Damage-induced lncRNAs control the DNA damage response through interaction with DDRNAs at individual double-strand breaks

Abstract

The DNA damage response (DDR) preserves genomic integrity. Small non-coding RNAs termed DDRNAs are generated at DNA double-strand breaks (DSBs) and are critical for DDR activation. Here we show that active DDRNAs specifically localize to their damaged homologous genomic sites in a transcription-dependent manner. Following DNA damage, RNA polymerase II (RNAPII) binds to the MRE11–RAD50–NBS1 complex, is recruited to DSBs and synthesizes damage-induced long non-coding RNAs (dilncRNAs) from and towards DNA ends. DilncRNAs act both as DDRNA precursors and by recruiting DDRNAs through RNA–RNA pairing. Together, dilncRNAs and DDRNAs fuel DDR focus formation and associate with 53BP1. Accordingly, inhibition of RNAPII prevents DDRNA recruitment, DDR activation and DNA repair. Antisense oligonucleotides matching dilncRNAs and DDRNAs impair site-specific DDR focus formation and DNA repair. We propose that DDR signalling sites, in addition to sharing a common pool of proteins, individually host a unique set of site-specific RNAs necessary for DDR activation.

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Figure 1: Sequence-specific localization of DDRNAs at DNA damage sites is transcription-dependent.
Figure 2: DSBs induce dilncRNAs that interact with DDRNAs.
Figure 3: Active RNAPII is recruited to DSBs in mammalian cells and in cell extracts.
Figure 4: DSBs induce bidirectional transcription in cell-free extracts.
Figure 5: The MRN complex binds to RNAPII following DNA damage and is necessary for RNAPII transcription at DSBs in mammalian cells.
Figure 6: RNAPII transcription is necessary for DDR focus formation and DNA repair, and 53BP1 interacts with DDRNA and dilncRNA through its Tudor domain.
Figure 7: ASOs preventing dilncRNA–DDRNA interaction affect 53BP1 focus formation.
Figure 8: Site-specific inhibition of 53BP1 focus formation and DNA repair by ASOs.

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References

  1. 1

    Polo, S. E. & Jackson, S. P. Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Genes Dev. 25, 409–433 (2011).

    CAS  Article  Google Scholar 

  2. 2

    Sharma, V. & Misteli, T. Non-coding RNAs in DNA damage and repair. FEBS Lett. 587, 1832–1839 (2013).

    CAS  Article  Google Scholar 

  3. 3

    Derr, L. K. & Strathern, J. N. A role for reverse transcripts in gene conversion. Nature 361, 170–173 (1993).

    CAS  Article  Google Scholar 

  4. 4

    Storici, F., Bebenek, K., Kunkel, T. A., Gordenin, D. A. & Resnick, M. A. RNA-templated DNA repair. Nature 447, 338–341 (2007).

    CAS  Article  Google Scholar 

  5. 5

    Keskin, H. et al. Transcript-RNA-templated DNA recombination and repair. Nature 515, 436–439 (2014).

    CAS  Article  Google Scholar 

  6. 6

    Wei, W. et al. A role for small RNAs in DNA double-strand break repair. Cell 149, 101–112 (2012).

    CAS  Article  Google Scholar 

  7. 7

    Wang, Q. & Goldstein, M. Small RNAs recruit chromatin-modifying enzymes MMSET and Tip60 to reconfigure damaged DNA upon double-strand break and facilitate repair. Cancer Res. 76, 1904–1915 (2016).

    CAS  Article  Google Scholar 

  8. 8

    Gao, M. et al. Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination. Cell Res. 24, 532–541 (2014).

    CAS  Article  Google Scholar 

  9. 9

    Qi, Y., Zhang, Y., Baller, J. A. & Voytas, D. F. Histone H2AX and the small RNA pathway modulate both non-homologous end-joining and homologous recombination in plants. Mutat. Res. 783, 9–14 (2016).

    CAS  Article  Google Scholar 

  10. 10

    Yang, Y. G. & Qi, Y. RNA-directed repair of DNA double-strand breaks. DNA Repair 32, 82–85 (2015).

    CAS  Article  Google Scholar 

  11. 11

    Ohle, C. et al. Transient RNA-DNA hybrids are required for efficient double-strand break repair. Cell 167, 1001–1013 e1007 (2016).

    CAS  Article  Google Scholar 

  12. 12

    Chakraborty, A. et al. Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes. Nat. Commun. 7, 13049 (2016).

    CAS  Article  Google Scholar 

  13. 13

    Mochizuki, K., Fine, N. A., Fujisawa, T. & Gorovsky, M. A. Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in tetrahymena. Cell 110, 689–699 (2002).

