ARTICLE

https://doi.org/10.1038/s41467-019-08863-1 OPEN SFPQ and NONO suppress RNA:DNA-hybrid- related telomere instability

Eleonora Petti1,2,6, Valentina Buemi1,2, Antonina Zappone1,2, Odessa Schillaci1, Pamela Veneziano Broccia1,2, Roberto Dinami1,2,6, Silvia Matteoni 3, Roberta Benetti4,5 & Stefan Schoeftner1,2

In vertebrates, the telomere repeat containing long, non-coding RNA TERRA is prone to form RNA:DNA hybrids at telomeres. This results in the formation of R-loop structures, replication

1234567890():,; stress and telomere instability, but also contributes to alternative lengthening of telomeres (ALT). Here, we identify the TERRA binding NONO and SFPQ as novel regulators of RNA:DNA hybrid related telomere instability. NONO and SFPQ locate at telomeres and have a common role in suppressing RNA:DNA hybrids and replication defects at telomeres. NONO and SFPQ act as heterodimers to suppress fragility and homologous recombination at telo- meres, respectively. Combining increased telomere fragility with unleashing telomere recombination upon NONO/SFPQ loss of function causes massive recombination events, involving 35% of telomeres in ALT cells. Our data identify the RNA binding proteins SFPQ and NONO as novel regulators at telomeres that collaborate to ensure telomere integrity by suppressing telomere fragility and homologous recombination triggered by RNA:DNA hybrids.

1 Genomic Stability Unit, Laboratorio Nazionale—Consorzio Interuniversitario per le Biotecnologie (LNCIB), Padriciano 99, 34149 Trieste, Italy. 2 Department of Life Sciences, Università degli Studi di Trieste, Via E. Weiss 2, 34127 Trieste, Italy. 3 Cellular Networks and Molecular Therapeutic Targets, Proteomics Unit, IRCCS—Regina Elena National Cancer Institute, via Elio Chianesi 53, 00144 Rome, Italy. 4 Dipartimento di Area Medica (Dame), Università degli Studi di Udine, p.le Kolbe 1, 33100 Udine, Italy. 5 Cancer Epigenetics Unit, Laboratorio Nazionale—Consorzio Interuniversitario per le Biotecnologie (LNCIB), Padriciano 99, 34149 Trieste, Italy. 6Present address: Oncogenomic and Epigenetic Unit, IRCCS—Regina Elena National Cancer Institute, via Elio Chianesi 53, 00144 Rome, Italy. These authors contributed equally: Eleonora Petti, Valentina Buemi, Antonina Zappone. Correspondence and requests for materials should be addressed to S.S. (email: [email protected])

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elomeres are heterochromatic structures that protect but also TERRA, a telomere repeat [UUAGGGn] containing long Teukaryotic ends from an unwanted elicitation non-coding RNA5,6. In human cancer cells, DNA methylation of a DNA damage response, telomere degradation and sensitive promoters located in subtelomeres recruit RNA Poly- aberrant recombination1–3. Incomplete replication of chromo- merase II that uses the CCCTAA-repeat containing telomere some ends results in progressive shortening of telomeres, finally strand as template for the transcription of TERRA5–11. TERRA is leading to telomere dysfunction and irreversible cell cycle arrest, heterogeneous in size and a subfraction of TERRA has been also referred to as replicative senescence4. Core components of shown to localize to telomeric chromatin5,6,11. Accordingly, vertebrate telomeric chromatin comprise the multi- TERRA is reported to act as scaffold that promotes the con- complex “shelterin” that has a key role in telomere protection centration of proteins or enzymatic activities at telomeres, thereby

a 6 b c 6 6 MW NE Biotin- r[EGFP] Biotin- r[UUAGGG] Beads

150 kDa

100 kDa hnRNP U * SFPQ NE Beads Biotin-r[EGFP] Biotin-r[UUAGGG] Beads NE Biotin-r[UUAGGG] * Bbiotin-r[EGFP] 75 kDa Not * indentified 100 kDa SFPQ 100 kDa SFPQ FUS NONO NONO * NONO 50 kDa 50 kDa 50 kDa * hnRNP U FUS FUS * hnRNP H1 50 kDa 50 kDa * hnRNP H2 Actin Actin * hnRNP F 37 kDa 37 kDa * hnRNP D0 * mESCs H1299 37 kDa * DAZAP1 * hnRNP A/B * hnRNP A3 mESCs hnRNP A3 hnRNP A2/B1

