Biology subjects

Chiolo, I.

Publications and source records attributed to Chiolo, I..

6 recordsLinked to original sources

bioRxiv2023

'Off-pore' nucleoporins relocalize heterochromatic breaks through phase separation

Phase separation forms membraneless compartments in the nuclei, including by establishing heterochromatin "domains" and repair foci. Pericentromeric heterochromatin mostly comprises repeated sequences prone to aberrant recombination, and "safe" homologous recombination (HR) repair of these sequences requires the movement of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. How this mobilization initiates is unknown, and the contribution of phase separation to these dynamics is unclear. Here, we show that Nup98 nucleoporin is recruited to heterochromatic repair sites before relocalization through Sec13 or Nup88 nucleoporins, and downstream from the Smc5/6 complex and SUMOylation. Remarkably, the phase separation properties of Nup98 are required and sufficient to mobilize repair sites and exclude Rad51, thus preventing aberrant recombination while promoting HR repair. Disrupting this pathway results in heterochromatin repair defects and widespread chromosome rearrangements, revealing a novel "off-pore" role for nucleoporins and phase separation in nuclear dynamics and genome integrity in a multicellular eukaryote. HighlightsO_LINup88 and Sec13 recruit Nup98 to heterochromatic DSBs downstream from Smc5/6 C_LIO_LINup88, Sec13 and Nup98 promote repair focus mobilization in heterochromatin off-pore C_LIO_LINup98 excludes Rad51 from repair sites inside the heterochromatin domain C_LIO_LIPhase separation by Nup98 is required and sufficient for relocalization and Rad51 exclusion C_LI

cell biology↗

bioRxiv2023

Replication fork plasticity upon replication stress requires rapid nuclear actin polymerization

Cells rapidly respond to replication stress actively slowing fork progression and inducing fork reversal. How replication fork plasticity is achieved in the context of nuclear organization is currently unknown. Using nuclear actin probes in living and fixed cells, we visualized nuclear actin filaments in unperturbed S phase, rapidly extending in number and thickness upon genotoxic treatments, and taking frequent contact with replication factories. Chemically or genetically impairing nuclear actin polymerization shortly before these treatments prevents active fork slowing and abolishes fork reversal. Defective fork plasticity is linked to reduced recruitment of RAD51 and SMARCAL1 to nascent DNA. Conversely, PRIMPOL gains access to replicating chromatin, promoting unrestrained and discontinuous DNA synthesis, which is associated with increased chromosomal instability and decreased cellular resistance to replication stress. Hence, nuclear F-actin orchestrates replication fork plasticity and is a key molecular determinant in the rapid cellular response to genotoxic treatments.

cancer biology↗

bioRxiv2023

Mutagenic-end joining results in smaller deletions in heterochromatin relative to euchromatin

Pericentromeric heterochromatin is highly enriched for repetitive sequences prone to aberrant recombination. Previous studies showed that homologous recombination (HR) repair is uniquely regulated in this domain to enable safe repair while preventing aberrant recombination. In Drosophila cells, DNA double-strand breaks (DSBs) relocalize to the nuclear periphery through nuclear actin-driven directed motions before recruiting the strand invasion protein Rad51 and completing HR repair. End-joining (EJ) repair also occurs with high frequency in heterochromatin of fly tissues, but how alternative EJ (alt-EJ) pathways operate in heterochromatin remains largely uncharacterized. Here, we induce DSBs in single euchromatic and heterochromatic sites using a new system that combines the DR-white reporter and I-SceI expression in spermatogonia of flies. Using this approach, we detect higher frequency of HR repair in heterochromatin, relative to euchromatin. Further, sequencing of mutagenic repair junctions reveals the preferential use of different EJ pathways across distinct euchromatic and heterochromatic sites. Interestingly, synthesis-dependent microhomology-mediated end joining (SD-MMEJ) appears differentially regulated in the two domains, with a preferential use of motifs close to the cut site in heterochromatin relative to euchromatin, resulting in smaller deletions. Together, these studies establish a new approach to study repair outcomes in fly tissues, and support the conclusion that heterochromatin uses more HR and less mutagenic EJ repair relative to euchromatin.

cell biology↗

bioRxiv2019

Arp2/3 and Unc45 maintain heterochromatin stability in Drosophila polytene chromosomes.