    CAS  Article  Google Scholar 

  14. 14

    Schmidts, I., Bottcher, R., Mirkovic-Hosle, M. & Forstemann, K. Homology directed repair is unaffected by the absence of siRNAs in Drosophila melanogaster. Nucleic Acids Res. 44, 8261–8271 (2016).

    CAS  Article  Google Scholar 

  15. 15

    Miki, D. et al. Efficient generation of diRNAs requires components in the posttranscriptional gene silencing pathway. Sci. Rep. 7, 301 (2017).

    Article  Google Scholar 

  16. 16

    Francia, S. et al. Site-specific DICER and DROSHA RNA products control the DNA-damage response. Nature 488, 231–235 (2012).

    CAS  Article  Google Scholar 

  17. 17

    Francia, S., Cabrini, M., Matti, V., Oldani, A. & d’Adda di Fagagna, F. DICER, DROSHA and DNA damage response RNAs are necessary for the secondary recruitment of DNA damage response factors. J. Cell Sci. 129, 1468–1476 (2016).

    CAS  Article  Google Scholar 

  18. 18

    Rossiello, F. et al. DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs. Nat. Commun. 8, 13980 (2017).

    CAS  Article  Google Scholar 

  19. 19

    Michalik, K. M., Bottcher, R. & Forstemann, K. A small RNA response at DNA ends in Drosophila. Nucleic Acids Res. 40, 9596–9603 (2012).

    CAS  Article  Google Scholar 

  20. 20

    Soutoglou, E. et al. Positional stability of single double-strand breaks in mammalian cells. Nat. Cell Biol. 9, 675–682 (2007).

    CAS  Article  Google Scholar 

  21. 21

    Pitchiaya, S., Androsavich, J. R. & Walter, N. G. Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly. EMBO Rep. 13, 709–715 (2012).

    CAS  Article  Google Scholar 

  22. 22

    Pitchiaya, S., Krishnan, V., Custer, T. C. & Walter, N. G. Dissecting non-coding RNA mechanisms in cellulo by single-molecule high-resolution localization and counting. Methods 63, 188–199 (2013).

    CAS  Article  Google Scholar 

  23. 23

    Pitchiaya, S., Heinicke, L. A., Park, J. I., Cameron, E. L. & Walter, N. G. Resolving subcellular miRNA trafficking and turnover at single-molecule resolution. Cell Rep. 19, 630–642 (2017).

    CAS  Article  Google Scholar 

  24. 24

    Bensaude, O. Inhibiting eukaryotic transcription: which compound to choose? How to evaluate its activity? Transcription 2, 103–108 (2011).

    Article  Google Scholar 

  25. 25

    Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    CAS  Article  Google Scholar 

  26. 26

    Lemaitre, C. et al. The nucleoporin 153, a novel factor in double-strand break repair and DNA damage response. Oncogene 31, 4803–4809 (2012).

    CAS  Article  Google Scholar 

  27. 27

    Lemaitre, C. et al. Nuclear position dictates DNA repair pathway choice. Genes Dev. 28, 2450–2463 (2014).

    Article  Google Scholar 

  28. 28

    Roukos, V. et al. Spatial dynamics of chromosome translocations in living cells. Science 341, 660–664 (2013).

    CAS  Article  Google Scholar 

  29. 29

    Pankotai, T., Bonhomme, C., Chen, D. & Soutoglou, E. DNAPKcs-dependent arrest of RNA polymerase II transcription in the presence of DNA breaks. Nat. Struct. Mol. Biol. 19, 276–282 (2012).

    CAS  Article  Google Scholar 

  30. 30

    Iacovoni, J. S. et al. High-resolution profiling of γH2AX around DNA double strand breaks in the mammalian genome. EMBO J. 29, 1446–1457 (2010).

    CAS  Article  Google Scholar 

  31. 31

    Asada, K. et al. Rescuing dicer defects via inhibition of an anti-dicing nuclease. Cell Rep. 9, 1471–1481 (2014).

    CAS  Article  Google Scholar 

  32. 32

    Feuerhahn, S., Iglesias, N., Panza, A., Porro, A. & Lingner, J. TERRA biogenesis, turnover and implications for function. FEBS Lett. 584, 3812–3818 (2010).

    CAS  Article  Google Scholar 

  33. 33

    Wang, Y., Maharana, S., Wang, M. D. & Shivashankar, G. V. Super-resolution microscopy reveals decondensed chromatin structure at transcription sites. Sci. Rep. 4, 4477 (2014).

    PubMed  PubMed Central  Google Scholar 

  34. 34

    Dupre, A. et al. A forward chemical genetic screen reveals an inhibitor of the Mre11–Rad50–Nbs1 complex. Nat. Chem. Biol. 4, 119–125 (2008).

