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SCAI promotes DNA double-strand break repair in distinct chromosomal contexts

Nature Cell Biology volume 18, pages 1357–1366 (2016)Cite this article

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Abstract

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose accurate repair by non-homologous end-joining (NHEJ) or homologous recombination (HR) is crucial for genome integrity and is strongly influenced by the local chromatin environment1,2. Here, we identify SCAI (suppressor of cancer cell invasion) as a 53BP1-interacting chromatin-associated protein that promotes the functionality of several DSB repair pathways in mammalian cells. SCAI undergoes prominent enrichment at DSB sites through dual mechanisms involving 53BP1-dependent recruitment to DSB-surrounding chromatin and 53BP1-independent accumulation at resected DSBs. Cells lacking SCAI display reduced DSB repair capacity, hypersensitivity to DSB-inflicting agents and genome instability. We demonstrate that SCAI is a mediator of 53BP1-dependent repair of heterochromatin-associated DSBs, facilitating ATM kinase signalling at DSBs in repressive chromatin environments. Moreover, we establish an important role of SCAI in meiotic recombination, as SCAI deficiency in mice leads to germ cell loss and subfertility associated with impaired retention of the DMC1 recombinase on meiotic chromosomes. Collectively, our findings uncover SCAI as a physiologically important component of both NHEJ- and HR-mediated pathways that potentiates DSB repair efficiency in specific chromatin contexts.

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Figure 1: SCAI is recruited to DSB-surrounding chromatin via interaction with 53BP1.
Figure 2: SCAI is required for optimal DSB repair.
Figure 3: SCAI deficiency leads to meiotic recombination defects, germ cell loss and subfertility in mice.
Figure 4: ATM, 53BP1 and SCAI operate in a common pathway to mediate repair of heterochromatin-associated DSBs.
Figure 5: SCAI mediates ATM signalling from DSBs in heterochromatin.

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Acknowledgements

We thank C. Obuse (University of Hokkaido, Japan) and H. Kimura (University of Osaka, Japan) for sharing unpublished data, A. Nussenzweig (National Institutes of Health, USA), J. Rouse (University of Dundee, UK), V. Gorbunova (University of Rochester, USA), A. Hyman and I. Poser (both Max Planck Institute of Cell Biology and Genetics, Germany) for providing reagents, G. Karemore for statistical analysis support and J. Bulkescher (both University of Copenhagen, Denmark) for technical assistance with imaging. Work in the laboratory of N.M. was supported by grants from the Novo Nordisk Foundation (Grants no. NNF14CC0001 and NNF15OC0016926), European Research Council (ERC), The Danish Cancer Society, Danish Medical Research Council, and the Lundbeck Foundation. Work in the laboratory of M.M. was supported by the Center for Integrated Protein Research Munich (CIPSM). Work in the laboratory of J.A.D. was supported by grants from the Novo Nordisk Foundation (Grant no. NNF14CC0001) and the Danish Medical Research Council, and A.M. is supported by a Marie Curie Intra-European Fellowship for Career Development (Project no. 627187). The laboratory of A.A.G. is supported by the Canadian Institutes of Health Research. A.A.G is currently the Canada Research Chair for Genome Damage and Instability Disease and this work was undertaken, in part, thanks to funding from the Canada Research Chairs programme. Work in the laboratory of F.C. is supported by the Cancer Prevention and Research Institute of Texas (R1213), the Jeanne F. Shelby Scholarship Fund for the R. Lee Clark Fellowship, and the National Institutes of Health (DP2HD087943). Work in the laboratory of R.G. is supported by the German Research Foundation (Grant GR 2111/2-2). All experiments were performed in full compliance with the ethical guidelines for biological research in Denmark. Work with all animals has been approved by the Department of Experimental Medicine (University of Copenhagen), the Danish Working Environment Authority, the Danish Animal Experiment Inspectorate, the MDACC Institutional Animal Care and Use Committee (IACUC) and the Regierungspräsidia Karlsruhe and Darmstadt.

