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E4 ligase–specific ubiquitination hubs coordinate DNA double-strand-break repair and apoptosis

Nature Structural & Molecular Biology volume 23, pages 995–1002 (2016)Cite this article

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Abstract

Multiple protein ubiquitination events at DNA double-strand breaks (DSBs) regulate damage recognition, signaling and repair. It has remained poorly understood how the repair process of DSBs is coordinated with the apoptotic response. Here, we identified the E4 ubiquitin ligase UFD-2 as a mediator of DNA-damage-induced apoptosis in a genetic screen in Caenorhabditis elegans. We found that, after initiation of homologous recombination by RAD-51, UFD-2 forms foci that contain substrate-processivity factors including the ubiquitin-selective segregase CDC-48 (p97), the deubiquitination enzyme ATX-3 (Ataxin-3) and the proteasome. In the absence of UFD-2, RAD-51 foci persist, and DNA damage-induced apoptosis is prevented. In contrast, UFD-2 foci are retained until recombination intermediates are removed by the Holliday-junction-processing enzymes GEN-1, MUS-81 or XPF-1. Formation of UFD-2 foci also requires proapoptotic CEP-1 (p53) signaling. Our findings establish a central role of UFD-2 in the coordination between the DNA-repair process and the apoptotic response.

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Figure 1: Ubiquitin ligase activity of UFD-2 is required for apoptosis execution.
Figure 2: UFD-2 forms foci late after IR treatment.
Figure 3: UPS factors accumulate in UFD-2 hubs and balance apoptotic signaling.
Figure 4: Loss of ufd-2 delays DSB-repair processing.
Figure 5: UFD-2 foci in repair and apoptosis after DNA damage.
Figure 6: UFD-2 coordinates communication between repair and apoptosis after DNA damage.

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Acknowledgements

We thank Y. Kohara (National Institute of Genetics, Shizuoka, Japan), the Caenorhabditis Genetics Center (funded by the NIH National Center for Research Resources), the Bloomington Stock Center, M. Vidal (Dana-Farber Cancer Institute), Addgene and Geneservice Ltd for antibodies, plasmids, cDNAs and strains. We thank K. Gödderz, A. Lisowski and E. Stellbrink for technical help. We thank A. Franz and A. Gutschmidt for critical reading of the manuscript. We thank K. Ramadan (CRVK/MRC Oxford Institute for Radiation Oncology) and Y. Shiloh (David and Inez Myers Laboratory for Cancer Genetics) for insightful discussions on the project and for exchange of unpublished results. This work was supported by a Wellcome Trust Senior Research award (090944/Z/09/Z) to A.G.; grants from the German-Israeli Foundation (GIF 1104-68.11/2010), the Deutsche Forschungsgemeinschaft (EXC 229, SFB 829, SFB 670 and KFO 286), the European Research Council (ERC Starting Grant 260383), Marie Curie Actions (FP7 ITN CodeAge 316354, aDDRess 316390 and MARRIAGE 316964) and the Bundesministerium für Forschung und Bildung (Sybacol FKZ0315893A-B) to B.S.; and the Deutsche Forschungsgemeinschaft (EXC 229, HO 2541/8-1 and KFO 286) and the European Research Council (consolidator grant 616499) to T.H. In addition, this work was supported by COST Action (PROTEOSTASIS BM1307), supported by COST (European Cooperation in Science and Technology).

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  1. Leena Ackermann and Michael Schell: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Genetics, CECAD Research Center,

    Leena Ackermann, Michael Schell, Wojciech Pokrzywa, Éva Kevei & Thorsten Hoppe

  2. , University of Cologne, Cologne, Germany

    Leena Ackermann, Michael Schell, Wojciech Pokrzywa, Éva Kevei & Thorsten Hoppe

  3. Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK

    Anton Gartner

  4. Institute for Genome Stability in Aging and Disease, Medical Faculty, University of Cologne, Cologne, Germany

    Björn Schumacher

  5. CECAD Research Center, Center for Molecular Medicine Cologne,

    Björn Schumacher

  6. , University of Cologne, Cologne, Germany

    Björn Schumacher

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Contributions

L.A. and M.S. designed, performed and analyzed the experiments. W.P. performed in vitro ubiquitination assays. É.K. generated anti-ATX-3. A.G. and B.S. designed and performed the RNAi screen. B.S. and T.H. supervised the design and data interpretation. L.A., B.S. and T.H. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Cell-cycle arrest and developmental and physiological apoptosis are functional in ufd-2-deleted worms.

