Damage-induced long non-coding RNAs (dilncRNA) synthesized at DNA double-strand breaks (DSBs) by RNA polymerase II are necessary for DNA-damage-response (DDR) focus formation. We demonstrate that induction of DSBs results in the assembly of functional promoters that include a complete RNA polymerase II preinitiation complex, MED1 and CDK9. Absence or inactivation of these factors causes a reduction in DDR foci both in vivo and in an in vitro system that reconstitutes DDR events on nucleosomes. We also show that dilncRNAs drive molecular crowding of DDR proteins, such as 53BP1, into foci that exhibit liquid–liquid phase-separation condensate properties. We propose that the assembly of DSB-induced transcriptional promoters drives RNA synthesis, which stimulates phase separation of DDR factors in the shape of foci.
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We thank N. G. Walter and S. Pitchiaya (Single Molecule Analysis Group and Center for RNA Biomedicine, Department of Chemistry, University of Michigan) for the generation of crucial preliminary data; all of the F.d’A.d.F. group members for support and discussions; E. Soutoglou (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France) and J. Lukas (Novo Nordisk Foundation Center for Protein Research, Copenhagen, Denmark) for reagents; C. Zeirhut (Laboratory of Chromosome and Cell Biology, The Rockefeller University, New York, USA) for reagents and discussions; F. Iannelli for bioinformatic analysis of repair products; S. Pasqualato (Biochemistry and Structural Biology Unit at the European Institute of Oncology IRCCS, Milan, Italy) for the purification of DNA fragments. F.P. was supported by a Marie Curie international mobility fellowship part of Structured International Post Doc Program; F.G. and R.C. were supported by the Regione Lombardia and Cariplo foundation (Project-2016-0998); F.G. was supported by the Associazione Italiana per Ricerca sul Cancro, (MFAG 2018-22083); F.d’A.d.F. was supported by AIRC (application numbers 12971 and 21091), the Cariplo Foundation (grant number 2014-0812), the Fondazione Telethon (GGP17111), PRIN 2010–2011 and 2015, the Italian Ministry of Education Universities and Research EPIGEN Project, InterOmics Project and AMANDA project Accordo Quadro Regione Lombardia-CNR, a European Research Council advanced grant (322726), AriSLA (project ‘DDRNA and ALS’) and AIRC Special Program 5 per mille metastases (Project-21091).
F.d’A.d.F. is listed as an inventor on the patent application: PCT/EP2013/059753.
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Integrated supplementary information
Supplementary Figure 1 RNAP II and PIC are recruited to DSBs as detected by ChIP in vivo and in vitro.
(a) Bar plot shows enrichment relative to the input of γH2AX, POLR2A, POLR1A, POLR3A, PIC components, MED1 and CDK9 as detected by ChIP at actin promoter. N=1. (b) Bar plot shows enrichment relative to the input of γH2AX, POLR2A, POLR1A, POLR3A, PIC components, MED1 and CDK9 as detected by ChIP at 100 bp, 2000bp, 3000bp and 10000bp distances upstream from the DSB induced by I-PpoI at an endogenous site within the DAB1 gene. N=3 (100bp, 2000bp), N=2 (3000bp) and N=1 (10000bp) independent experiments. (c) Schematic representing 100bp DNA oligonucleotide with both ends biotinylated and bound to the beads and thus not available, and 100bp DNA oligonucleotide with only one end biotinylated and one free end. (d) Analysis by immunoblotting with the indicated antibodies of pull-down experiments with biotinylated 100bp DNA oligonucleotides bound to streptavidin beads either at both ends (lane 1: blocked ends) or one end only (lane 2: free ends) incubated with HeLa nuclear extract. One tenth of the beads was used to purify DNA and it was run on a gel to demonstrate equal amounts, N=3 independent experiments. (e) Analysis by immunoblotting with the indicated antibodies of pull-down carried out in Hela nuclear extract with biotinylated 50bp DNA oligonucleotides bound to streptavidin beads either at both ends (lane 1: blocked ends), on only one end (lane 2: blunt ends), and exposing different DNA ends (lane 3: 3’ protruding, lane 4: 5’ protruding; both 10nt protruding). One tenth of the beads was used to purify DNA and it was run on a gel to demonstrate equal amounts of DNA, N=2 independent experiments. Statistics source data are provided in Supplementary Table 2.
