Developmental Biology

Genetic and physical interactions reveal overlapping and distinct contributions to meiotic double-strand break formation in C. elegans

Marilina Raices
Fabiola Balmir
Nicola Silva
Wei Li
McKenzie K. Grundy
Dane K. Hoffman
Elisabeth Altendorfer
Carlos Jaime Camacho
Kara A. Bernstein
Monica P. Colaiácovo
Judith Yanowitz author has email address

Magee-Womens Research Institute, Pittsburgh, PA 15213 USA
Department of Biology, Masaryk University, Czech Republic;
Tsinghua U. Medical School, China;
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, UPMC Hillman Cancer Center, Pittsburgh, Pennsylvania;
Department of Genetics, Blavatnik Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Room 334, Boston, MA 02115, USA;
Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213 USA;
Department of Biochemistry and Biophysics, University of Pennsylvania, Penn Center for Genome Integrity, Philadelphia, Pennsylvania;
Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213 USA

https://doi.org/10.7554/eLife.96458.1

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Figures and data

XND-1 and HIM-17 interact and are predominantly localized away from the DNA axis.
A) Summary of the yeast two-hybrid assay results. The complete set of results is given in Figure S1A. B) Western blot analysis of endogenous XND-1 (top) and HIM-5::GFP::FLAG (bottom) on HA pull-downs performed in him-17::3xHA;him-5::GFP::3xFLAG strain. Analysis was performed in biological duplicates. C) Mass spectrometry results of GFP pulldowns of HIM-17::GFP. IP/MS analysis was performed in duplicate and corrected for nonspecific binding of proteins bound to GFP trap beads. D) Quantification of embryonic viability (hatching rates) and frequencies of male offspring among the progeny of the indicated genotypes. Data are shown as mean +/- SD; NS stands for not significant; **** p < 0.0001, n= number of P₀ parents whose brood was examined. E) Representative STED images of mid-pachytene stage nuclei showing substantial overlap of XND-1 and HIM-17 on DNA not at the chromosome axis (stained with HTP-3). The X chromosome is not stained by either XND-1 and HIM-17 (red arrow). F) Representative confocal images showing co-localization of XND-1 and HIM-17 with an autosome-enriched variant histone, HTZ-1. Red arrow indicates the X chromosome. Scale bars = 2μm.

DSB-1 associates with HIM-5 and regulates its localization
A) Summary of the yeast two-hybrid assay results. The complete set of results is given in Figure S1A. B) Western blot analysis of GFP pull-downs performed in GFP::dsb-1;him-5::3XHA strain showing co-IP of HA-tagged HIM-5 proteins. Analysis was performed in biological duplicates. C) Quantification of DAPI-stained bodies at diakinesis for the indicated genotypes. Colors correspond to the number of DAPI-stained bodies shown in the key below. D) Quantification of embryonic viability (hatching rates) and frequencies of male offspring among the progeny of the indicated genotypes. Data are shown as mean +/- SD; NS stands for not significant; **** p < 0.0001, n= number of P₀ parents whose brood was examined. E) Top: C. elegans gonad fixed and stained with DAPI to show the organization and distribution of the nuclei along the Prophase I. Bottom: live images of nuclei in the transition zone (leptotene-zygotene) and middle-pachytene. eaIs15 (Ppie-1::him-5) is visualized in freshly dissected gonads by GFP fluorescence (green), and DNA by DRAQ5 (red). In dsb-1 mutants, HIM-5 is located is nuclear in the transition zone and becomes located in cytoplasmic puncta by middle pachytene.

Genetic and protein-protein interaction studies show evidence of DSB protein sub-complexes in C. elegans.
A) Summary of the genetic interaction results. The complete set of results is given in Figure S3. B) Genetic groups are based on data presented here and previously published (Chung et al., 2015; Mateo et al., 2016; McClendon et al., 2016; Janisiw et al., 2020). C) Summary of the yeast two-hybrid assay results. The complete set of results is given in Figure S1A. D) Schematic representation of the interaction network based on Y2H interactions. Thick line: Strong interaction between two proteins. Thin line: weak interaction between two proteins. E) Different DSB-1 constructs (detailed in F) were used to analyze yeast-two-hybrid interactions with SPO-11, DSB-2, and DSB-3. F) Scheme representation of the wild-type DSB-1 structure, and the different truncations used in E) For more details about the DSB-1 sequence, protein structure, and deletions used in these experiments, see Figure S5.

Model of the DSB formation complex compared in S. cerevisiae, C. elegans, and A. thaliana.
The results of this paper and previous work discussed in this article allow us to propose a model to explain the known interactions and localization patterns of the DSB regulatory factors in C. elegans (center) where SPO-11 forms a subcomplex with DSB-1/-2/-3 that is located initially on the DNA loops and that we propose is brought to the chromosome axes by association with HIM-5 and its binding to MRE-11 and PARG-1 which both associate with HTP-3. SPO-11 could also be recruited to the DSB-1 after axis association. Other actors of the complex are also initially on the chromatin loops and also get recruited to the axes by HIM-5, allowing the formation of DSBs and coupling of DSBs to downstream repair by the MRN complex and others. This model shares some similarities to what was proposed for S. cerevisiae (Bouuaert et al., 2021b; Sommermeyer et al., 2013)and A. thaliana (Vrielynck et al., 2021). For more details see the Discussion.

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