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 utilizing 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 employ 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 find 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.

FAM111A induces nuclear dysfunction in disease and viral restriction

“…A key difference is that U2OS cells maintain their telomeres via a recombination‐based replication process called alternative lengthening of telomeres (ALT), as they do not have active telomerase (Bryan et al , 1997; Cesare & Reddel, 2010). ALT telomeres undergo DNA synthesis in the G2/M phases of the cell cycle at ALT‐associated PML bodies (APBs) that contain PCNA among other DNA replication and repair factors (Dilley et al , 2016; Min et al , 2017; Pan et al , 2017; Zhang et al , 2019; Sobinoff & Pickett, 2020; Loe et al , 2020). Further analysis revealed that the atypical FAM111A foci in U2OS cells colocalize with both PCNA and PML (Fig 1C).…”

“…Human embryonic kidney cell lines HEK293 and HEK293T, human osteosarcoma cell line U2OS, human colon cancer line HCT116, and human cervical adenocarcinoma cell line HeLa were obtained from American Type Culture Collection (ATCC). Human epithelial cells immortalized with hTERT cell line RPE was obtained from Denchi lab (Loe et al , 2020). These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS).…”

Protein phase separation and its role in tumorigenesis

“…A recent study suggests that the formation of APBs is driven by phase separation, thus promoting the clustering of telomere repeats and telomere lengthening ( Zhang et al, 2020 ; Figure 6 ). Consequently, knocking down the PML body component PML inhibited APB formation and caused telomere shortening ( Draskovic et al, 2009 ; Loe et al, 2020 ; Osterwald et al, 2015 ). Together, this suggests that APBs facilitate ALT telomere maintenance, eventually allowing cancer cells to grow indefinitely and become immortal.…”

Amyloid precursor protein causes fusion of promyelocytic leukemia nuclear bodies in human hippocampal areas with high plaque load

“…APP-CT was either uniformly distributed or concentrated at the inner wall of the nuclear body (Figure 2L). These results suggest that upon translocation of APP-CT into the nucleus, there is a regulated interaction between the APP-CT/FE65/TIP60 complex, p53 and PML bodies 40 .…”