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.

In vivo tracking of functionally tagged Rad51 unveils a robust strategy of homology search.

Homologous recombination (HR) is a major pathway to repair DNA double-strand breaks (DSB). HR uses an undamaged homologous DNA sequence as a template for copying the missing information, which requires identifying a homologous sequence among megabases of DNA within the crowded nucleus. In eukaryotes, the conserved Rad51-single-stranded DNA nucleoprotein filament (NPF) performs this homology search. Although NPFs have been extensively studied in vitro by molecular and genetic approaches, their in vivo formation and dynamics could not thus far be assessed due to the lack of functional tagged versions of Rad51. Here we develop and characterize in budding yeast the first fully functional, tagged version of Rad51. Following induction of a unique DSB, we observe Rad51-ssDNA forming exceedingly long filaments, spanning the whole nucleus and eventually contacting the donor sequence. Emerging filaments adopt a variety of shapes not seen in vitro and are modulated by Rad54 and Srs2, shedding new light on the function of these factors. The filaments are also dynamic, undergoing rounds of compaction and extension. Our biophysical models demonstrate that formation of extended filaments, and particularly their compaction-extension dynamics, constitute a robust search strategy, allowing DSB to rapidly explore the nuclear volume and thus enable efficient HR.

When fixation creates fiction.

A chemical regularly used to image cells can dramatically alter the way cellular compartments called condensates look under the microscope.

Repair Foci as Liquid Phase Separation: Evidence and Limitations.

In response to DNA double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less condensates or "foci". The formation of these foci and their disassembly within the proper time window are essential for genome integrity. However, how these membrane-less sub-compartments are formed, maintained and disassembled remains unclear. Recently, several studies across different model organisms proposed that DNA repair foci form via liquid phase separation. In this review, we discuss the current research investigating the physical nature of repair foci. First, we present the different models of condensates proposed in the literature, highlighting the criteria to differentiate them. Second, we discuss evidence of liquid phase separation at DNA repair sites and the limitations of this model to fully describe structures formed in response to DNA damage. Finally, we discuss the origin and possible function of liquid phase separation for DNA repair processes.

Physical observables to determine the nature of membrane-less cellular sub-compartments.

The spatial organization of complex biochemical reactions is essential for the regulation of cellular processes. Membrane-less structures called foci containing high concentrations of specific proteins have been reported in a variety of contexts, but the mechanism of their formation is not fully understood. Several competing mechanisms exist that are difficult to distinguish empirically, including liquid-liquid phase separation, and the trapping of molecules by multiple binding sites. Here, we propose a theoretical framework and outline observables to differentiate between these scenarios from single molecule tracking experiments. In the binding site model, we derive relations between the distribution of proteins, their diffusion properties, and their radial displacement. We predict that protein search times can be reduced for targets inside a liquid droplet, but not in an aggregate of slowly moving binding sites. We use our results to reject the multiple binding site model for Rad52 foci, and find a picture consistent with a liquid-liquid phase separation. These results are applicable to future experiments and suggest different biological roles for liquid droplet and binding site foci.

Single molecule microscopy reveals key physical features of repair foci in living cells.

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus reflecting the existence of a liquid droplet around damaged DNA.

Complex Chromatin Motions for DNA Repair.

A number of studies across different model systems revealed that chromatin undergoes significant changes in dynamics in response to DNA damage. These include local motion changes at damage sites, increased nuclear exploration of both damaged and undamaged loci, and directed motions to new nuclear locations associated with certain repair pathways. These studies also revealed the need for new analytical methods to identify directed motions in a context of mixed trajectories, and the importance of investigating nuclear dynamics over different time scales to identify diffusion regimes. Here we provide an overview of the current understanding of this field, including imaging and analytical methods developed to investigate nuclear dynamics in different contexts. These dynamics are essential for genome integrity. Identifying the molecular mechanisms responsible for these movements is key to understanding how their misregulation contributes to cancer and other genome instability disorders.

[When science and music meet]. / Quand science et musique se rencontrent.

TITLE: Quand science et musique se rencontrent. ABSTRACT: Musique et sciences ont souvent été liées dans l'histoire de la musique. Cependant, rares sont les compositeurs qui ont eu l'occasion d'interagir avec un scientifique pour s'imprégner de son travail et l'utiliser comme source d'inspiration. Le projet Muse-IC se propose de donner cette opportunité à des compositeurs d'aujourd'hui, en leur commandant une œuvre inspirée par une découverte scientifique récente. À la suite d'un double appel réalisé auprès de chercheurs et de compositeurs, des compositeurs se sont immergés dans l'univers scientifique de chercheurs afin de repousser les limites de leur démarche créative. Entre 2017 et 2019, six compositeurs ont écrit une pièce inspirée de la découverte d'un chercheur avec lequel ils ont interagit directement. L'aboutissement de ce projet a donné lieu à une conférence-concert à la salle Cortot, à Paris, une occasion unique de sensibiliser le public à l'importance de la recherche fondamentale au travers d'une rencontre originale entre compositeurs, musiciens et chercheurs.

