Stochiometry of the SUMO-SIM interactions controls recruitment of BLM or of the full BLM–TOP3A–RMI (BTR) complex at telomeric ends in APBs ( 33 , 207 ). The BTR complex is essential for telomere lengthening and its artificial tethering to telomeres can bypass the absence of PML to induce ALT ( 207 ).…”