Super-resolution imaging reveals higher-order structures within common fragile sites in human mitotic chromosomes

Common fragile sites (CFSs) are large genomic loci that are frequently deleted under replication stress and are thought to play a role in carcinogenesis as well as developmental disorders during early embryogenesis. They often appear as gaps or breaks in mitotic chromosomes by conventional optical microscopy and are also loci that are replicated in mitosis in a process called mitotic DNA synthesis (MiDAS). However, we still have a very poor understanding of the potential mechanisms underlying their genomic instability. We recently found that in normal mitotic chromosomes, there is a two-level hierarchy in the higher-order chromatin structure, with mechanically resistant ~90 nm Mitotic NanoDomains (MNDs) assembling into ~125 nm compact granules (CGs). In this work, we combine multiple super-resolution imaging techniques, including binding-activated localization microscopy (BALM), atomic force microscopy (AFM), and stochastic optical reconstruction microscopy (STORM), to characterize CFSs. Our super-resolution data indicate that sites that appear as gaps by conventional microscopy can be filled with chromatin of the size and shape of MNDs but not CGs. Moreover, we find that MiDAS loci only form an array of MNDs with no CGs. Taken together, our work suggests that under replication stress, CFS loci can not only be replicated but also assembled into higher-order chromatin. However, this organization is limited to the level of MNDs, which suggests that a failure to progress beyond MNDs to form CGs constitutes a key structural deficiency at these loci.