    CAS  Article  Google Scholar 

  35. 35

    Kumar, R. & Cheok, C. F. RIF1: a novel regulatory factor for DNA replication and DNA damage response signaling. DNA Repair (Amst) 15, 54–59 (2014).

    CAS  Article  Google Scholar 

  36. 36

    Goodarzi, A. A. & Jeggo, P. A. The heterochromatic barrier to DNA double strand break repair: how to get the entry visa. Int. J. Mol. Sci. 13, 11844–11860 (2012).

    CAS  Article  Google Scholar 

  37. 37

    Smeenk, G. & Mailand, N. Writers, readers, and erasers of histone ubiquitylation in DNA double-strand break repair. Front. Genet. 7, 122 (2016).

    Article  Google Scholar 

  38. 38

    Pryde, F. et al. 53BP1 exchanges slowly at the sites of DNA damage and appears to require RNA for its association with chromatin. J. Cell Sci. 118, 2043–2055 (2005).

    CAS  Article  Google Scholar 

  39. 39

    Fradet-Turcotte, A. et al. 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark. Nature 499, 50–54 (2013).

    CAS  Article  Google Scholar 

  40. 40

    Burger, K. et al. Nuclear phosphorylated Dicer processes double-stranded RNA in response to DNA damage. J. Cell Biol. 216, 2373–2389 (2017).

    CAS  Article  Google Scholar 

  41. 41

    Holoch, D. & Moazed, D. RNA-mediated epigenetic regulation of gene expression. Nat. Rev. Genet. 16, 71–84 (2015).

    CAS  Article  Google Scholar 

  42. 42

    Guil, S. & Esteller, M. RNA-RNA interactions in gene regulation: the coding and noncoding players. Trends Biochem. Sci. 40, 248–256 (2015).

    CAS  Article  Google Scholar 

  43. 43

    Vitelli, V. et al. Recent advancements in DNA damage-transcription crosstalk and high-resolution mapping of DNA breaks. Annu. Rev. Genomics Hum. Genet. 18, 87–113 (2017).

    CAS  Article  Google Scholar 

  44. 44

    Proudfoot, N. J. Transcriptional termination in mammals: stopping the RNA polymerase II juggernaut. Science 352, aad9926 (2016).

    Article  Google Scholar 

  45. 45

    Skourti-Stathaki, K., Kamieniarz-Gdula, K. & Proudfoot, N. J. R-loops induce repressive chromatin marks over mammalian gene terminators. Nature 516, 436–439 (2014).

    CAS  Article  Google Scholar 

  46. 46

    Capozzo, I., Iannelli, F., Francia, S. & d’Adda di Fagagna, F. Express or repress? The transcriptional dilemma of damaged chromatin. FEBS J. 284, 2133–2147 (2017).

    CAS  Article  Google Scholar 

  47. 47

    Iannelli, F. et al. A damaged genome’s transcriptional landscape through multilayered expression profiling around in situ-mapped DNA double-strand breaks. Nat. Commun. 8, 15656 (2017).

    CAS  Article  Google Scholar 

  48. 48

    Britton, S. et al. DNA damage triggers SAF-A and RNA biogenesis factors exclusion from chromatin coupled to R-loops removal. Nucleic Acids Res. 42, 9047–9062 (2014).

    CAS  Article  Google Scholar 

  49. 49

    Boeing, S. et al. Multiomic analysis of the UV-induced DNA damage response. Cell Rep. 15, 1597–1610 (2016).

    CAS  Article  Google Scholar 

  50. 50

    Kakarougkas, A. et al. Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin. Mol. Cell 55, 723–732 (2014).

    CAS  Article  Google Scholar 

  51. 51

    Nojima, T., Gomes, T., Carmo-Fonseca, M. & Proudfoot, N. J. Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide. Nat. Protoc. 11, 413–428 (2016).

    CAS  Article  Google Scholar 

  52. 52

    Cawthon, R. M. Telomere measurement by quantitative PCR. Nucleic Acids Res. 30, e47 (2002).

    Article  Google Scholar 

  53. 53

    Berkovich, E., Monnat, R. J. Jr & Kastan, M. B. Roles of ATM and NBS1 in chromatin structure modulation and DNA double-strand break repair. Nat. Cell Biol. 9, 683–690 (2007).

    CAS  Article  Google Scholar 

  54. 54

    Dignam, J. D., Lebovitz, R. M. & Roeder, R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983).