(a) Developmental apoptosis in head region of newly hatched L1 larvae of indicated genotypes. Data show means ± s.e.m. of 3 independent experiments. n = 38 animals for ced-1(e1735) and n = 33 animals for ced-1(e1735);ufd-2(tm1380). n.s. not significant in two-tailed Student’s t-test. (b) Physiological apoptosis in pachytene region of day 1 adults of indicated genotypes. Data show means ± s.e.m. of 3 independent experiments. n varied from 44-45 animals, see Supplementary Table 2. The triple asterisk indicates P values of ≤ 0.0001 in two-tailed Student’s t-test, n.s. not significant. (c) Representative DIC images of cell cycle arrest in mitotic region of germlines of indicated genotypes 16 hrs after IR treatment(60 Gy). Filled arrowheads mark enlarged arrested mitotic cells. Representative images of 3 independent experiments. (d) Quantification of cell cycle arrest in mitotic region of germlines 16 hrs after IR treatment (60 Gy). Data show means ± s.e.m. of 3 independent experiments. n varied from 23-33 animals, see Supplementary Table 2. The triple asterisk indicates P value of ≤ 0.0001 in two-tailed Student’s t-test, n.s. not significant. (e) Autoubiquitylation of UFD-2 using UFD-2 (wild-type), UFD-2P951A, or UFD-2C448Y as ubiquitin ligases and ubiquitin (wild-type), or ubiquitin mutants that contain only one lysine for conjugation (ubiquitinonly K29, ubiquitinonly K48). Representative immunoblot of 3 independent experiments.

Supplementary Figure 2 UFD-2 forms foci late after damage induction.

(a) α-UFD-2 and α-TBG-1 western blot. Samples are lysates of wild-type, ufd-2(tm1380) and ufd-(hh1) worms. Representative immunoblot of 3 independent experiments. (b) Schematic illustration of C. elegans germline and illustrative images of a IR treated (60 Gy) germline stained with α-UFD-2 and DAPI. Two slides from a z-stack (z-slide 1 and z-slide b), one from upper and one from lower part of the germline are shown. Filled arrowheads mark nuclei positive for UFD-2 foci. Scale bar, 10 µm. (c) Quantification of UFD-2 foci in pachytene region of wild-type germlines treated with IR (0, 60 Gy) isolated 5 and 24 hrs later. Data show means of ± s.e.m. of 3 experiments. n varied from 37-61 animals, see Supplementary Table 2.

Supplementary Figure 3 UFD-2 forms IR-dependent foci and interacts with CDC-48.

(a) Quantification of UFD-2 foci in pachytene region of wild-type germlines 24 hrs after treatment with IR (0, 60 Gy) or UV (600 J/m2). Data show means ± s.e.m. of 3 experiments. n varied from 69-96 animals, see Supplementary Table 2. (b) Embryonic survival after IR treatment (0, 30 or 60 Gy). Data show means ± s.e.m. of five experiments. n varied from 75-147 animals, see Supplementary Table 2. The single, double and triple asterisks indicate P values of ≤ 0.05, 0.001, 0.0001 in two-tailed Student’s t-test. (c) Representative images of worm germlines of indicated genotypes stained with α-ATX-3 antibody and DAPI 24 hrs after IR treatment (60 Gy). Scale bar, 5 µm. Representative images of 3 independent experiments. (d) α-ATX-3 and α-TBG-1 western blot. Samples are lysates of wild-type and atx-3(gk193) worms. Representative immunoblot of 3 independent experiments. (e) Quantification of number of UFD-2 foci that co-localize with ubiquitin in wild-type germlines 24 hrs after IR treatment (60 Gy). Data show means ± s.e.m. of 3 experiments. n = 32 animals. (f) Sequence alignment shows UFD-2 from C. elegans and other species, the conserved residue C448 is highlighted. (g) in vivo co-immunoprecipitation of CDC-48 with UFD-2 from indicated worm lysates. Representative immunoblot of 3 independent experiments. (h) in vitro co-immunoprecipitation of CDC-48 with UFD-2 with purified recombinantly expressed protein. Representative immunoblot of 3 independent experiments. (i) Autoubiquitylation of UFD-2 using UFD-2 (wild-type), UFD-2P951A or UFD-2C448Y as ubiquitin ligases. Representative immunoblot of 3 independent experiments. (j) Quantification of UFD-2 foci in pachytene region of indicated germlines 24 hrs after IR treatment (0, 60 Gy). Data show means of ± s.e.m. of three experiments. n varied from 40-43 animals, see Supplementary Table 2. (k) Quantification of ubiquitin foci in pachytene region of indicated germlines 24 hrs after IR treatment (60 Gy). Data show means of ± s.e.m. of three experiments. n varied from 41-45 animals, see Supplementary Table 2.