Author information

Author notes

  1. Rebecca Kring Hansen, Andreas Mund and Sara Lund Poulsen: These authors contributed equally to this work.

Authors and Affiliations

  1. Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark

    Rebecca Kring Hansen, Sara Lund Poulsen, Maxim A. X. Tollenaere, Niels Mailand & Simon Bekker-Jensen

  2. Chromatin Structure and Function Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark

    Andreas Mund, Rebeca Soria & Jeremy A. Daniel

  3. Epigenetics and Molecular Carcinogenesis Department, The University of Texas MD Anderson Cancer Center, Smithville, Texas 78957, USA

    Maria Sandoval & Francesca Cole

  4. Departments of Biochemistry & Molecular Biology and Oncology, Robson DNA Science Centre, Arnie Charbonneau Cancer Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada

    Karolin Klement & Aaron A. Goodarzi

  5. Institut de Génétique et de Biologie Moléculaire et Celullaire (IGBMC), University of Strasbourg, 67404 Illkirch, France

    Katerina Tsouroula & Evi Soutoglou

  6. Department of Cellular and Molecular Medicine, Center for Healthy Aging, University of Copenhagen, DK-2200 Copenhagen, Denmark

    Maxim A. X. Tollenaere & Simon Bekker-Jensen

  7. Department of Molecular Genetics, TU Kaiserslautern, Paul-Ehrlich Str. 24, 67663, Kaiserslautern, Germany

    Markus Räschle

  8. Department of Pharmacology, Max-Planck-Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany

    Stefan Offermanns & Thomas Worzfeld

  9. Institute of Pharmacology, University of Marburg, 35032 Marburg, Germany

    Thomas Worzfeld, Robert Grosse & Dominique T. Brandt

  10. Department of Experimental Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark

    Björn Rozell

  11. Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany

    Matthias Mann

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  1. Rebecca Kring Hansen
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Contributions

R.K.H., A.M., S.L.P., K.T. and K.K. performed the biochemical and cell biological experiments. R.S., A.M. and M.S. carried out mouse experiments. B.R. performed and analysed the mouse histology experiments. M.R. performed and analysed the proteomics experiments. R.K.H. and M.T. designed and generated CRISPR-based knockout cell lines. S.O., T.W., R.G. and D.T.B. generated the SCAI knockout mouse. M.R., M.M., F.C., E.S., A.A.G., J.A.D., N.M. and S.B.-J. designed the experiments, and N.M. and S.B.-J. conceived the project and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Jeremy A. Daniel, Niels Mailand or Simon Bekker-Jensen.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 4 SCAI is enriched on DNA damage-containing chromatin and interacts with 53BP1.

(a) Protein intensity of SCAI on psoralen (PSO)-crosslinked (red) or undamaged chromatin (blue) plotted against time. Data was replotted from ref. 1. (b) Protein intensity of SCAI on psoralen-crosslinked chromatin in the absence (red) or presence of the replication inhibitor geminin (GEM) (black) plotted against time. Data was replotted from ref. 1. (c) Soluble and chromatin-enriched (CHR) fractions of lysates from WT and SCAI-deleted (KO) U2OS cells were analyzed by immunoblotting with the indicated antibodies. (d) U2OS cells were treated with doxycycline (DOX) to induce expression of Strep-HA-tagged SCAI, and SCAI-containing complexes were isolated by Strep-tactin pull-down from total cell lysates. Associated proteins were analyzed by immunoblotting with antibodies to 53BP1 and HA. (e) U2OS cells stably expressing GFP-SCAI transfected with control (CTRL) or 53BP1 siRNAs were subjected to laser micro-irradiation, fixed 1 h later and immunostained with γ-H2AX antibody. (f) U2OS cells stably expressing GFP-SCAI were transfected with control (CTRL) or 53BP1 siRNA, exposed to ionizing radiation (IR, 5 Gy) as indicated and fixed 4 h later. (g) U2OS cells were transfected with control (CTRL) or one of two independent SCAI siRNAs (si#9, si#10). Chromatin-enriched fractions of the cells were analyzed by immunoblotting with antibodies against SCAI and MCM6. (h) U2OS cells stably expressing GFP-SCAI (U2OS/GFP-SCAI) were treated with proteasome inhibitor MG132 for 3 h and subjected to laser micro-irradiation. Cells were fixed after 1 h and immunostained with γ-H2AX antibody. (i) Cells from (g) were exposed to ionizing radiation (IR, 5 Gy) as indicated, fixed 1 h later and immunostained with antibodies against 53BP1 and γ-H2AX. All scale bars, 10 μm. Unprocessed original scans of blots (c,d,g) are shown in Supplementary Fig. 6.