    CAS  Article  Google Scholar 

  55. 55

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  Article  Google Scholar 

  56. 56

    Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

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Acknowledgements

We thank E. Soutoglou (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France), T. Misteli (National Cancer Institute, Bethesda, USA), G. Legube (Centre de Biologie Intégrative, Toulouse, France), M. Kastan (Duke Cancer Institute, Durham, USA), A. Aguilera, (Centro Andaluz de Biología Molecular y Medicina Regenerativa, Sevilla, Spain), Y. Xu (University of California, San Diego, USA), S. P. Jackson (Gurdon Institute, Cambridge, UK), D. Durocher (The Lunenfeld-Tanenbaum Research Institute, Toronto, Canada), B. Amati (European Institute of Oncology, Milan, Italy), A. Verrecchia (European Institute of Oncology, Milan, Italy), P. Pellanda (European Institute of Oncology, Milan, Italy) for reagents; Single Molecule Analysis in Real-Time Center (University of Michigan, USA) for instruments; M. Bedford (MD Anderson Cancer Center, Texas, USA), C. A. Sagum (MD Anderson Cancer Center, Texas, USA) and M. Roncador (Italian National Research Council, Pavia, Italy) for their help during the revision of the manuscript and all F.d’A.d.F. group members for reading the manuscript, support and constant discussions. F.M. was supported by Fondazione Italiana Ricerca Sul Cancro (FIRC, 12491). S.F. was supported by Collegio Ghislieri and Fondazione Cariplo (Grant rif. 2014-1215). G.V.S. is supported by Mechanobiology Institute (MBI) and Singapore Ministry of Education Academic Research Fund Tier3 (MOE2012-T3-1-001). N.G.W. is supported by NIH grants 2R01 GM062357, 1R01 GM098023 and 1R21 AI109791. F.d’A.d.F. was supported by the Associazione Italiana per la Ricerca sul Cancro, AIRC (application 12971), Human Frontier Science Program (contract RGP 0014/2012), Cariplo Foundation (grant 2010.0818 and 2014-0812), Marie Curie Initial Training Networks (FP7 PEOPLE 2012 ITN (CodAge)), Fondazione Telethon (GGP12059), Association for International Cancer Research (AICR-Worldwide Cancer Research Rif. N. 14-1331), Progetti di Ricerca di Interesse Nazionale (PRIN) 2010–2011, the Italian Ministry of Education Universities and Research EPIGEN Project, a European Research Council advanced grant (322726) and AriSLA (project ‘DDRNA and ALS’).

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Contributions

S.P. conceived and performed all microinjection assays, intracellular single-molecule imaging of DDRNA, FISH and kinetics experiments. V.V. generated the lentiviral I-SceI-GR construct, produced strand-specific RT–qPCR data for dilncRNA detection (NIH2/4 cells, NIH3T3duo cells, U2OS19ptight cells, HeLa111 cells, HeLa I-PpoI cells cut in the DAB1 gene) and qPCR analyses of ChIP experiments in NIH2/4 and determined dilncRNA polyadenylation status. S.S. performed all in vitro DSB-induced transcription assays and 5′ RACE and produced samples for sequencing. U.G. performed the RNA pulldown experiments without ASOs and RIP experiments, performed RT–qPCR analyses of DDRNA and generated the GFP-53BP1ΔTUD construct. M.C. performed ChIP experiments of RNAPII and the phosphorylated forms in NIH2/4 cells and the ChIP in AsiSI-ER BJ-5Ta cells treated with DRB. F.P. performed the in vitro binding assay of RNAPII to DNA ends, the co-immunoprecipitations of RNAPII with MRN and the ChIP of RNAPII in HeLa I-PpoI cells. Y.W. performed confocal and super-resolution analyses of RNAPII localization on damaged chromatin. F.I. conducted bioinformatics analyses of next-generation sequencing data. I.C. detected dilncRNA in the AsiSI system. V.M. contributed with technical support. S.F. supervised M.C. and I.C. G.V.S. supervised Y.W. N.G.W. initiated the single-molecule experiments, advised S.P. in their execution, provided critical input in experimental design and result interpretation, and edited the manuscript. F.M. designed and performed all of the remaining experiments and wrote the manuscript. F.d’A.d.F. conceived the study and, together with F.M., assembled and revised the manuscript. All authors commented on the manuscript.

Corresponding author

Correspondence to Fabrizio d’Adda di Fagagna.

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Competing interests

F.d’A.d.F., F.M. and S.F. are inventors on the following patent application: PCT/EP2013/059753.

Integrated supplementary information

Supplementary Figure 1 Localization of DDRNAs at sites of DNA damage is not dependent on H2AX or DDRNA:DNA pairing.

