Abstract
SummaryCells coordinate interphase-to-mitosis transition, but recurrent cytogenetic lesions appear at common fragile sites (CFSs), termed CFS expression, in a tissue-specific manner after replication stress, marking regions of instability in cancer. Despite such a distinct defect, no model fully provides a molecular explanation for CFSs. We show that CFSs are characterized by impaired chromatin folding, manifesting as disrupted mitotic structures visible with molecular fluorescence in situ hybridization (FISH) probes in the presence and absence of replication stress. Chromosome condensation assays reveal that compaction-resistant chromatin lesions persist at CFSs throughout the cell cycle and mitosis. Cytogenetic and molecular lesions are marked by faulty condensin loading at CFSs, a defect in condensin-I-mediated compaction, and are coincident with mitotic DNA synthesis (MIDAS). This model suggests that, in conditions of exogenous replication stress, aberrant condensin loading leads to molecular defects and CFS expression, concomitantly providing an environment for MIDAS, which, if not resolved, results in chromosome instability.
Highlights
The folding of chromosomes in preparation for mitosis is the most profound structural change the genome undergoes throughout a cell’s lifetime (Antonin and Neumann, 2016)
common fragile sites (CFSs) frequency and repertoire in RPE1 and HCT116 cells To analyse the relationship between chromosome architecture and CFS structure we characterised the CFS repertoire and frequency in two epithelial chromosomally near-normal diploid cell lines (HCT116 and RPE1), using DAPI banding, after inducing replication stress with aphidicolin (APH). 372 lesions across 371 metaphases for APH concentrations ranging from 0.1 to 0.6 μM were observed, showing that higher APH concentration led to increased rate of breakage and more severe CFS phenotypes (Supplementary Figure 1A-B), with a concomitantly delayed cell cycle (Supplementary Figure 1C)
Chromatin at CFS regions is not remodelled for mitosis As the mechanism(s) giving rise to aberrant chromatin compaction observed at metaphase (Figure 1A, 2B) were unclear we investigated the possibility that they might arise as interphase chromatin defects
Summary
The folding of chromosomes in preparation for mitosis is the most profound structural change the genome undergoes throughout a cell’s lifetime (Antonin and Neumann, 2016). Condensation defects have been shown to underlie HR-deficiency mediated mitotic lesions, and if not resolved lead to DNA damage and chromosomal instability (Chan et al, 2018). The effectors of such condensation failures are likely to be proteins that drive mitotic folding such as the condensin I and II complexes which are crucial for chromosome compaction (Gibcus et al, 2018; Lipp et al, 2007; Samejima et al, 2012). CFS are characterised by aberrant condensin loading, leading to molecular lesions, whilst in the extreme conditions of exogenous replication stress cytological chromosome abnormalities are apparent
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