Physical principles and functional consequences of nuclear compartmentalization in budding yeast.

One striking feature of eukaryotic nuclei is the existence of discrete regions, in which specific factors concentrate while others are excluded, thus forming microenvironments with different molecular compositions and biological functions. These domains are often referred to as subcompartments even though they are not membrane enclosed. Despite their functional importance the physical nature of these structures remains largely unknown. Here, we describe how the Saccharomyces cerevisiae nucleus is compartmentalized and discuss possible physical models underlying the formation and maintenance of chromatin associated subcompartments. Focusing on three particular examples, the nucleolus, silencing foci, and repair foci, we discuss the biological implications of these different models as well as possible approaches to challenge them in living cells.

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways.

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

High-resolution visualization of H3 variants during replication reveals their controlled recycling.

DNA replication is a challenge for the faithful transmission of parental information to daughter cells, as both DNA and chromatin organization must be duplicated. Replication stress further complicates the safeguard of epigenome integrity. Here, we investigate the transmission of the histone variants H3.3 and H3.1 during replication. We follow their distribution relative to replication timing, first in the genome and, second, in 3D using super-resolution microscopy. We find that H3.3 and H3.1 mark early- and late-replicating chromatin, respectively. In the nucleus, H3.3 forms domains, which decrease in density throughout replication, while H3.1 domains increase in density. Hydroxyurea impairs local recycling of parental histones at replication sites. Similarly, depleting the histone chaperone ASF1 affects recycling, leading to an impaired histone variant landscape. We discuss how faithful transmission of histone variants involves ASF1 and can be impacted by replication stress, with ensuing consequences for cell fate and tumorigenesis.

Multi-scale tracking reveals scale-dependent chromatin dynamics after DNA damage.

The dynamic organization of genes inside the nucleus is an important determinant for their function. Using fast DNA tracking microscopy in S. cerevisiae cells and improved analysis of mean square displacements, we quantified DNA motion at time scales ranging from 10 milliseconds to minute and found that following DNA damage, DNA exhibits distinct sub-diffusive regimes. In response to double-strand breaks, chromatin is more mobile at large time scales but, surprisingly, its mobility is reduced at short time scales. This effect is even more pronounced at the site of damage. Such a pattern of dynamics is consistent with a global increase in chromatin persistence length in response to DNA damage. Scale-dependent nuclear exploration is regulated by the Rad51 repair protein, both at the break and throughout the genome. We propose a model in which stiffening of the damaged ends by the repair complex, combined with global increased stiffness, act like a "needle in a ball of yarn", enhancing the ability of the break to traverse the chromatin meshwork.

DNA in motion during double-strand break repair.

DNA organization and dynamics profoundly affect many biological processes such as gene regulation and DNA repair. In this review, we present the latest studies on DNA mobility in the context of DNA damage. Recent studies demonstrate that DNA mobility is dramatically increased in the presence of double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae. As a consequence, chromosomes explore a larger nuclear volume, facilitating homologous pairing but also increasing the rate of ectopic recombination. Increased DNA dynamics is dependent on several homologous recombination (HR) proteins and we are just beginning to understand how chromosome dynamics is regulated after DNA damage.

Increased chromosome mobility facilitates homology search during recombination.

Homologous recombination, an essential process for preserving genomic integrity, uses intact homologous sequences to repair broken chromosomes. To explore the mechanism of homologous pairing in vivo, we tagged two homologous loci in diploid yeast Saccharomyces cerevisiae cells and investigated their dynamic organization in the absence and presence of DNA damage. When neither locus is damaged, homologous loci occupy largely separate regions, exploring only 2.7% of the nuclear volume. Following the induction of a double-strand break, homologous loci co-localize ten times more often. The mobility of the cut chromosome markedly increases, allowing it to explore a nuclear volume that is more than ten times larger. Interestingly, the mobility of uncut chromosomes also increases, allowing them to explore a four times larger volume. We propose a model for homology search in which increased chromosome mobility facilitates homologous pairing. Finally, we find that the increase in DNA dynamics is dependent on early steps of homologous recombination.