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1. Quantitative Methods to Investigate the 4D Dynamics of Heterochromatic Repair Sites in Drosophila Cells

   https://doi.org/10.1016/bs.mie.2017.11.033  

   Caridi, C; Delabaere, L; Tjong, H; Hopp, H; Das, D; Alber, F; Chiolo, I. (March 2018, Methods in enzymology)

   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 postimaging using SoftWorX and Imaris software. We present a method to derive mean square displacement curves revealing some of the biophysical properties of the motion. Finally, we describe a method in R to identify tracts of directed motions (DMs) 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. 

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2. Nup153 is not required for anchoring heterochromatic DSBs to the nuclear periphery

   Taehyun, Ryu; Chiara, Merigliano; Irene, Chiolo (April 2024, microPublication biology)

   Pericentromeric heterochromatin mostly comprises repeated DNA sequences prone to ectopic recombination. In Drosophila cells, ‘safe’ homologous recombination repair requires relocalization of heterochromatic repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. DSBs are anchored to the nuclear periphery through the Nup107/160 nucleoporin complex. Previous studies suggested that the nuclear pore ‘basket’ protein Nup153 could also mediate anchoring, but Nup153 RNAi depletion also affects Nup107 association with the pores, preventing a direct assessment of Nup153 role. Using a separation of function mutant, here we show that Nup153 is not required for anchoring heterochromatic DSBs to the nuclear periphery. 

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3. Alternative end-joining results in smaller deletions in heterochromatin relative to euchromatin

   Jacob, Miller M; Sydney, Prange; Huanding, Ji; Alesandra, Rau R; Varandt, Khodaverdian Y; Xiao, Li; Avi, Patel; Nadejda, Butova; Avery, Lutter; Helen, Chung; et al (December 2023, eLife)

   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. 

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4. “Off-pore” nucleoporins relocalize heterochromatic breaks through phase separation

   https://doi.org/10.1101/2023.12.07.570729  

   Merigliano, Chiara; Ryu, Taehyun; See, Colby D.; Caridi, Christopher P.; Li, Xiao; Butova, Nadejda L; Reynolds, Trevor; Deng, Changfeng; Chenoweth, David M.; Capelson, Maya; et al (December 2023, bioRxiv)

   SUMMARY 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. HighlightsNup88 and Sec13 recruit Nup98 to heterochromatic DSBs downstream from Smc5/6Nup88, Sec13 and Nup98 promote repair focus mobilization in heterochromatin ‘off-pore’Nup98 excludes Rad51 from repair sites inside the heterochromatin domainPhase separation by Nup98 is required and sufficient for relocalization and Rad51 exclusion 

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5. Replication forks associated with long nuclear actin filaments in mild stress conditions display increased dynamics

   Merigliano, Chiara; Palumbieri, Maria Dilia; Lopes, Massimo; Chiolo, Irene (November 2024, microPublication biology)

   Nuclear actin filaments (F-actin) form during S-phase and in response to replication stress to promote fork remodeling and repair. In mild replication stress conditions, nuclear actin polymerization is required to limit PrimPol recruitment to the fork while promoting fork reversal. Both short and long filaments form during this response, but their function in the nuclear dynamics of replication sites was unclear. Here, we show that replication centers associated with long nuclear actin filaments become more mobile than the rest of the forks, suggesting relocalization of replication sites as a response to prolonged fork stalling and/or fork breakage, even in response to mild replication stress. 

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