Abstract
Common fragile sites (CFSs) are conserved genomic regions prone to break under conditions of replication stress (RS). Thus, CFSs are hotspots for rearrangements in cancer and contribute to its chromosomal instability. Here, we have performed a global analysis of proteins that recruit to CFSs upon mild RS to identify novel players in CFS stability. To this end, we performed Chromatin Immunoprecipitation (ChIP) of FANCD2, a protein that localizes specifically to CFSs in G2/M, coupled to mass spectrometry to acquire a CFS interactome. Our strategy was validated by the enrichment of many known regulators of CFS maintenance, including Fanconi Anemia, DNA repair and replication proteins. Among the proteins identified with unknown functions at CFSs was the chromatin remodeler ATRX. Here we demonstrate that ATRX forms foci at a fraction of CFSs upon RS, and that ATRX depletion increases the occurrence of chromosomal breaks, a phenotype further exacerbated under mild RS conditions. Accordingly, ATRX depletion increases the number of 53BP1 bodies and micronuclei, overall indicating that ATRX is required for CFS stability. Overall, our study provides the first proteomic characterization of CFSs as a valuable resource for the identification of novel regulators of CFS stability.
Highlights
Common fragile sites (CFSs) are chromosomal regions highly susceptible to break, and pose a serious threat to genomic integrity [1]
Some CFSs encompass repetitive sequences and regions that are A-T rich, which can form secondary DNA structures, further complicating DNA replication and contributing to fragility [12]. It is well-known that CFSs complete replication very late in S-phase [13], limiting the time available to repair challenged CFSs prior to mitosis, thereby further increasing their fragility. These inherent complications of replicating CFSs are exacerbated in conditions of mild replication stress (RS) which occurs upon oncogenic transformation or experimentally with low doses of the polymerase inhibitor aphidicolin (APH), that slows down replication [13,14,15]
To create conditions of mild RS that mimic those of oncogenic transformation and that cause CFS instability, we treated the cells with low doses of APH (0.2–0.4 M)
Summary
Common fragile sites (CFSs) are chromosomal regions highly susceptible to break, and pose a serious threat to genomic integrity [1] These genomic regions are present in all individuals and can give rise to chromosomal abnormalities, including small deletions and insertions, and translocations [2,3]. Some CFSs encompass repetitive sequences and regions that are A-T rich, which can form secondary DNA structures, further complicating DNA replication and contributing to fragility [12] It is well-known that CFSs complete replication very late in S-phase [13], limiting the time available to repair challenged CFSs prior to mitosis, thereby further increasing their fragility. These inherent complications of replicating CFSs are exacerbated in conditions of mild RS which occurs upon oncogenic transformation or experimentally with low doses of the polymerase inhibitor aphidicolin (APH), that slows down replication [13,14,15]
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