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

Abstract

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.

1. Introduction

To maintain their proper functioning, eukaryotic cells must faithfully duplicate their genomic DNA and accurately fold their chromatin during the cell cycle. However, this can be significantly challenging, as cells often encounter various forms of replication stress from both exogenous and endogenous sources [1]. These stresses can lead to a wide range of chromosomal alterations, from single- or few-base pair mutations to larger changes that span hundreds to millions of base pairs that can likewise lead to significant changes in cell behavior [2–5]. However, how precisely these replication stresses lead to such changes, particularly those that span incredibly large genomic regions, is very poorly understood.

Arguably, the best characterized large-scale changes occur at so-called common fragile sites (CFSs) [6]. These regions, which can be up to a few mega base pairs in length, frequently form cytogenetically defined chromosomal gaps or breaks in G-banding assays under replication stress [6,7]. Importantly, CFSs are known to frequently overlap with chromosomal breakpoints in many cancers, where they are hotspots for chromosomal rearrangements, contributing to tumor development and progression [8,9]. In addition, large-scale changes in CFSs are also found in the embryo during development, perhaps owing to replication stress during the rapid cell division of early embryogenesis [10,11]. However, despite these observations, it is not understood how replication stress leads to deletions, rearrangements, or any particular manifestation of genomic changes at CFSs.