d NONO TRF2 TRF2/NONO

f 50.7 ± 6.4 80 n = 100 N = 3 60 19.0 ± 2.1 40 n = 100 N = 3

1 μm 20

U-2 OS events per nucleus

Number of co-localization 0

e SFPQ TRF1 TRF1/SFPQ

NONO/TRF2 SFPQ/TRF1

1 μm U-2 OS

g h p = 0.002

p = 0.005 1:10 1:100 IgG M TRF2 NONO FUS H3 Tot 1:1000 IgG R SFPQ 15 p = 0.04 Telomere probe s.e. 7.6 ± 1.2 N = 3 10 5.7 ± 0.7 Telomere probe l.e. 3.3 ± 0.8 N = 3 N = 3 5 1.0 telomeric repeats

AluY probe Immunoprecipitated N = 3 (control IgG R set «1») 0

FUS IgG R NONO SFPQ

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Fig. 1 SFPQ and NONO interact with TERRA and localize to telomere repeats. a Silver staining of SDS-PAGE gels of eluates obtained from RNA-pull-down experiments using biotinylated r[UUAGGG]6, biotinylated r[EGFP] RNA oligonucleotides or empty beads. Candidate TERRA interacting proteins identified by mass spectrometry are indicated. NE, nuclear extract used as input; MW, molecular weight marker. b, c Western blotting analysis of RNA-pull-down eluates using specific anti-NONO and anti-SFPQ antibodies confirms binding specificity of NONO and SFPQ for UUAGGG RNA repeats in mESCs (b) and H1299 cells (c). FUS was previously reported to interact with TERRA42. Actin was used as loading control; NE, nuclear extract was used as input. Source blots are available as Supplementary Figure 6 and 7. d Representative image of co-localization events (arrowheads) between NONO and the shelterin protein TRF2 as determined by confocal microscopy in U-2 OS cells. e Representative image of co-localization events (arrowheads) between SFPQ and the shelterin protein TRF1 by confocal microscopy in U-2 OS cells. f Quantification of d and e. Mean number of NONO-TRF2 and SFPQ-TRF1 co-localization events per nucleus is shown. Error bars indicate standard deviation. n = number of analyzed nuclei. g Chromatin immunoprecipitation experiments (ChIP) using U-2 OS cells and mouse anti-TRF2, rabbit anti-histone H3, rabbit anti-FUS, rabbit anti-NONO, and rabbit anti-SFPQ antibodies. Mouse and rabbit control IgGs (IgG M/IgG R) were used as negative control. Serial dilutions of chromatin extract (input) prepared from U-2 OS cells were loaded. h Quantification of three independent ChIP experiments, average enrichment of telomeric repeats is indicated; s.e.: short exposure; l.e.: long exposure. f, h N, number of independent experiments, whiskers indicate standard deviation; a two-tailed Student’s t-test was used to calculate p-values. Source data is provided as a Supplementary Information File. Scale bar, 1 μm. siRNAs listed in Supplementary Table 1