Supplementary Figure 5 53BP1-dependent and -independent recruitment of SCAI to DNA damage sites.

(a) Schematic representation of human 53BP1 constructs used in the complementation assay in (Fig. 2a). WT; wild type, ΔBRCT; deletion of tandem BRCT domains; ΔN-term; deletion of N-terminus, S28A; alanine substitution of all putative N-terminal ATM phosphorylation sites. Ability to support SCAI recruitment when transfected into 53BP1−/− MEFs is indicated for each construct on the right. (b) U2OS cells transfected with control (CTRL) or siRNA targeting SCAI or 53BP1 were subjected to laser micro-irradiation, fixed 1 h later and immunostained with antibodies against RIF1 and γ-H2AX. (c) U2OS/GFP-SCAI cells were transfected with control (CTRL), 53BP1 or RNF8 siRNAs, subjected to laser micro-irradiation, and pre-extracted and fixed 1 h later. Inserts show larger magnifications of the highlighted nuclear regions, revealing accumulation of SCAI in the DSB-surrounding chromatin compartment dependent on 53BP1 and RNF8, and in the ssDNA compartment, independently of 53BP1 and RNF8. Lower panel: Schematic of compartmentalization of IRIF. ‘Chromatin compartment’ denotes regions of DDR-modified chromatin, into which 53BP1 accumulates and recruits SCAI. ‘ssDNA compartment’ denotes areas of resected DNA to which SCAI is recruited by a 53BP1-independent mechanism. (d) U2OS/GFP-SCAI cells were transfected with control (CTRL) or siRNAs targeting BRCA1 or BRCA2, and treated with proteasome inhibitor MG132 for 3 h to interfere with ubiquitin-dependent recruitment of SCAI and 53BP1 to chromatin regions. Cells were the subjected to laser micro-irradiation, pre-extracted and fixed 1 h later and immunostained with γ-H2AX antibody. All scale bars, 10 μm. (e) SCAI interacts with ssDNA. Untagged or Biotinylated ssDNA oligos were incubated with purified GST or GST-SCAI as indicated and subjected to pull-down (PD) with Streptavidin beads. Beads were washed extensively and resolved on SDS-PAGE along with input (5%) samples. Uncropped blots (e) are shown in Supplementary Fig. 6.

Supplementary Figure 6 SCAI is required for optimal DSB repair but is dispensable for accrual of HR factors.

(a) U2OS cells (WT) or derivate lines with targeted knockout of SCAI (SCAI KO) and reconstituted expression of ectopic SCAI (rescue) were incubated in the presence of EdU, exposed to IR (1Gy) and fixed at the indicated time points. Cell cycle position of individual cells was determined by detection of EdU and DAPI signal. (b) U2OS cells lacking SCAI (SCAI KO) were obtained by CRISPR-mediated knockout and used to generate a derivative cell line in which expression of SCAI was stably reconstituted (‘rescue’). Chromatin-enriched fractions of these cell lines were analyzed by immunoblotting with SCAI and MCM6 antibodies. (c) U2OS cells deleted for 53BP1 were generated by CRISPR-mediated knockout. Lysates from parental and 53BP1 KO derivatives of U2OS cells were analyzed by immunoblotting with 53BP1 and MCM6 antibodies. (d) U2OS cells and derivative cell lines from Figure S3b were fixed at indicated times after exposure to IR (1 Gy) and stained with 53BP1 antibody. The number of foci per cell was measured by high content microscopy. Centre indicates the median and whiskers the borders of the 95% quantiles. 1,000 cells (n = 1,000 independent measurements) were measured per condition and P-values were calculated from a non-parametric two-tailed Mann-Whitney U test. (e) Parental U2OS cells and two independently derived derivative lines with targeted SCAI knockout were co-transfected with either circular (negative control) or linearized GFP-NHEJ reporter plasmid and construct encoding RFP. Cells were analyzed by flow cytometry for GFP and RFP positivity 48 h later, and the proportion of GFP-/RFP-positive cells compared to RFP positive cells only cells was determined. The NHEJ efficiency of WT cells was set to 1. Values indicate the mean and error bars the standard deviation from 3 independent experiments (n = 3). P-values were calculated from a two-tailed t-test. (f) Cells from Fig. 3b were treated and analyzed as in (e), expect cells were transfected with either circular or linearized GFP-HR reporter plasmid. The HR efficiency of WT cells was set to 1. Error bars indicate the standard deviation from 3 independent experiments (n = 3) and P-values were calculated from a two-tailed t-test. (g) U2OS WT and SCAI KO cells were exposed to IR, fixed 6 h later and immunostained with antibodies against RAD51 and γ-H2AX. (h) As in (g), except that WT or SCAI−/− MEFs and the indicated dose of IR were used. All scale bars, 10 μm. (i) Analysis of RAD51 foci in WT and SCAI−/− primary MEFs at different cell cycle stages. Cells were incubated in the presence of EdU for 1 h, exposed to IR and fixed after another 3.5 h, then immunostained with RAD51 antibody and counterstained for EdU and DAPI. Cells were analyzed by automated high-content microscopy using EdU positivity to identify S phase cells and DAPI intensity to discriminate between G1 and G2 phase in EdU-negative cells. Four individual MEF preparations were analyzed for each genotype (n = 4 biologically independent samples), and 1,500 cells were analyzed per sample. Values indicate the mean and error bars are the standard deviation. Uncropped blots (b,c) are shown in Supplementary Fig. 6.