(A) Mapping of the synthetic DDRNA pairs (in blue) L1:L2, U1:U2, U3:U4, T1:T2 relative to the I-SceI cut site. (BD) Examples of stepwise photobleaching traces of individual DDRNA particles co-localizing with GFP-LacR (relative to Fig. 1B). Raw intensity is depicted in red. Chung-Kennedy non-linear filter is represented in black. (E) Deconvolved DDRNAs display the same localization pattern of the DDRNA pool (relative to Fig. 1B). Plot represents the number of DDRNA molecules at the LacR spot as measured by single-molecule counting based on stepwise photobleaching of fluorescent probes. Dots represent individual cells. The black line represents the mean ± SEM (n = 3 independent experiments). (F,G) Mean fold change of Dicer and Drosha mRNA in NIH2/4 cells by RT-qPCR. Error bars indicate SEM (relative to Fig. 1C, n = 3 independent experiments). (H) DDRNA-Cy5 localization to the damaged site does not depend on H2AX in NIH2/4 cells. The bar plot shows the percentage of cells with DDRNA-TetR co-localization. Error bars indicate SEM (n = 3 independent experiments, ≥80 cells analysed in total per condition). (I) Immunofluorescence analysis (relative to H) confirms reduced γH2AX and 53BP1 foci co-localization with YFP-TetR upon H2AX knockdown in cut NIH2/4 cells (mean of n = 2 independent experiments, ≥40 cells analysed in total per condition). (J,K) DDRNA-Cy5 localization does not depend on DDRNA:DNA pairing. NIH2/4 cells were expressing YFP-TetR and I-SceI and RNase H1-expressing vector or an empty control vector (E.V.) were incubated with DDRNA-Cy5. Cells overexpressing RNase H1 were scored by staining with anti-RNase H1 antibody. In K, the bar plot shows the percentage of cells DDRNA-TetR co-localization. Error bars indicate SEM (n = 3 independent experiments, ≥80 cells analysed in total per condition). (L) Mean fold change of the indicated RNAs in NIH2/4 cells by RT-qPCR. Error bars indicate SEM (relative to Fig. 1D, n = 3 independent experiments). P values were calculated using chi-squared test. ns indicates not significant. Statistical source data are provided in Supplementary Table 4.

Supplementary Figure 2 Characterization of DSB-induced transcripts.

(A) Representative images of FISH signal (red) on NIH2/4 cells transfected with GFP-LacR (green), 53BP1-mCherry (yellow) and pLacZ (uncut) or pI-SceI (cut). Scale bar 5 μm. Inset is a magnified view (relative to Fig. 2B, representative of 3 independent experiments). (B) Plot shows the relative intensity of Uni Lac-from FISH probe. When indicated, RNase A or RNase H treatment was performed prior or after DNA probe hybridization, respectively. Dots represent individual cells. Black bar represents mean ± SEM (n = 3 independent experiments). (C) Uni Lac-from probe at various concentrations (0.1–1 nM) was used with the same acquisition parameters as FISH samples. Linear fit is depicted as a grey dotted line (R2 = 0.92). Error bars show SD (n = 5 regions/slide, 2 slides). (D) Plot represents the number of transcripts at locus, based on the FISH signal intensity of Uni Lac-from and Uni Lac-to probes. Dots represent individual cells. Black bar represents mean ± SEM (n = 3 independent experiments). (E) DilncRNAs are non-polyadenylated RNAs. Total RNA extracted from NIH2/4 cells was reverse-transcribed with oligo-dT or sequence-specific primers and dilncRNA and GAPDH mRNA were detected by RT-qPCR. RT with sequence-specific primer was used as reference. Error bars indicate SEM (n = 3 independent experiments). (F) Strand-specific RT-qPCR shows that DSB-dependent transcription is sensitive to RNAPII inhibition. Bar plot shows the fold change relative to vehicle-treated samples of the indicated RNAs in cells treated AM, DRB, ACTD at low or high doses for 2 h before I-SceI induction. Error bars indicate SEM (n = 3 independent experiments). (G) FISH signal relative intensity of Uni Lac-from probe upon DRB treatment. Dots represent individual cells. Black bar represents the mean ± SEM (n = 3 independent experiments). (H) DSB-induced transcription is not dependent on ATM kinase activity. Bar plot shows the mean fold change respect to uncut of the indicated dilncRNAs in NIH2/4 cells treated with ATM inhibitor (KU60019 10 μM) or DMSO for 16 h before cut. Error bars indicate SEM (n = 3 independent experiments). (IM) Strand-specific-RT-qPCR showing dilncRNA-from and dilncRNA-to in different cell lines upon DSB induction. Bar plots show the mean fold change respect to uncut. Error bars indicate SEM. (I) NIH3T3duo cells cut by I-SceI (n = 3 independent experiments); (J) HeLa cells cut by I-PpoI in an intergenic site (n = 3 independent experiments); (K) U2OS cells cut by AsiSI in a genic site (n = 3 independent experiments); (L) BJ cells cut by AsiSI in an intergenic site (n = 3 independent experiments); (M) NIH2/4 cells cut by CRISPR/Cas9 at the c-Myc locus (n = 4 independent experiments). (N) Mean fold change of Drosha, Dicer and Translin mRNA by RT-qPCR in NIH2/4 cells. Error bars indicate SEM (n = 3 independent experiments). (O) Pre-DDRNA and pre-miRNA have a similar biogenesis. NIH2/4 cells knocked-down for Drosha (siDro), Dicer (siDic) or Luciferase (siLuc) were transfected with a vector expressing I-SceI (+) or an empty vector (−). RNA fractions of 40–200 nt in length were recovered by gel-extraction. Bar plots show the relative enrichment of let7a pre-miRNA and Lac pre-DDRNA. Error bars indicate SEM (n = 3 independent experiments). (P,Q) DilncRNAs and the chromatin-bound ncRNA-TERRA are resistant to RNase A. Bar plots show the mean fold change of the indicated RNAs upon RNase A treatment relative to BSA-treated sample. Error bars indicate SEM (n = 3 independent experiments). P value was calculated using two-tailed t-test. P < 0.05, P < 0.01, P < 0.001, P < 0.0001. Statistical source data are provided in Supplementary Table 4.