impacting on chromatin structure, chromosome end protection, telomere fragility; in contrast SFPQ represents a powerful barrier replication but also telomere maintenance by telomerase12–14. to homologous recombination at telomeres. Accordingly, com- Importantly, due to its G-rich sequence content, TERRA is prone bined loss of NONO and SFPQ results in a massive increase of to form RNA:DNA hybrids with the C-rich telomeric strand. This telomere recombination events and rapid alterations in telomere results in the displacement of the TTAGGG repeat containing length in both, telomerase-positive and negative cells. Our study telomere strand, giving rise to so-called “R-loop” structures15–19. introduces NONO and SFPQ as novel regulators of RNA:DNA- In a natural context, R-loops are relevant for the regulation of hybrid management that may open new inroads in defining expression, chromatin structure and IgG class switch strategies that aim to target recombination-based pathways of recombination20–29. However, R-loops can represent obstacles to telomere maintenance in human cancer. DNA replication resulting in replicative stress, chromosome fra- gility, DNA lesions and the activation of recombination-mediated DNA repair that can finally lead to chromosome Results rearrangements28,30–34. Recent studies showed that several bio- NONO and SFPQ are TERRA interacting proteins.Inorderto logical pathways prevent or resolve RNA:DNA-hybrid structures identify novel TERRA interacting proteins, we performed at eukaryotic telomeres. In yeast, loss of the 5′ to 3′ exonuclease, TERRA RNA-pull-down experiments using mouse embryonic Rat1p was linked with increased abundance of telomeric RNA: stem cells that maintain telomeres via telomerase dependent DNA hybrids35. Loss of the THO complex in yeast results in and independent telomere maintenance pathways39. Biotiny- TERRA accumulation at telomeres, R-loop formation and lated RNA oligonucleotides containing six UUAGGG repeats or increased telomere recombination rates16. Further, lack of the flap control oligonucleotides were incubated with nuclear extracts endonuclease 1 (FEN1), a canonical lagging strand DNA repli- obtained from mouse embryonic stem cells and recovered by cation protein, leads to replicative stress at telomeres, caused by using stretpavidine coated agarose beads. Eluates from TERRA increased telomeric RNA:DNA hybrids36. In yeast and human and control RNA-pull-down experiments were processed by gel cells, a particular relevance has been attributed to RNaseH that electrophoresis. Silver staining revealed a series of proteins that antagonize R-loop formation by degrading RNA paired with were specifically eluted from TERRA RNA oligonucleotides DNA15,17,35. In yeast, loss of RNaseH function results increased (Fig. 1a). Mass spectrometric analysis revealed the identity of a abundance of telomeric R-loops, higher telomere recombination large set of reported TERRA interacting proteins, such as a rates, and a delayed onset of replicative senescence in telomerase- series of hnRNPs, as well as Fused in Sarcoma (FUS)40–42. negative cells15. Telomerase-negative human ALT cells, that Importantly, the TERRA-pull-down approach identified the maintain telomere function by homologous recombination-based Non-POU domain-containing octamer-binding protein “alternative lengthening of telomeres” (ALT), have been (NONO) and Splicing factor proline- and glutamine-rich demonstrated to display an increased abundance of R-loops and (SFPQ) as TERRA interacting proteins that have not yet been RNaseH1 localization at telomeres17. Depletion of RNaseH1 in studied in the context of telomere function. The specificity of these cells results in enhanced R-loop formation, replicative SFPQ and NONO for TERRA was validated by western blotting stress, selective fragility at the telomeric CCCTAA-repeat con- using TERRA-pull-down eluates obtained from mouse taining strand and increased extra-chromosomal telomere repeat embryonic stem cells or H1299 non-small cell lung cancer cells content17. This suggests that R-loop-related activation of (Fig. 1b, c). NONO and SFPQ belong to the Drosophila beha- recombination-based DNA repair mechanisms can translate into vior/human splicing (DBHS) family protein that are defined by improved telomere repeat maintenance. This may be of special highly conserved tandem N-terminal RNA recognition motifs relevance in telomerase-negative tumors that represent approxi- (RRMs), a NonA/paraspeckle domain (NOPS) and a C-terminal mately 10–15% of all human tumors37,38. coiled-coil43. NONO and SFPQ function is reported to depend Here, we set out to investigate novel pathways that impact on on the obligatory formation of homo- or heterodimers of both RNA:DNA-hybrid-related regulation of telomere function. We proteins43. In line with this, immunoprecipitation experiments show that the TERRA RNA binding proteins NONO and SFPQ using extracts from telomerase-positive H1299 and telomerase- are novel protein components of telomeric chromatin that have a negative U-2 OS osteosarcoma cells that maintain telomeres via central role suppressing RNA:DNA-hybrid formation, DNA the recombination-based “alternative lengthening of telomeres” replication defects, recombination, and DNA damage at telo- (ALT) pathway, confirm a direct interaction between NONO meres of telomerase-positive and telomerase-negative cancer cells. and SFPQ in the cell model systems used in this study (Sup- Remarkably, NONO and SFPQ antagonize different downstream plementary Figure 1A, B). Confocal microscopy revealed that a effects of telomeric RNA:DNA hybrids: NONO suppresses significant fraction of nuclear restricted SFPQ and NONO foci

