Multi-Scale Imaging of the Dynamic Organization of Chromatin.

Chromatin is now regarded as a heterogeneous and dynamic structure occupying a non-random position within the cell nucleus, where it plays a key role in regulating various functions of the genome. This current view of chromatin has emerged thanks to high spatiotemporal resolution imaging, among other new technologies developed in the last decade. In addition to challenging early assumptions of chromatin being regular and static, high spatiotemporal resolution imaging made it possible to visualize and characterize different chromatin structures such as clutches, domains and compartments. More specifically, super-resolution microscopy facilitates the study of different cellular processes at a nucleosome scale, providing a multi-scale view of chromatin behavior within the nucleus in different environments. In this review, we describe recent imaging techniques to study the dynamic organization of chromatin at high spatiotemporal resolution. We also discuss recent findings, elucidated by these techniques, on the chromatin landscape during different cellular processes, with an emphasis on the DNA damage response.

Global chromatin mobility induced by a DSB is dictated by chromosomal conformation and defines the HR outcome.

Repair of DNA double-strand breaks (DSBs) is crucial for genome integrity. A conserved response to DSBs is an increase in chromatin mobility that can be local, at the site of the DSB, or global, at undamaged regions of the genome. Here, we address the function of global chromatin mobility during homologous recombination (HR) of a single, targeted, controlled DSB. We set up a system that tracks HR in vivo over time and show that two types of DSB-induced global chromatin mobility are involved in HR, depending on the position of the DSB. Close to the centromere, a DSB induces global mobility that depends solely on H2A(X) phosphorylation and accelerates repair kinetics, but is not essential. In contrast, the global mobility induced by a DSB away from the centromere becomes essential for HR repair and is triggered by homology search through a mechanism that depends on H2A(X) phosphorylation, checkpoint progression, and Rad51. Our data demonstrate that global mobility is governed by chromosomal conformation and differentially coordinates repair by HR.

The Dynamic Behavior of Chromatin in Response to DNA Double-Strand Breaks.

The primary functions of the eukaryotic nucleus as a site for the storage, retrieval, and replication of information require a highly dynamic chromatin organization, which can be affected by the presence of DNA damage. In response to double-strand breaks (DSBs), the mobility of chromatin at the break site is severely affected and, to a lesser extent, that of other chromosomes. The how and why of such movement has been widely studied over the last two decades, leading to different mechanistic models and proposed potential roles underlying both local and global mobility. Here, we review the state of the knowledge on current issues affecting chromatin mobility upon DSBs, and highlight its role as a crucial step in the DNA damage response (DDR).

Modified chromosome structure caused by phosphomimetic H2A modulates the DNA damage response by increasing chromatin mobility in yeast.

In budding yeast and mammals, double-strand breaks (DSBs) trigger global chromatin mobility together with rapid phosphorylation of histone H2A over an extensive region of the chromatin. To assess the role of H2A phosphorylation in this response to DNA damage, we have constructed strains where H2A has been mutated to the phosphomimetic H2A-S129E. We show that mimicking H2A phosphorylation leads to an increase in global chromatin mobility in the absence of DNA damage. The intrinsic chromatin mobility of H2A-S129E is not due to downstream checkpoint activation, histone degradation or kinetochore anchoring. Rather, the increased intrachromosomal distances observed in the H2A-S129E mutant are consistent with chromatin structural changes. Strikingly, in this context the Rad9-dependent checkpoint becomes dispensable. Moreover, increased chromatin dynamics in the H2A-S129E mutant correlates with improved DSB repair by non-homologous end joining and a sharp decrease in interchromosomal translocation rate. We propose that changes in chromosomal conformation due to H2A phosphorylation are sufficient to modulate the DNA damage response and maintain genome integrity.This article has an associated First Person interview with the first author of the paper.