Supplementary Figure 3 RNAPII localizes to DSBs in mammalian cells.

(A) Confocal microscopy reveals the enrichment of γH2AX and active RNAPII at the damaged locus. The panel shows representative confocal images of LacR loci (red, inset L), γH2AX (green, inset H), RNAPII pSer5 (purple, inset R) and merge (inset M) in uncut and cut NIH2/4 cells. The yellow-boxed inset images correspond to the yellow-boxed regions. White outline defines nuclear contour. Scale bar 10 μm. (B,C) Quantification of A. Plots show the enrichment of RNAPII pSer5 and γH2AX in uncut and cut cells, calculated on confocal image stacks. Dots represent individual cells. Black bar represents mean (data are shown as pool of 2 independent experiments, ≥25 cells per sample). (D) RNAPII pSer5 enrichment index was plotted as a function of γH2AX enrichment index at the Cherry-LacR signal. Linear fit is depicted as a red line (Pearson’s r = 0.6). Individual data points correspond to individual nuclei (data are shown as pool of 2 independent experiments, ≥50 nuclei). (E) Intensity profiles along the chromatin fibers in the yellow-boxed images in Fig. 3A. (F) Bar plot shows the mean fold change relative to uncut cells of RNAPII pSer5 and pSer2 and total RNAPII enrichment at the Lac sequences in NIH2/4. Error bars indicate SEM (n = 3 independent experiments). (GI) ChIP controls in NIH2/4 cells. RNAPII pSer5 and pSer2 and total RNAPII are enriched in the coding and promoter regions of the beta-actin (Act) gene, but not in an intergenic region. Data are shown as percentage of input. Data are shown as one representative of 2 independent experiments. (J) ChIP controls in HeLa cells. Total RNAPII is enriched in the coding and promoter regions of the beta-actin (Act) gene, but not in an intergenic region. Data are shown as percentage of input. Data are shown as one representative of 3 independent experiments. (K) Biotinylated DNA on streptavidin beads was cut or not by recombinant I-SceI and incubated with nuclear cell extract. Input and pull-down samples were probed for total RNAPII pSer5 and pSer2 and total RNAPII. P value was calculated using two-tailed t-test. P < 0.05, P < 0.01, P < 0.0001. Statistical source data are provided in Supplementary Table 4. Unprocessed original blots are shown in Supplementary Figure 9.

Supplementary Figure 4 In vitro DSB-induced transcription is not dependent on DDR kinases.

(AC) Denaturing PAGE showing the products of in vitro transcription reactions in the presence of [α32P]UTP with pLacTet plasmid (A) or pUC19 plasmid (B,C) and appropriate controls. CFE indicates cell free extract. M indicates radiolabeled DNA ladder, nt indicates nucleotides. (D) Denaturing PAGE showing the products of in vitro transcription reactions in the presence of [α32P]UTP with pBluescript plasmid in its circular form or digested with different restriction enzymes generating one DSB with 5’-protruding, blunt or 3’-protruding DNA ends. pLacTet plasmid is used as control. CFE indicates cell free extract. M indicates radiolabeled DNA ladder, nt indicates nucleotides. (E) Agarose gel shows equal amounts of DNA used in D. (F) Denaturing PAGE showing the products of in vitro transcription reactions in the presence of [α32P]UTP with pUC19 plasmid in its circular form or digested with different restriction enzymes, as in D. (GL) Analysis of the role of DDR upstream kinases in the control of transcription from DSBs. Denaturing PAGE showing in vitro transcription assays performed by incubating cell-free extract (CFE) with linear pLacTet plasmid and increasing concentrations (0, 1, 10, 100 nM, 1, 10 μM) of the ATM inhibitor KU60019 (G); with cell-free extract (CFE) prepared from ATM wild-type or knockout embryonic stem (ES) cells (H); with increasing concentrations (0, 1, 10, 100, 1000 μM) of the ATR inhibitor VE-821 (I); with increasing concentrations (0,1, 5, 10, 20, 50 μM) of the PI3K-like kinases inhibitor Wortmannin (J); with increasing concentrations of the following DNA-PKcs inhibitors: (K) NU7441 (0, 10, 100, 1000 nM) and (L) NU7026 (0, 1, 10, 100, 1000 μM). M indicates radiolabeled DNA ladder, nt indicates nucleotides.

