Abstract
Alternative Lengthening of Telomeres (ALT) is a Break-Induced Replication (BIR)-based mechanism elongating telomeres in a subset of human cancer cells. While the notion that spontaneous DNA damage at telomeres is required to initiate ALT, the molecular triggers of this physiological telomere instability are largely unknown. We previously proposed that the telomeric long noncoding RNA TERRA may represent one such trigger; however, given the lack of tools to suppress TERRA transcription in cells, our hypothesis remained speculative. We have developed Transcription Activator-Like Effectors able to rapidly inhibit TERRA transcription from multiple chromosome ends in an ALT cell line. TERRA transcription inhibition decreases marks of DNA replication stress and DNA damage at telomeres and impairs ALT activity and telomere length maintenance. We conclude that TERRA transcription actively destabilizes telomere integrity in ALT cells, thereby triggering BIR and promoting telomere elongation. Our data point to TERRA transcription manipulation as a potentially useful target for therapy.
Highlights
Alternative Lengthening of Telomeres (ALT) is a Break-Induced Replication (BIR)-based mechanism elongating telomeres in a subset of human cancer cells
Because RNaseH1 and FANCM inactivation increase telomere instability and ALT activity, while their over-expression alleviates ALT8,9,30,31, we proposed that physiological damage triggered by TERRA/telR-loops at ALT telomeres may provide the substrate for BIR-mediated telomere elongation[8,9,32,33]
T-Transcription Activator-Like Effectors (TALEs) were C-terminally fused to a strong nuclear localization signal (NLS), four transcription repressor domains of the mSIN3 interaction domain (Enhanced Repressor Domain, SID4X), and a human influenza hemagglutinin (HA) epitope (Fig. 1a)
Summary
Alternative Lengthening of Telomeres (ALT) is a Break-Induced Replication (BIR)-based mechanism elongating telomeres in a subset of human cancer cells. We conclude that TERRA transcription actively destabilizes telomere integrity in ALT cells, thereby triggering BIR and promoting telomere elongation. Transcription of telomeric DNA into the long noncoding RNA TERRA is an evolutionarily conserved feature of eukaryotic cells with linear chromosomes[1]. RNA polymerase II produces TERRA proceeding from subtelomeric regions towards chromosome ends and using the C-rich telomeric strand as a template. The molecular mechanisms mediating TERRA retention on chromosomes still need to be fully elucidated; the propensity of TERRA to form RNA:DNA hybrids with its template DNA strand (telomeric R-loops or telR-loops)[8,9,10,11] and the physical interaction of human TERRA with the shelterin factors TRF1 and TRF212,13 suggest that TERRA association with telomeric DNA-containing loci involves RNA–DNA and RNA–protein interactions. Supporting the proposed origin of TERRA, 20q-TERRA KO cells displayed substantially diminished total TERRA levels when compared to parental cells[23,24]
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