Telomere crisis occurs during tumorigenesis when depletion of the telomere reserve leads to frequent telomere fusions. The resulting dicentric chromosomes have been proposed to drive genome instability. Here we examine the fate of dicentric human chromosomes in telomere crisis. We observed that dicentric chromosomes invariably persisted through mitosis and developed into 50-200 μm chromatin bridges connecting the daughter cells. Before their resolution at 3-20 h after anaphase, the chromatin bridges induced nuclear envelope rupture in interphase, accumulated the cytoplasmic 3' nuclease TREX1, and developed RPA-coated single stranded (ss) DNA. CRISPR knockouts showed that TREX1 contributed to the generation of the ssDNA and the resolution of the chromatin bridges. Post-crisis clones showed chromothripsis and kataegis, presumably resulting from DNA repair and APOBEC editing of the fragmented chromatin bridge DNA. We propose that chromothripsis in human cancer may arise through TREX1-mediated fragmentation of dicentric chromosomes formed in telomere crisis.

SUMMARY Modeling late onset disorders such as Parkinson’s disease (PD) using iPSC technology remains a challenge as current differentiation protocols yield cells with the properties of fetal-stage cells. Here we tested whether it is possible to accelerate aging in vitro to trigger late onset disease phenotypes in an iPSC model of PD. In order to manipulate a factor that is involved in natural aging as well as in premature aging syndromes, we used telomere shortening as an age-inducing tool. We show that shortened telomeres result in age-associated as well as potentially disease-associated phenotypes in human pluripotent stem cell (hPSC) derived midbrain dopamine (mDA) neurons. Our approach provides proof of concept for the further validation of telomere shortening as an induced aging tool for late onset diseases modeling.

Mouse telomeres have been suggested to resemble common fragile sites (CFS), showing disrupted TTAGGG fluorescent in situ hybridization signals after aphidicolin treatment. This “fragile” telomere phenotype is induced by deletion of TRF1, a shelterin protein that binds telomeric DNA and promotes efficient replication of the telomeric ds[TTAGGG]n tracts. Here we show that the chromosome-internal TTAGGG repeats present at human chromosome 2q14 form an aphidicolin-induced CFS. TRF1 binds to and stabilizes CFS 2q14 but does not affect other CFS, establishing 2q14 as the first CFS controlled by a sequence-specific DNA binding protein. The data show that telomeric DNA is inherently fragile regardless of its genomic position and imply that CFS can be caused by a specific DNA sequence.

The mammalian chromosomes present specific sites of gaps or breaks, the common fragile sites (CFSs), when the cells are exposed to DNA replication stress or to some DNA binding compounds. CFSs span hundreds or thousands of kilobases. The analysis of these sequences has not definitively clarified the causes of their fragility. There is considerable evidence that CFSs are regions of late or slowed replication in the presence of sequence elements that have the propensity to form secondary structures, and that the cytogenetic expression of CFSs may be due to unreplicated DNA. In order to analyse the relationship between DNA replication time and fragility, in this work we have investigated the timing of replication of sequences mapping within two CFSs (FRA1H and FRA2G), of syntenic non-fragile sequences and of early and late replicating control sequences by using fluorescent in situ hybridization on interphase nuclei, conventional fluorescence microscopy and confocal microscopy. Our results indicate that the fragile sequences are slow replicating and that they enter G2 phase unreplicated with very high frequency. Thus these regions could sometimes reach mitosis unreplicated or undercondensed and be expressed as chromosome gaps/breakages.

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