Supplementary Figure 5 The MRN complex is necessary for RNAPII recruitment to DSBs and DDR foci are sensitive to RNAPII inhibition.

(A) Analysis by RT-qPCR shows a significant reduction in Mre11, Rad50 and Nbs1 mRNA relative levels upon knockdown by siRNA (siMRN) in NIH2/4 cells. Error bars represent SEM (n = 3 independent experiments). (B,C) The bar plots show the enrichment of RNAPII pSer5 and γH2AX in uncut and cut cells treated with DMSO or Mirin, calculated on confocal image stacks. Data is shown as mean. Error bars represent SEM (n = 3 independent experiments). (D) Focal accumulation of pATM, but not γH2AX, at the damage site is reduced in cut NIH2/4 cells treated with RNAPII inhibitor. The panel shows representative images of NIH2/4 cells treated with vehicle or RNAPII inhibitor before induction of I-SceI. Scale bar 5 μm. (E) Specific inhibition of RNAPII by a 6 h treatment with AM before cut induction strongly inhibits 53BP1 focus formation at the locus in NIH2/4 cells. The bar plot shows the percentage of cells positive for 53BP1-LacR co-localization. Error bars indicate SEM (n = 3 independent experiments, ≥60 cells per sample). (FI) Efficacy as well as specificity of AM, DRB, ACTD treatments in NIH2/4 cells were evaluated by RT-qPCR by quantifying short-lived RNAs specifically transcribed by individual RNA polymerases, as indicated, as well as 53bp1 and Atm mRNA levels. The bar plots show the levels of the indicated RNA. Data are shown as one representative of 2 independent experiments. (J) NIH2/4 cells transfected with GFP-LacR and Cherry-53BP1 were microinjected either with BSA, I-SceI or I- SceI together with AM. The plot shows the relative intensity of 53BP1 focus over time (min) in the different conditions. Data are normalized to the 0 min time point for each sample (≥10 cells per time point, per sample). (B,C) P value was calculated using two-tailed t-test. (E) P value was calculated using chi-squared test. P < 0.05, P < 0.01, P < 0.0001. ns indicates not significant. Statistical source data are provided in Supplementary Table 4.

Supplementary Figure 6 IR-induced DDR foci are sensitive to RNAPII inhibition.

(AC) Focal accumulation of 53BP1 and pATM, but not γH2AX, upon IR is reduced in HeLa cells treated with RNAPII inhibitors (relative to Fig. 6E, F). Representative images of HeLa treated with vehicle or RNAPII inhibitors (AM in these images) before IR and probed for γH2AX, 53BP1 and pATM. Scale bar 10 μm. (DG) HeLa cells were treated with AM for 6 h, DRB, ACTD at low or high doses for 2 h before IR and RNA was extracted 1 h post IR. Efficacy of the treatments were evaluated by RT-qPCR by quantifying short-lived RNAs specifically transcribed by individual RNA polymerases, as indicated, as well as 53BP1 and ATM mRNA levels. The bar plots show the levels of the indicated RNA. Data are shown as one representative of 4 (D,F) or 2 (E,G) independent experiments. (H) Bar plot shows the percentage of irradiated HeLa cells positive for γH2AX, 53BP1 and pATM upon different treatments with AM (cells with >10 foci were scored positive, >100 cells per sample). (I,J) Efficacy as well as specificity of the treatments with AM for 2, 6 and 8 h were evaluated by RT-qPCR by quantifying short-lived RNAs specifically transcribed by individual RNA polymerases, as indicated, as well as 53BP1 and ATM mRNA levels. The bar plots show the levels of the indicated RNA. Data are shown as one representative of 2 independent experiments. (K) pATM foci are reduced in irradiated normal human fibroblasts (BJ) treated with RNAPII inhibitors. The panel shows representative images of cells treated with vehicle or RNAPII inhibitor (DRB in these images) 2 h before IR. Scale bar 5 μm. (L) BJ cells were treated with AM, DRB or vehicle 2 h before IR. Bar plot (quantification of Fig. 6D and Supplementary Fig. 6K) shows the percentage of cells positive for γH2AX, 53BP1 and pATM (cells with >10 foci were scored positive). Error bars indicate SEM (n = 3, n > 100 cells per sample). P value was calculated using two-tailed t-test. P < 0.01, P < 0.001, P < 0.0001. Statistical source data are provided in Supplementary Table 4.