Supplementary Figure 4 Apoptosis induction in ufd-2-deleted worms is functional.

(a) Representative images of worm germlines of indicated genotypes stained with α-UFD-2 antibody and DAPI 24 hrs after IR treatment (60 Gy). Filled and empty arrowhead indicated nuclei positive or negative for UFD-2 foci, respectively. Scale bar, 5 µm. Representative images of 3 independent experiments. (b) α-CEP-1 and α-TBG-1 western blot of worm lysates of indicated genotypes. Representative immunoblot of 3 independent experiments and (c) corresponding quantification. (d) Relative expression levels of egl-1 target gene in wild-type and ufd-2(tm1380) worms at indicated time points after IR treatment (0 or 60 Gy). mRNA levels were normalized to 0 Gy samples. Data show means ± s.e.m. of 3 independent experiments.

Supplementary Figure 5 UFD-2 foci are dependent on apoptosis and DNA-damage signaling.

(a) Representative images of worm germlines of indicated genotypes stained with α-UFD-2 antibody and DAPI 24 hrs after IR treatment (60 Gy). Filled and empty arrowhead indicated nuclei positive or negative for UFD-2 foci, respectively. Scale bar, 5 µm. Representative images of 3 independent experiments. (b) α-UFD and α-TBG-1 western blot of worm lysates of indicated genotypes 24 hrs after IR treatment (0, 60 Gy). Representative immunoblot of 3 independent experiments. (c) Quantification of germ cells positive for RAD-51 staining of indicated genotypes 24 hrs after IR treatment (0 or 20 Gy). Data show means ± s.e.m. of 3 independent experiments. n varied from 30-33 animals, see Supplementary Table 2. n.s. not significant in two-tailed Student’s t-test.

Supplementary Figure 6 RAD-51 in DSB repair.

(a) Embryonic survival after IR treatment (0, 30 or 60 Gy) of indicated genotypes. Data show means ± s.e.m. of 3 independent experiments. n varied from 90-95 animals, see Supplementary Table 2. The single asterisk indicates P value of ≤ 0.05 in two-tailed Student’s t-test. (b) Quantification of germ cells positive for RAD-51 staining of indicated genotypes 24 hrs after IR treatment (0 or 20 Gy). Data show means ± s.e.m. of 3 independent experiments. n varied from 35-48 animals, see Supplementary Table 2. The single and double asterisks indicate P values of ≤ 0.05 and 0.001 in two-tailed Student’s t-test. (c) Embryonic survival after IR treatment (0, 30 or 60 Gy) of indicated genotypes treated with rad-51 or control RNAi. Data show means ± s.e.m. of one experiment, n varied from 15-25 animals, see Supplementary Table 2. The double asterisk indicates P value of ≤ 0.001 in two-tailed Student’s t-test.

Supplementary Figure 7 Full-sized images of immunoblots in figures.

(a) Presented in Figure 1e. (b) Presented in Supplementary Figure 1e. (c) Presented in Supplementary Figure 2a. (d) Presented in Supplementary Figure 3d. (e) Presented in Supplementary Figure 3g. (f) Presented in Supplementary Figure 3h. (g) Presented in Supplementary Figure 3i. (h) Presented in Supplementary Figure 4b. (i) Presented in Supplementary Figure 5b.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 1616 kb)

Supplementary Table 1

n values for Figures 1–6 (XLSX 13 kb)

Supplementary Table 2

n values for Supplementary Figures 1–6 (XLSX 10 kb)

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Ackermann, L., Schell, M., Pokrzywa, W. et al. E4 ligase–specific ubiquitination hubs coordinate DNA double-strand-break repair and apoptosis. Nat Struct Mol Biol 23, 995–1002 (2016). https://doi.org/10.1038/nsmb.3296

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  • DOI: https://doi.org/10.1038/nsmb.3296

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