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The NBS1–Treacle complex controls ribosomal RNA transcription in response to DNA damage

Nature Cell Biology volume 16, pages 792–803 (2014)Cite this article

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Abstract

Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks.

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Figure 1: In trans inhibition of Pol I transcription in response to DNA damage is associated with NBS1 recruitment into the nucleoli.
Figure 2: NBS1 is enriched at sites of rRNA transcription in response to DNA damage.
Figure 3: The NBS1 FHA domain mediates ATM-dependent nucleolar accumulation of NBS1.
Figure 4: Treacle is an interaction partner of the NBS1 FHA-BRCT region.
Figure 5: Conserved acidic SDT-like phospho-motifs in Treacle mediate the binding to NBS1.
Figure 6: Phosphorylated Thr 210 in Treacle is the main interaction site for NBS1.
Figure 7: Treacle mediates NBS1 nucleolar translocation in response to DNA damage and a fraction is transiently recruited to sites of DNA damage.
Figure 8: Treacle-dependent NBS1 recruitment into the nucleoli mediates rRNA silencing in response to DNA damage.

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Acknowledgements

We thank S. Jackson (The Gurdon Institute, Cambridge, UK) for reagents, R. Santoro for technical support in relation to rRNA transcription, C. Dinant for critical discussion of the FRAP data and members of the Department of Gynecology, the Novo Nordisk Foundation Center for Protein Research and the National Institute of Medical Research for helpful discussions. This work was supported by grants from the Swiss National Foundation (3100A0-111818 and 31003A-144284), Promedica Foundation, Lundbeckfonden (R93-A8863), Novo Nordisk Foundation and by the Kanton of Zürich. S.J.S. is supported by the Medical Research Council UK (U117584228).

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Authors and Affiliations

  1. Department of Gynecology, University Hospital and University of Zurich, Wagistrasse 14, CH-8952 Schlieren, Switzerland

    Dorthe H. Larsen, Myriam Gwerder, Katrin Gutsche, Daniel Fink & Manuel Stucki

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

    Dorthe H. Larsen, Matthias Altmeyer, Stephanie Jungmichel, Luis I. Toledo, Maj-Britt Rask, Merete Grøfte, Claudia Lukas, Michael L. Nielsen & Jiri Lukas

  3. Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland

    Flurina Hari & Manuel Stucki

  4. Division of Protein Structure, MRC National Institute of Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

    Julie A. Clapperton & Stephen J. Smerdon

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  1. Dorthe H. Larsen
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Contributions

D.H.L. designed and performed most of the experiments, analysed data and contributed to writing the paper; F.H. designed the mass spectrometry screen that identified TCOF1 and performed some of the biochemical analysis; S.J. and M.L.N. designed and performed most of the mass spectrometry analysis, analysed data and contributed to writing the paper; J.A.C. and S.J.S. designed and performed the ITC experiments, analysed data and contributed to writing the paper; M.A. provided feedback in experimental design, performed some of the microscopic analysis and revised the written manuscript; L.I.T. provided feedback in experimental design and performed some of the microscopic analysis; K.G. performed some of the ChIP experiments; M. Gwerder purified proteins, generated reagents and performed in vitro phosphorylation assays and pulldown analysis; M-B.R. and M. Grøfte generated reagents; D.F. supervised the project; C.L. discovered NBS1 nucleolar localization and provided feedback in the design of the microscopic analysis and interpretation of the data; J.L. and M.S. coordinated and supervised the project, designed experiments, analysed data and wrote the paper.

Corresponding authors

Correspondence to Jiri Lukas or Manuel Stucki.

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Integrated supplementary information

Supplementary Figure 3 Additional data on rRNA silencing after DNA damage and NBS1 and MRE11 nucleolar localization.

(a) Measurement of rRNA synthesis after laser-irradiation by 5-EU incorporation. A larger field of cells is shown here as compared to Fig. 1A (2 independent experiments; one representative image shown). (b) U2OS cells stably transfected with NBS1–GFP were presensitized with 10 μM BrdU for 24 h and exposed to laser micro-irradiation. To mark nucleoli, cells were immunostained with antibodies against Fibrillarin (2 independent experiments; 1 representative experiment shown). (c) Translocation of NBS1–GFP was followed by timelapse microscopy (5 independent experiments; 1 representative experiment shown). (d) U2OS cells stably transfected with GFP-MRE11 were presensitized with 10 μM BrdU for 24 h and exposed to laser micro-irradiation. Cells were immunostained with antibodies against γH2AX (2 independent experiments; 1 representative experiment shown). Scale bars, 10 μm.

