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1. Live Cell Imaging of Nuclear Actin Filaments and Heterochromatic Repair foci in Drosophila and Mouse Cells

   https://doi.org/10.7287/peerj.preprints.27900v1  

   See, C ; Arya, D ; Lin, E ; Chiolo, I ( July 2021 , Methods in molecular biology)

   null (Ed.)

   Pericentromeric heterochromatin is mostly composed of repeated DNA sequences, which are prone to aberrant recombination during double-strand break (DSB) repair. Studies in Drosophila and mouse cells revealed that ‘safe’ homologous recombination (HR) repair of these sequences relies on the relocalization of repair sites to outside the heterochromatin domain before Rad51 recruitment. Relocalization requires a striking network of nuclear actin filaments (F-actin) and myosins that drive directed motions. Understanding this pathway requires the detection of nuclear actin filaments that are significantly less abundant than those in the cytoplasm, and the imaging and tracking of repair sites for long time periods. Here, we describe an optimized protocol for live cell imaging of nuclear F-actin in Drosophila cells, and for repair focus tracking in mouse cells, including: imaging setup, image processing approaches, and analysis methods. We emphasize approaches that can be applied to identify the most effective fluorescent markers for live cell imaging, strategies to minimize photobleaching and phototoxicity with a DeltaVision deconvolution microscope, and image processing and analysis methods using SoftWoRx and Imaris software. These approaches enable a deeper understanding of the spatial and temporal dynamics of heterochromatin repair and have broad applicability in the fields of nuclear architecture, nuclear dynamics, and DNA repair. 

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2. ‘Off-pore’ nucleoporins relocalize heterochromatic breaks through phase separation

   Merigliano, Chiara ; Ryu, Taehyun ; See, Colby D. ; Caridi, Christopher P. ; Butova, Nadejda L ; Reynolds, Trevor ; Deng, Changfeng ; Chenoweth, David M. ; Capelson, Maya ; Chiolo Irene. ( December 2023 , Biorxiv)

   Heterochromatin mostly comprises repeated DNA sequences prone to ectopic recombination. In Drosophila cells, ‘safe’ homologous recombination (HR) repair of heterochromatic double-strand breaks (DSBs) requires relocalization of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. Relocalization is driven by nuclear actin filaments and myosins, while anchoring is mediated by the Nup107 complex at nuclear pores. Here, we show an additional ‘off pore’ role of nucleoporins in heterochromatin repair. Sec13 and Nup88 independently recruit Nup98 to DSBs before relocalization and downstream from the Smc5/6 complex and SUMOylation. Remarkably, the phase separation properties of Nup98 are required and sufficient to induce the mobilization of repair sites and to exclude Rad51, thus preventing aberrant recombination while facilitating HR repair. Disrupting this pathway results in heterochromatin repair defects, revealing a novel role for nucleoporins and phase separation in nuclear dynamics and genome integrity in a multicellular eukaryote. 

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

   Miller, Jacob M ; Prange, Sydney ; Ji, Huanding ; Rau, Alesandra R ; Khodaverdian, Varandt Y ; Li, Xiao ; Patel, Avi ; Butova Nadejda L ; Lutter, Avery ; Chung, Helen ; 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. Diffusion and distal linkages govern interchromosomal dynamics during meiotic prophase

   https://doi.org/10.1073/pnas.2115883119  

   Newman, Trent A. ; Beltran, Bruno ; McGehee, James M. ; Elnatan, Daniel ; Cahoon, Cori K. ; Paddy, Michael R. ; Chu, Daniel B. ; Spakowitz, Andrew J. ; Burgess, Sean M. ( March 2022 , Proceedings of the National Academy of Sciences)

   The pairing of homologous chromosomes (homologs) in meiosis is essential for distributing the correct numbers of chromosomes into haploid gametes. In budding yeast, pairing depends on the formation of 150 to 200 Spo11-mediated double-strand breaks (DSBs) that are distributed among 16 homolog pairs, but it is not known if all, or only a subset, of these DSBs contribute to the close juxtaposition of homologs. Having established a system to measure the position of fluorescently tagged chromosomal loci in three-dimensional space over time, we analyzed locus trajectories to determine how frequently and how long loci spend colocalized or apart. Continuous imaging revealed highly heterogeneous cell-to-cell behavior of foci, with the majority of cells exhibiting a “mixed” phenotype where foci move into and out of proximity, even at late stages of prophase, suggesting that the axial structures of the synaptonemal complex may be more dynamic than anticipated. The observed plateaus of the mean-square change in distance (MSCD) between foci informed the development of a biophysical model of two diffusing polymers that captures the loss of centromere linkages as cells enter meiosis, nuclear confinement, and the formation of Spo11-dependent linkages. The predicted number of linkages per chromosome in our theoretical model closely approximates the small number (approximately two to four) of estimated synapsis-initiation sites, suggesting that excess DSBs have negligible effects on the overall juxtaposition of homologs. These insights into the dynamic interchromosomal behavior displayed during homolog pairing demonstrate the power of combining time-resolved in vivo analysis with modeling at the granular level. 

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5. Recovery of Alternative End-Joining Repair Products From Drosophila Embryos

   https://doi.org/10.1016/bs.mie.2017.11.027  

   Hanscom, T ; Khodaverdian, VY ; McVey, M ( February 2018 , Methods in enzymology)

   In this chapter, we describe a method for the recovery and analysis of alternative end-joining (alt-EJ) DNA double-strand break repair junctions following I-SceI cutting in Drosophila melanogaster embryos. Alt-EJ can be defined as a set of Ku70/80 and DNA ligase 4-independent end-joining processes that are typically mutagenic, producing deletions, insertions, and chromosomal rearrangements more frequently than higher-fidelity repair pathways such as classical nonhomologous end joining or homologous recombination. Alt-EJ has been observed to be upregulated in HR-deficient tumors and is essential for the survival and proliferation of these cells. Alt-EJ shares many initial processing steps with homologous recombination, specifically end resection; therefore, studying alt-EJ repair junctions can provide useful insight into aborted HR repair. Here, we describe the injection of plasmid constructs with specific cut sites into Drosophila embryos and the subsequent recovery of alt-EJ repair products. We also describe different analytical approaches using this system and how amplicon sequencing can be used to provide mechanistic information about alt-EJ. 

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