Supplementary Figure 7 IR-induced DDR foci are sensitive to RNAPII inhibition in normal human fibroblasts.

(A,B) Accumulation of 53BP1, but not γH2AX, to an AsiSI-induced DSB is impaired in AsiSI-ER BJ-5Ta cells treated with DRB at a genic site. The bar plots show the percentage of ChIP enrichment relative to the input of γH2AX (A) and 53BP1 (B) associated with genomic DNA. Data are shown as one representative of 3 independent experiments. (C) Fold change relative to uninduced cells of γH2AX and 53BP1 enrichment at a genic site (relative to A,B). Values are shown as mean. Error bars indicate SEM (n = 3 independent experiments). (D,E) Same analyses as in A,B at an AsiSI-ER intergenic site. Data are shown as one representative of 3 independent experiments. (F) Fold change relative to uninduced cells of γH2AX and 53BP1 enrichment at an intergenic site (relative to D,E). Values are shown as mean. Error bars indicate SEM (n = 3 independent experiments). (GJ) Foci of RIF1 (G), pKAP1 (H), RNF168 (I) and ubiquitinylated proteins detected by FK2 antibody (J) are reduced in irradiated normal human fibroblasts (BJ) treated with DRB for 2 h at RT before IR. The panel shows representative images. Scale bar 5 μm. The bar plot shows the percentage of positive cells (cells with >10 foci for RIF1, RNF168 and FK2 and >3 foci for pKAP1 were considered positive). Error bars indicate SEM (n = 3 independent experiments, >150 cells per sample). P value was calculated using two-tailed t-test. P < 0.05, P < 0.01, P < 0.0001. ns indicates not significant. Statistical source data are provided in Supplementary Table 4.

Supplementary Figure 8 RNAPII inhibition impairs DNA repair and sequence-specific ASOs reduce DDR foci formation at individual DSBs.

(A) Representative images of BJ cells pre-treated with DMSO or DRB for 2 h, irradiated (2Gy) and fixed at the indicated time points (relative to Fig. 6G). Scale bar 5 μm (B) Representative images of neutral comet assay. HeLa cells were pre-treated with DMSO or DRB for 2 h, irradiated (5Gy) and collected at the indicated time points (relative to Fig. 7H, I). Scale bar 20 μm (C) Quantitative analysis of 53BP1 focus intensity, normalized on Mock sample, of the experiments shown in Fig. 7D. Dots represent individual cells. Black bar represents mean. Error bars indicate SEM (data are shown as pool of n = 3 independent experiments, ≥100 cells analysed for each sample). (D) NIH2/4 cells were transfected with the indicated ASOs, irradiated (2Gy) and fixed 1 h post IR. Bar plot shows the percentage of DDR-positive cells (mean of n = 2 independent experiments, >50 cells per sample). (E) Schematic representation of ASOs (A and B in red) preventing the interaction between dilncRNAs (light blue) and DDRNAs (dark blue) originating from Tet sequences in NIH3T3duo cell line. The black arrows indicate the head-to-head configuration of Tet sequences flanking the I-SceI site. (F) Bar plot shows mean fold change normalized to uncut CTL ASO of enrichment relative to input of γH2AX at DAB1 locus at 50, 1000 bp from DSB. Error bars indicate SEM (n = 3 independent experiments). (G) Bar plot shows the percentage of enrichment relative to the input of Mock sample at DAB1 locus at 50, 1000 bp from DSB. Data are shown as one representative of 3 independent experiments. (H) Bar plot shows the percentage of enrichment relative to the input of 53BP1 or Mock at RYR2 locus cut by I-PpoI in HeLa cells. Data are shown as one representative of 2 independent experiments. (I) Bar plot shows the percentage of enrichment relative to the input of 53BP1 or Mock at an unrelated region on chromosome 22 not cut by I-PpoI in HeLa cells. Data are shown as one representative of 2 independent experiments. P value was calculated using two-tailed t-test. P < 0.0001. ns indicates not significant. Statistical source data are provided in Supplementary Table 4.

Supplementary Figure 9 Unprocessed blots.

Unprocessed blots relative to Figures 3E, 5A and Supplementary Figure 3K.

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Michelini, F., Pitchiaya, S., Vitelli, V. et al. Damage-induced lncRNAs control the DNA damage response through interaction with DDRNAs at individual double-strand breaks. Nat Cell Biol 19, 1400–1411 (2017). https://doi.org/10.1038/ncb3643

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