Telomerase-negative cancer cells maintain their telomeres via the Alternative Lengthening of Telomeres (ALT) pathway1–3. Although a growing body of evidence demonstrates that the ALT mechanism is a post-replicative telomere recombination process, molecular details of this pathway are largely unknown. Here we demonstrate that MUS81, a DNA structure–specific recombination endonuclease, plays a key role in the maintenance of telomeres in human ALT cells. We find that MUS81 specifically localizes to ALT-associated promyelocytic leukemia nuclear bodies (APBs) and associates with telomeric DNA in ALT cells, which is enriched during G2 phase of the cell cycle. Depletion of MUS81 results in reduction of ALT specific-telomere recombination and leads to proliferation arrest of ALT cells. In addition, the endonuclease activity of MUS81 is required for recombination-based ALT cell survival, and the interaction of MUS81 with TRF2 regulates this enzymatic activity to maintain telomere recombination. Thus, our results suggest that MUS81 is involved in the maintenance of ALT cell survival at least in part by telomere-HR process.
High telomerase activity is a characteristic of human embryonic stem cells (hESCs), however, the regulation and maintenance of correct telomere length in hESCs is unclear. In this study we investigated telomere elongation in hESCs in vitro and found that telomeres lengthened from their derivation in blastocysts through early expansion, but stabilized at later passages. We report that the core unit of telomerase, hTERT, was highly expressed in hESCs in blastocysts and throughout long-term culture; furthermore, this was regulated in a Wnt-b-catenin-signaling-dependent manner. Our observations that the alternative lengthening of telomeres (ALT) pathway was suppressed in hESCs and that hTERT knockdown partially inhibited telomere elongation, demonstrated that high telomerase activity was required for telomere elongation. We observed that chromatin modification through trimethylation of H3K9 and H4K20 at telomeric regions decreased during early culture. This was concurrent with telomere elongation, suggesting that epigenetic regulation of telomeric chromatin may influence telomerase function. By measuring telomere length in 96 hESC lines, we were able to establish that telomere length remained relatively stable at 12.0261.01 kb during later passages (15-95). In contrast, telomere length varied in hESCs with genomic instability and hESC-derived teratomas. In summary, we propose that correct, stable telomere length may serve as a potential biomarker for genetically stable hESCs.
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