Repairing DNA double-strand breaks (DSBs) is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination. In Drosophila Kc cells, accurate homologous recombination (HR) repair of heterochromatic DSBs relies on the relocalization of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. This movement is driven by Arp2/3-dependent nuclear actin filaments and myosins ability to walk along them. Conserved mechanisms enable the relocalization of heterochromatic DSBs in mouse cells, and their defects lead to massive ectopic recombination in heterochromatin and chromosome rearrangements. In Drosophila polytene chromosomes, extensive DNA movement is blocked by a stiff structure of chromosome bundles. Repair pathways in this context are poorly characterized, and whether heterochromatic DSBs relocalize in these cells is unknown. Here, we show that damage in heterochromatin results in relaxation of the heterochromatic chromocenter, consistent with a dynamic response in this structure. Arp2/3, the Arp2/3 activator Scar, and the myosin activator Unc45, are required for heterochromatin stability in polytene cells, suggesting that relocalization enables heterochromatin repair in this tissue. Together, these studies reveal critical roles for actin polymerization and myosin motors in heterochromatin repair and genome stability across different organisms and tissue types.\n\nImpact StatementHeterochromatin relies on dedicated pathways for safe recombinational repair. In mouse and fly cultured cells, DNA repair requires the movement of repair sites away from the heterochromatin domain via nuclear actin filaments and myosins. Here, we explore the importance of these pathways in Drosophila salivary gland cells, which feature a stiff bundle of endoreduplicated polytene chromosomes. Repair pathways in polytene chromosomes are largely obscure and how nuclear dynamics operate in this context is unknown. We show that heterochromatin relaxes in response to damage, and relocalization pathways are necessary for repair and stability of heterochromatic sequences. This deepens our understanding of repair mechanisms in polytenes, revealing unexpected dynamics. It also provides a first understanding of nuclear dynamics responding to replication damage or rDNA breaks, providing a new understanding of the importance of the nucleoskeleton in genome stability. We expect these discoveries to shed light on tumorigenic processes, including therapy-induced cancer relapses.

cell biology↗

bioRxiv2017

Quantitative methods to investigate the 4D dynamics of heterochromatic repair sites in Drosophila cells

Heterochromatin is mostly composed of long stretches of repeated DNA sequences prone to ectopic recombination during double-strand break (DSB) repair. In Drosophila, safe homologous recombination (HR) repair of heterochromatic DSBs relies on a striking relocalization of repair sites to the nuclear periphery. Central to understanding heterochromatin repair is the ability to investigate the 4D dynamics (movement in space and time) of repair sites. A specific challenge of these studies is preventing phototoxicity and photobleaching effects while imaging the sample over long periods of time, and with sufficient time points and Z-stacks to track repair foci over time. Here we describe an optimized approach for high-resolution live imaging of heterochromatic DSBs in Drosophila cells, with a specific emphasis on the fluorescent markers and imaging setup used to capture the motion of repair foci over long time periods. We detail approaches that minimize photobleaching and phototoxicity with a DeltaVision widefield deconvolution microscope, and image-processing techniques for signal recovery post-imaging using SoftWorX and Imaris software. We present a method to derive mean square displacement (MSD) curves revealing some of the biophysical properties of the motion. Finally, describe a method in R to identify tracts of directed motions in mixed trajectories. These approaches enable a deeper understanding of the mechanisms of heterochromatin dynamics and genome stability in the three-dimensional context of the nucleus, and have broad applicability in the field of nuclear dynamics.

cell biology↗

bioRxiv2017

The 3D genome organization of Drosophila melanogaster through data integration

Genome structures are dynamic and non-randomly organized in the nucleus of higher eukaryotes. To maximize the accuracy and coverage of 3D genome structural models, it is important to integrate all available sources of experimental information about a genomes organization. It remains a major challenge to integrate such data from various complementary experimental methods. Here, we present an approach for data integration to determine a population of complete 3D genome structures that are statistically consistent with data from both genome-wide chromosome conformation capture (Hi-C) and lamina-DamID experiments. Our structures resolve the genome at the resolution of topological domains, and reproduce simultaneously both sets of experimental data. Importantly, this framework allows for structural heterogeneity between cells, and hence accounts for the expected plasticity of genome structures. As a case study we choose Drosophila melanogaster embryonic cells, for which both data types are available. Our 3D geome structures have strong predictive power for structural features not directly visible in the initial data sets, and reproduce experimental hallmarks of the D. melanogaster genome organization from independent and our own imaging experiments. Also they reveal a number of new insights about the genome organization and its functional relevance, including the preferred locations of heterochromatic satellites of differnet chromosomes, and observations about homologous pairing that cannot be directly observed in the original Hi-C or lamina-DamID data. To our knowledge our approach is the first that allows systematic integration of Hi-C and lamina DamID data for complete 3D genome structure calculation, while also explicitly considering genome structural variability.

bioinformatics↗