Supplementary Figure 4 Measurement of rRNA synthesis after ionizing radiation by EU labelling.

(a) Dose titration of rRNA synthesis after ionizing radiation. Error bars represent s.e.m. (n = 3 independent experiments). (b) rRNA synthesis after ionizing radiation on depletion of MRE11 by two different siRNAs (measured by EU labelling 20 min after 5 Gy). The bars in the graph represent the average of 2 independent experiments.

Supplementary Figure 5 Measurement of rRNA synthesis after ionizing radiation and CPT treatment by qRT-PCR.

(a) rRNA synthesis after ionizing radiation and CPT treatment in the presence of ATM inhibitor (KU55933). Error bars represent s.e.m. (n = 4 independent experiments; samples were run in duplicates). (b) rRNA synthesis after ionizing radiation and CPT treatment on depletion of NBS1 by two different siRNAs. Error bars represent s.e.m. (n = 4 independent experiments for siNBS1-1; n = 3 independent experiments for siNBS1-2; all samples were run in duplicates). (c) rRNA synthesis after ionizing radiation and CPT treatment on depletion of MDC1. Error bars represent s.e.m. (n = 4 independent experiments; samples were run in duplicates).

Supplementary Figure 6 Treacle is heavily phosphorylated by CK2 in vitro.

Bacterially purified GST–Treacle fragments were incubated with recombinant CK2 and γ-[32P]-ATP, followed by SDS-PAGE and autoradiography. MDC1 fragments were used as positive and negative control, respectively 6. Note that fragment T-2 (aa 109-330) that contains the NBS1-interacting region is efficiently phosphorylated by CK2 in vitro (2 independent experiments; 1 representative experiment shown).

Supplementary Figure 7 Treacle localization and mobility in the nucleoli.

(a) Hela cells were immunostained with antibodies against Treacle and UBF (2 independent experiments; 1 representative experiment shown). Scale bar, 10 μm. (b) ChIP experiment with antibodies against mouse IgG or GFP in stably transfected U2OS GFP–Treacle cells. GAPDH was used as an unspecific target. Error bars represent s.e.m. (n = 4 independent experiments). (c) FRAP experiment in GFP–Treacle-expressing Hela cells. Bleach pulse was directed against one single nucleolus and fluorescence recovery was measured in the same nucleolus. T1/2 = 210 ± 40 s. The black line represents the median of 17 FRAP experiments included in the graph.

Supplementary Figure 8 Additional data about Treacle localization to sites of DNA damage.

(a) Schematic representation of Treacle-c. Note that the C-terminal region comprising the nucleolar localization signal is missing from this variant. (b) U2OS cells stably expressing GFP–Treacle-c were pre-sensitized with 10 μM BrdU for 24 h and exposed to laser micro-irradiation. Cells were treated with control siRNA and siRNA against NBS1 and immunostained with antibodies against γH2AX (2 independent experiments; 1 representative experiment shown). Scale bar, 10 μm (c) U2OS cells stably transfected with NBS1–GFP were pre-treated with PARP inhibitors and exposed to laser micro-irradiation. NBS1–GFP positive nucleoli were quantified. Error bars represent s.e.m. (n = 3 independent experiments; at least 45 cells with laser stripes probed for nucleolar NBS1 foci in each experiment, for statistical source data see Supplementary Table 2).

Supplementary Figure 9 Treacle phosphorylation in response to DNA damage.

293T cells were treated with the indicated siRNAs for 72 h and subsequently transiently transfected with HA-Treacle as indicated. 1 h before irradiation indicated samples were treated with 10 μM ATMi (KU55933). Cells were exposed to 10 Gy of ionizing radiation and immunoprecipitation was performed against HA (Treacle). Blots were probed with the indicated antibodies, including an antibody against pSQ (pS61 Bid; 2 independent experiments; 1 representative experiment shown).

Supplementary Figure 10 Full scans.

Supplementary Table 1 Primer and siRNA sequences.
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Supplementary Table 2 Statistical source data.
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Larsen, D., Hari, F., Clapperton, J. et al. The NBS1–Treacle complex controls ribosomal RNA transcription in response to DNA damage. Nat Cell Biol 16, 792–803 (2014). https://doi.org/10.1038/ncb3007

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