ChIP-Seq anti gH2AX to investigate DNA damage and Telomeres senescence

The DNA damage response (DDR) coordinates DNA repair and transiently arrests cell-cycle progression until DNA damage has been removed1. We observed cellular senescence establishment and persistent DDR foci in human diploid fibroblasts (HDFs) long time after exposure to exogenous DNA damaging agents such as ionizing radiations (IR), indicating that a small fraction of DNA double-strand breaks (DSBs) is not repaired and fuels sustained DDR signalling. The molecular bases that distinguish transient from persistent DDR foci are unknown. We hypothesized that DDR persistency is caused by DSBs irreparability dictated by their chromosomal location. We reasoned that telomeric repeats, by the same mechanisms that inhibit DNA end joining at chromosome ends to prevent chromosomal fusions2, may also inhibit DNA repair within repeats. Here, we show both by confocal imaging and chromatin immunoprecipitations (ChIPs) that a large fraction of exogenously-induced persistent DDR markers are associated with telomeric DNA, both in cultured cells and in in vivo tissues. Whole-genome analysis performed by ChIPseq confirmed that telomeres are the preferential chromosomal domains associated with persistent DDR signals. The observed DDR activity is not triggered by telomere shortening. To demonstrate that telomeric DNA damage is irreparable, we induced a chromosomal DSB next to a stretch of telomeric DNA in Saccharomyces cerevisiae and we showed that telomeric DNA endows the locus with DNA repair resistance and impaired recruitment of DNA ligase 4. In mammalian cells, the ectopic recruitment of the telomere-binding protein TRF2 next to a DSB, in the absence of telomeric DNA, is sufficient to induce DDR persistence. In addition, introduction of telomeric DNA ends in HDFs induces a prolonged checkpoint arrest not observed with scrambled linear DNA or circular telomeric DNA. Ageing is associated with accumulation of 2 markers of DDR activation3-5. In terminally differentiated tissues of old primates, such as brain neurons and in liver hepatocytes, we observed markers of DDR activation accumulating at telomeres. These markers, however, are not associated with critically short telomeres. We propose that linear genomes are not uniformly reparable and telomeric DNA tracts, if exogenously or endogenously damaged, are irreparable.