Supplementary Figure 7 Characterization of SCAI−/− mice and MEFs.

(a) Proliferation of MEFs. Primary WT (n = 9 biologically independent samples from 3 cell lines) and SCAI−/− (n = 12 biologically independent samples from 4 cell lines) MEFs were plated at passages 2, 3, 4 and 5, and trypsinized, counted and replated every second day. Values indicate the mean and error bars are the standard deviation. (b) Overall DSB repair proficiency of 3 immortalized pairs of WT and SCAI−/− MEF cell lines was evaluated by enumeration of residual 53BP1 foci in G2 phase (H3-pS10-positive) cells at the indicated times after exposure to IR (2 Gy). Values indicate the mean and error bars the standard deviation across 3 independent experiments. P-value was calculated from a one-tailed t-test using Welch correction (n = 9 independent measurements across 3 MEF lines). Bars indicate mean ± SD. IRIF: Ionizing Radiation Induced Foci. (c) Examples of cells from the experiment in (b). MEFs were fixed 8 h after exposure to IR (2 Gy), immunostained with antibodies to 53BP1 and H3-pS10 and counterstained for nuclear content with DAPI. (d) 12 WT and 16 SCAI−/− age-matched male mice were subjected to whole-body gamma-irradiation with a one-time dose of 8 Gy. Survival was monitored over a 28-day period, after which remaining animals were euthanized. (e) As in (d) but with female mice. 7 WT and 6 SCAI−/−mice were used. Data from (d) and (e) have been combined in Fig. 3b. (f) Primary B cells isolated from WT and SCAI−/− mice were labeled with CellTrace Violet and analyzed by flow cytometry to monitor cell proliferation by dye dilution. (g) Flow cytometric quantification of splenic B220 + IgM + B cell populations from one WT and one SCAI−/− mouse. A representative of multiple mice. (h) Graphical representation of B220 + CD19 + total B cell number from 6 WT and 6 SCAI−/− mice. (i) B cells isolated from 6 WT and 6 SCAI−/− mice were stimulated for 4 days to undergo IgH class-switching and IgG1 (left) and IgG3 (right) frequencies were measured by flow cytometry. (j) Serum levels of IgG1, IgG3, and IgM immunoglobulins in 6 WT and 6 SCAI−/− mice as measured by ELISA. Each dot in the quantifications in (h), (i) and (j) represents data from individual mice and all lines indicate the mean. For each genotype, 3 males and 3 females were analyzed. (k) Breeding experiment of WT (male n = 19 and female n = 15 animals) and SCAI−/− (male n = 7 and female n = 4 animals) mice. Quantification of litter sizes. Bars indicate mean ± SD and P-value was calculated with a two-tailed t-test. Source data in Supplementary Table 2. (l) Spermatocyte spreads from WT and SCAI−/− mice were stained with SYCP3 and DMC1 antibodies. Representative pachynema spermatocytes are shown. Quantified in Fig. 3j. (m) Examples of the types of chromosomal aberrations observed in SCAI−/− B cells treated with PARP inhibitor and quantified in Fig. 3m.

