3D genome organization, transcription and aberrant DNA replication contribute to genome instability at fragile sites

Common fragile sites (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features characterizing CFSs have been associated with their instability, however, these features are prevalent across the genome and do not account for all known CFSs. Here we explored the transcriptional profile and DNA replication timing under mild replication stress in the context of the 3D genome organization. We report the analysis of nascent RNA-seq for high-throughput profiling of gene expression in human fibroblasts (BJ-hTERT), grown with or without aphidicolin, a DNA polymerase inhibitor used to induce CFS expression. We find that aphidicolin treatment does not affect the transcriptional program. However, large expressed genes are susceptible to replication timing delay under stress. We further study the mechanism leading to recurrent chromosomal instability at CFSs and find that the 3D genome organization underlies fragility at large expressed genes. We report a fragility signature at the core of CFSs comprised of a large expressed gene spanning over a TAD-boundary with delayed DNA replication. The fragility signature allows for mapping of the core fragile site and the identification of novel fragile sites in BJ cells. Nascent RNA-seq profiles of immortalized human foreskin fibroblasts (BJ-hTERT) with or without 200nM aphidicolin treatment for 24 hours (2 biological replicates per condition).