Transcription-mediated organization of the replication initiation program across large genes sets up common fragile sites genome-wide

Abstract

Running title (50 characters or less, including spaces)CFSs: a view through Repli-Seq, GRO-seq and OK-Seq ABSTRACT Common Fragile Sites (CFSs) are chromosome regions prone to breakage under replication stress, known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription elicits their instability but the underlying mechanisms remained elusive. Analyses of genome-wide replication timing of human lymphoblasts here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, thus replicated by long-traveling forks. In contrast to formation of sequence-dependent fork barriers or head-on transcription-replication conflicts, travelinglong in late S phase explains CFS replication features. We further show that transcription inhibition during the S phase, which excludes the setting of new replication origins, fails to rescue CFS stability. Altogether, results show that transcription-dependent suppression of initiation events delays replication of large gene body, committing them to instability.

DNA Replication Stress Generates Distinctive Landscapes of DNA Copy Number Alterations and Chromosome Scale Losses

“…Therefore single cell RNA sequencing may give an indication of whether, despite similarities at the bulk population level, variability in gene expression at the single cell level could explain why a subset of single cells are prone to specific aCNAs. Lastly, although we did not detect any obvious differences in replication timing at these large genes it is possible that delayed replication induced by replication stress could enhance fragility, as recently shown 21,26 . In our study we analysed replication timing from control-treated cells but it would be interesting to determine whether fragility of these regions correlates with aphidicolin-specific delays in replication timing.…”

“…Large and giant genes have been proposed to cause fragility under replication stress due to a number of mechanisms including late 13,[23][24][25] or delayed replication timing 21,26 , lack of origin density 27 , replicationtranscription collisions during S-phase 5,13,28 and gene expression in G1 that removes licensing complexes and reduces origin density 26,29,30 . We showed that large terminal aCNAs were generally close to large or giant genes, and that in the case of the most highly recurrent site of aCNAs in RPE1 cells this was likely due to the RPE1-specific gene expression of the nearby giant gene AUTS2.…”

“…There may be other factor(s) involved in predisposing these specific genomic sites to fragility. For example these genes may lie in regions that are replicated early or mid S-phase in normal cells but could be delayed in timing in the presence of aphidicolin 21,26 which would explain their fragility. The timing or rate of active gene transcription may also be an important factor that we were unable to detect with RNA sequencing.…”