Supplementary Figure 8 SCAI promotes DSB repair in heterochromatin.

(a) GFP-SCAI was affinity-purified on GFP-Trap beads from chromatin-enriched fractions of U2OS and U2OS/GFP-SCAI cells, and co-purifying proteins and input lysates were analyzed by immunoblotting with antibodies against HP1γ, GFP and Tubulin. (b) As in (a), except that co-purifying proteins and input lysates were analyzed with antibodies against HP1β, GFP and Tubulin. (c) Extended version of Fig. 4a. Independent and immortalized WT and SCAI−/− MEF cell lines were arrested in G0/G1 by growing to full confluency. Cultures were mock-treated or exposed to IR (2 Gy), fixed either 0.5 or 24 h later and stained with 53BP1 antibody. Images were acquired as Z-stacks and the number of 53BP1 foci per cell was counted through the entire nuclear volume. Bars indicate mean ± SD (n = 9 independent measurements across 3 MEF lines). IRIF: Ionizing Radiation Induced Foci (d) Extended version of (Fig. 4b). Immortalized WT and SCAI−/− MEFs were grown to full confluency while transfecting with 53BP1 siRNA for 72 h or incubating with ATM inhibitor for 1 h prior to irradiation. Cells were treated and analyzed as in (c), except that they were immunostained for γ-H2AX as a marker of unrepaired DSB. (n = 3 biologically independent samples). (e) Immortalized WT and SCAI−/− MEFs were treated and analyzed as in (c), except that cells were incubated with ATM inhibitor and co-stained with antibodies to γ-H2AX and the heterochromatin marker H3K9me3 to determine chromatin context (n = 3 biologically independent samples). HC (heterochromatin). (f) Representative images from the analysis in (Fig. 4e, f). (g) Mouse NIH-3T3 fibroblasts were transfected with GFP-SCAI, CAS9 and guide RNA (gRNA) targeting the major satellite repeats in heterochromatin containing chromocenters. Cells were fixed after 16 h and immunostained with 53BP1 antibodies and DNA stain DAPI. (h) Immortalized WT and SCAI−/− MEFs were transfected with Cas9-GFP and gRNAs targeting the major satellite repeats to induce CRISPR-mediated DSBs in heterochromatin-containing chromocenters. After 8 h cells were fixed and immunostained with antibodies against H3K9me3. Cells were analyzed by high content microscopy using DAPI signal as a mask for chromocenters. 220 cells (n = 220 independent measurements) were measured per condition and P-values were calculated from two-tailed t-tests using Welch correction. Centre indicates the median and whiskers the borders of the 95% quantiles. Y-axis on the left side corresponds to the non-transfected conditions, while y-axis on the right side corresponds to the transfected conditions. (i) As in (h), except cells were immunostained with HP1β antibodies. (n = 120 independent measurements) (j) As in (h), except cells were immunostained with HP1γ antibodies. (n = 130 independent measurements) (k) As in (h), except cells were only stained with DAPI. (n = 320 independent measurements) (l) Cells from (g) were treated with ATM inhibitor and transfected as in (g) to induce heterochromatin associated DSBs by CRISPR. Lysates were analyzed for markers of ATM signaling by immunoblotting with the indicated antibodies. (m) WT and SCAI−/− MEFs were exposed to ionizing radiation (IR, 1Gy) to induce DSBs predominantly in euchromatin. Lysates were collected at the indicated time points and analyzed for markers of ATM signaling by immunoblotting with the indicated antibodies. All scale bars, 10 μm. Uncropped blots (a,b,l,m) are shown in Supplementary Fig. 6.

Supplementary Table 1 Breeding of SCAI−/− mice.
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Supplementary Table 2 Fertility measurements of SCAI−/− mice.
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Supplementary Table 3 Genomic instability in SCAI−/− B cells.
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Hansen, R., Mund, A., Poulsen, S. et al. SCAI promotes DNA double-strand break repair in distinct chromosomal contexts. Nat Cell Biol 18, 1357–1366 (2016). https://doi.org/10.1038/ncb3436

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