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

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.

A new versatile system for rapid control of gene expression in the fission yeast Schizosaccharomyces pombe.

The ability to regulate the expression of a gene greatly aids the process of uncovering its functions. The fission yeast Schizosaccharomyces pombe has so far lacked a system for rapidly controlling the expression of chromosomal genes, hindering its full potential as a model organism. Although the widely used nmt1 promoter displays a wide dynamic range of activity, it takes > 14-15 h to derepress. The urg1 promoter also shows a large dynamic range and can be induced quickly (< 2 h), but its implementation requires laborious strain construction and it cannot be used to study meiosis. To overcome these limitations, we constructed a tetracycline-regulated system for inducible expression of chromosomal genes in fission yeast, which is easily established and implemented. In this system the promoter of a gene is replaced by simple one-step substitution techniques with a tetracycline-regulated promoter cassette (tetO(7) -TATA(CYC1) ) in cells where TetR/TetR'-based transcription activators/repressors are also produced. Using top1 and nse6 as reporter genes, we show that Top1 and Nse6 appear after just 30 min of activating tetO(7) -TATA(CYC1) and plateau after -4-6 h. The amount of synthesised protein is comparable to that produced from the attenuated nmt1 promoter P(nmt8) , which should be closer to wild-type levels for most genes than those generated from excessively strong promoters and can be controlled by changing the concentration of the effector antibiotic. This system also works efficiently during meiosis, thus making it a useful addition to the toolkit of the fission yeast community.