Supplementary Figure 2 RNAP II and PIC components localize to DSB as detected by super-resolution imaging and DI-PLA.
(a-f) Representative super-resolution images of indicated proteins in U2OS cells mock treated (a-c) or treated with NCS (d-f) for 30 min and labelled with EU (15 min pulse). (g) and (h) Cross-PC between POLR2A-pS5/TBP/CDK and γH2AX was plotted comparing to that with randomized nuclei (RND-ctrl). The box indicates mean +/- SD. n=189, 130, and 117 nuclei were collected from 4, 3, and 3 biologically independent experiments for cross-PC analyses between POLR2A-pS5/TBP/CDK7 and γH2AX, respectively, in (g) and n=184, 109, and 102 nuclei were collected from 4, 3, and 3 independent experimentsfor cross-PC analyses between POLR2A-pS5/TBP/CDK7 and γH2AX, respectively, in (h). P-values of the two-sample unpaired t-test were shown in the figure accordingly. (i-l) Co-Localization Occurrence between indicated protein pairs plotted as the function of the image density of γH2AX. The Co-Localization Occurrence was calculated as Corr(A, B)×ρ (A)×ρ (B) where Corr(A, B) is the Cross-PC between A and B in one image, and ρ(A) and ρ(B) are the image density of A and B, respectively. From i to l, n=180, 129, 104, and 391 nuclei were collected from 4, 3, 3, and 10 independent experimentsfor mock data, respectively, and n=175, 90, 98, and 320 nuclei were collected from 4, 3, 3, and 10 independent experiments for NC+S data. Note that the number of nuclei for co-localization analyses are slightly less than that submitted for Cross-PC analyses due to the failure in Auto-PC fit for some nuclei. (m) DI-PLA between CyclinA and biotin, in not irradiated Hela cells (No IR) or exposed to ionizing radiations (IR 2Gy) and fixed 30 minutes after treatment. DNA stained by DAPI. Quantifications show SEM of >75 nuclei from N=3 independent experiments. Statistics source data are provided in Supplementary Table 2.
Supplementary Figure 3 MRN, TBP and TFIIB knock-downs prevent dilncRNA synthesis without affecting other protein’s level.
(a) Immunoblotting analysis of proteins’ levels in response to siRNA treatment. Specific antibodies for target proteins are indicated. Experiment was repeated twice with similar results. (b) Immunoblotting analysis of proteins’ levels in response to siRNA treatment. Specific antibodies for target proteins are indicated. (right) dilncRNA induction upon DSB formation, at DAB1 endogenous site in HeLa cells treated with indicated siRNA, quantified by strand-specific RT-qPCR, is shown. Data shown are one representative of N=3 biologically independent experiments except for siTFIIB N=1. Statistics source data are provided in Supplementary Table 2.
Supplementary Figure 4 Inactivation by RNA interference or small molecules reduces DDR signalling in cultured cells.
(a-b) Representative figures of pATM and MDC1 staining in siLuc, siTBP and siTFIIB treated U2OS cells, irradiated with 1 Gy. Quantifications show SEM of: for pATM siLUC n=121, siTBP n=112, siTFIIB n=130; for MDC1 siLUC n=111, siTBP n=129, siTFIIB n=122. Each from 2 biologically independent experiments. γH2AX quantification included in Fig. 4a. (c) Vectors expressing knockdown-resistant TBP or TFIIB fused to GFP, or GFP alone, were transfected in U2OS cells treated with siLuc, siTBP or siTFIIB. Quantifications of 53BP1 and γH2AX foci performed on GFP positive cells are shown, error bars represent the SEM of (from top to bottom) 64, 30, 32, 53, 24, 27, 38 nuclei from 2 biologically independent experiments except for siLUC+TBPGFP and siLUC+TFIIBGFP, performed once. (d) Calculation of fraction of γH2AX foci associated with de novo transcription (EU pulse) in cells treated with siRNA against Luciferase (CTRL), TBP and TFIIB. The level of association is normalized on CTRL. The bars represent the SD and n=300, 295, and 297 nuclei from 4 independent experiments. P-values of two-sample unpaired t-test are shown in the figure accordingly. (e) Pearson correlation between EU signal in nuclei (global) and at DSB (local) upon siRNA treatment against Luciferase (CTRL), TBP and TFIIB. Canonical transcription dependent on PIC and transcription at DSB decrease proportionally. n=390, 385, and 386 nuclei from 4 independent experiments. (f) Representative figures of irradiated U2OS cells (1 Gy) and pATM staining in response to THZ1, DRB or ATM inhibitor (KU-60019) treatments are shown. Quantifications show SEM of DMSO n=114, THZ1 n=112, DRB n=114, ATMi n=110 nuclei scored from 2 biologically independent experiments γH2AX quantification included in Fig. 4b. In panels (a), (b), (c) and (f) P values were calculated by one-way ANOVA and significance are represented as ** P<0.01, **** P<0.0001. Statistics source data are provided in Supplementary Table 2.
