Genome-wide High Resolution Mapping of Mitotic DNA Synthesis Sites and Common Fragile Sites by Direct Sequencing [MiDA-seq]
Ontology highlight
ABSTRACT: Common fragile sites (CFSs) are genomic loci prone to the formation of breaks or gaps on metaphase chromosomes. Here, we seek to map human CFSs with high resolution on a genome-wide scale by sequencing the sites of mitotic DNA synthesis (MiDASeq) that are specific for CFSs. We generated a nucleotide-resolution atlas of MiDAS sites (MDSs) that covered most of know CFSs, and comprehensively analyzed their sequence characteristics and genomic features.
Project description:Common fragile sites (CFSs) are genomic loci prone to the formation of breaks or gaps on metaphase chromosomes. Here, we seek to map human CFSs with high resolution on a genome-wide scale by sequencing the sites of mitotic DNA synthesis (MiDASeq) that are specific for CFSs. We generated a nucleotide-resolution atlas of MiDAS sites (MDSs) that covered most of know CFSs, and comprehensively analyzed their sequence characteristics and genomic features.
Project description:Common fragile sites (CFSs) are genomic loci prone to the formation of breaks or gaps on metaphase chromosomes. Here, we seek to map human CFSs with high resolution on a genome-wide scale by sequencing the sites of mitotic DNA synthesis (MiDASeq) that are specific for CFSs. We generated a nucleotide-resolution atlas of MiDAS sites (MDSs) that covered most of know CFSs, and comprehensively analyzed their sequence characteristics and genomic features.
Project description: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 DNA replication timing (RT) and transcriptional profile under mild replication stress in the context of the 3D genome organization. We report the analysis of DNA replication timing profiling of human fibroblasts (BJ-hTERT), grown with or without aphidicolin, a DNA polymerase inhibitor used to induce CFS expression. We find that aphidicolin treatment affects the RT of a small portion of the genome. However, CFSs are enriched for delayed RT regions under stress. We further study the mechanism leading to recurrent chromosomal instability at CFSs and find that the 3D genome organization underlies fragility at RT delayed large expressed genes. We report a fragility signature at the core of CFSs comprised of delayed replication of large expressed gene spanning over a TAD-boundary. The fragility signature allows for mapping of the core fragile site and the identification of novel fragile sites in BJ cells.
Project description: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.
Project description:Multiple replication abnormalities cause cells lacking BRCA2 to enter mitosis with under-replicated DNA and to activate mitotic DNA synthesis (MiDAS). However, the precise position of these MiDAS sites, as well as their origin, remains unknown. Here we labelled mitotic nascent DNA and performed high-throughput sequencing to identify at high-resolution the sites where MiDAS occurs in the absence of BRCA2. This approach revealed 150 genomic loci affected by MiDAS, which map within regions replicating during early S-phase and are therefore distinct from the aphidicolin-induced common fragile sites. Moreover, these sites largely localise near early firing origins and within genes transcribed in early S, suggesting that they stem from transcription-replication conflicts (TCRs). Inhibiting transcription with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) during early S-phase abrogates MiDAS. Strikingly, MiDAS sites co-localise with genomic loci where R-loops form in unchallenged conditions, suggesting that R-loop accumulation caused by BRCA2 inactivation leads to DNA lesion which are repaired by MiDAS. RAD52 is required in this process, as its abrogation in BRCA2-deficient cells reduces the rate of MiDAS and causes DNA damage accumulation in G1. Furthermore, MiDAS sites triggered by BRCA2 inactivation are hotspots for genomic rearrangement in BRCA2-mutated breast tumours. These results indicate that BRCA2 acts in early S-phase to protect TRC- and R-loop-induced DNA lesions, thereby preventing them from becoming a source of genomic instability and tumorigenesis.
Project description:Chromosomal Common Fragile Sites (CFSs) are conserved regions of our genome prone to break in conditions of replication stress (RS. Here, we have performed Chromatin Immunoprecipitation (ChIP) with FACND2 antibodies coupled to Mass Spectrometry to isolate CFSs from HeLa cells and identify the proteins enriched at these loci.
Project description:Here, we identify that a human DNA helicase RTEL1, which is known to play a role in the maintenance of telomeres by interacting with proteins in the shelterin complex by resolving G4 quadruplexes, is essential for prevention of R-loop accumulation during replication and for MiDAS in mitosis at both CFSs and telomeres. Our findings indicate that RTEL1 plays a genome-wide role in preventing the collapse of replication forks at sites of G-quadruplexes and R-loop formation. Considering cancer cells rely on RTEL1 function to resolve the elevated transcription-replication conflicts caused by oncogene activation, RTEL1 is a potential drug target for cancer therapy.