(a) Schematic showing structured domains contained in 53BP1 proteins, together with Low Complexity Regions (adapted from S.M.A.R.T. EMBL software database https://smart.embl.de). (b) Graph plotting intrinsic disordered regions (y-axis) of human (left) and mouse (right) 53BP1 amino acid sequence (x-axis) analysed by PONDR VSL2. Scores higher than 0.5 indicate predicted disordered regions. (c) Fourier analysis of droplet shape fluctuations. (i) Differential image obtained by subtracting to an instantaneous snapshot of a large droplet its time-averaged profile. Positive regions (outlined in yellow) correspond to portions of the droplet that are outside the average contour, while negative regions (blue) corresponds to local inflections of the droplet membrane. For both axes, a unit corresponds to 1 μm. (ii) Spatial-temporal map of droplet shape fluctuation, obtained by considering, as a function of time, the value corresponding to the differential image (like the one shown in panel (a)) along a curvilinear coordinate s following the average droplet contour. Colormap is as in panel (a). (iii) Structure functions D(q,Δt) obtained from the spatio-temporal map shown in (b), and quantifying the temporal relaxation of Fourier modes of the droplet shape fluctuations. Five excitations modes are shown, corresponding to wave-vectors q=2πn/L, where L=11 μm is the length of the average droplet contour and n=1-5. Continuous lines are exponential fit to the data. (d) Visualization of turbidity associated with phase separation in in vitro reactions upon incubation at 4 °C for 48 h. Graph on the right shows quantifications of turbidity of the solutions as measured by absorbance at 350 nm wavelength. N=3 independent samples. Statistics source data are provided in Supplementary Table 2.
(a) schematic illustration of estimation of the fraction of protein A associated with protein B. The STORM data of the two proteins are submitted for DBSCAN clustering. Each cluster of protein A is scored as a paired one If its Nearest-Neighbouring Distance (NND) to any cluster of protein B is smaller than the average radius of protein A clusters. The average radius of clusters of protein A is estimated with the Pair-Correlation function (see section in Methods). Meanwhile, clusters of both protein A and protein B are randomly traslocted and orientated, and each cluster of protein A is scored whether it is paired to protein B at such randomization level. Fraction of protein A associated with protein B is then calculated as indicated. (b) Exambples of DBSCAN clustering and cluster randomization. (c) Fraction of γH2AX with either MCM (black, negative control) or EU (red). Box denotes mean +/- SD (n = 40 and 162 nuclei collected from 2 and 4 experimental replicates for MCM and EU, respectively. The p-value of the two-sample unpaired t-test is 2E-106).
Unprocessed blots for Fig. 3c.
Supplementary Figures 1–7, titles and legends for Supplementary Tables 1–3 and Supplementary Videos 1–6.
Primers and oligos.
Statistics source data.
53BP1 foci are sensitive to agents that target liquid compartments.
53BP1 foci nucleate, grow and coarse in time.
53BP1 foci coalescence as liquid droplets.
53BP1 liquid properties are dependent on transcription.
Extensive treatment with THZ1 is not lethal for cells.
53BP1 foci rely on RNA multi-interaction properties.
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Pessina, F., Giavazzi, F., Yin, Y. et al. Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors. Nat Cell Biol 21, 1286–1299 (2019). https://doi.org/10.1038/s41556-019-0392-4
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