NATURE COMMUNICATIONS | (2019) 10:1001 | https://doi.org/10.1038/s41467-019-08863-1 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08863-1 co-localize with telomere repeat binding factor 2 (TRF2) or co-localization between NONO or SFPQ with PML, suggesting telomere repeat binding factor 1 (TRF1) in U-2 OS interphase that the studied RNA binding proteins do not represent central cells (Fig. 1d–f). Immunoprecipitation experiments support the components of APBs (Supplementary Figure 1E, F). Performing interaction of NONO with TRF1 and TRF2 (Supplementary telomere ChIP we confirm association of SFPQ and NONO Figure 2C, D). Confocal microscopy did not reveal a significant with telomere repeat chromatin and exclude binding to AluY

b c p = 0.0001 p < 0.001 p = 0.0001 p < 0.001 93.33 ± 1.92 ± a si-CTRL si-NONO si-SFPQ n = 90 13,660 245.9 75.55 ± 1.1 10,820 ± 261 N = 3 80,000 n = 90 100 n = 90 n = 90 N = 1328 12,220 ± 188.4 N = 3 N = 667 80 42.5 ± 1 60,000 n = 90 n = 90 N = 891

TERRA 60 N = 3 40,000 1 μm 40 20,000 U-2 OS Cells with > 7 20 TERRA foci (%)

0 TERRA intensity (a.f.u.) 0

si-SFPQ d TERRA γ H2AX Merge si-control si-NONO si-SFPQ si-control si-NONO

e p = 0.022 p = 0.011 14.5 ± 3.0 n = 104 1 μm 25 ± 12.3 1.9 N = 4 n = 100 20 N = 4 15 4.6 ± 0.9 n = 101

si-NONO 10 N = 4 5 co-localizing with H2AX (%) per nucleus Fraction of TERRA foci γ 0 si-SFPQ si-control si-control si-NONO si-SFPQ U-2 OS

TRF1 RNA:DNA hybrids Merge f h TRF1 RNA:DNA hybrids Merge

1 μm 1 μm si-RNaseH1 si-control si-RNaseH1 si-control si-NONO si-NONO si-SFPQ si-SFPQ U-2 OS H1299 g p = 0.007 i p = 0.012 p = 0.013 p = 0.045 p = 0.01 p = 0.04 ± 2.97 ± 0.3 3.06 0.3 2.2 ± 0.2 n = 90 2.6 ± 0.3 n = 91 n = 90 2.2 ± 0.03 2.0 ± 0.04 4 N = 3 3 N = 3 n = 90 N = 3 n = 92 ± n = 90 1.6 0.1 N = 3 N = 3 n = 90 N = 3 3 ± 1.3 0.1 2 N = 3 n = 93 2 N = 3 1 per nucleus

1 per nucleus co-localization events co-localization events TRF1-RNA:DNA hybrids 0 TRF1-RNA:DNA hybrids 0

si-SFPQ si-control si-NONO si-SFPQ si-control si-NONO si-RNaseH1 si-RNaseH1

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Fig. 2 Depletion of NONO and SFPQ cause accumulation of RNA:DNA hybrids. a Representative images of TERRA RNA-FISH performed in U-2 OS cells transiently transfected with NONO- or SFPQ-specific siRNAs or with a non-targeting control siRNA. b Quantification of the percentage of cells with more than three TERRA foci per nucleus. c Quantification of TERRA signals intensity. Arbitrary fluorescence units (a.f.u.) are shown. Box plots: middle line represents median, and the box extends from the 25th to 75th percentiles. The whiskers mark the 10th and 90th percentiles. p-values were calculated using a two-tailed Mann–Whitney test. Median a.f.u. values and standard deviation are indicated; n = number of analyzed nuclei, N = number of analyzed TERRA signals. d Representative images of TERRA RNA-FISH combined with anti-γH2AX immunostaing performed in U-2 OS cells transfected with indicated siRNAs. Arrowheads indicate co-localization events. e Quantification of d; percentage of TERRA foci co-localizing with γH2AX per nucleus is indicated. f, h Representative confocal microscope images of combined immunofluorescence using S9.6 anti-RNA:DNA hybrid and anti-TRF1 antibodies in U-2 OS (f) or H1299 (h) cells transfected with indicated siRNAs. Arrowheads indicate co-localization events. g, i Quantification of telomeric RNA:DNA hybrids per nucleus in U-2 OS (i) or H1299 (h) cells. For quantifications in b, e, g, i mean values are indicated; error bars indicate standard deviation; N = number of independent experiments. n = number of analyzed nuclei. A two-tailed Student’s t-test was used to calculate statistical significance; p-values are shown. Source data is provided as a Supplementary Information File. Scale bar, 1 μm. siRNAs listed in Supplementary Table 1 repeats (Fig. 1g, h). These data indicate that SFPQ and NONO foci in both, U-2 OS and H1299 cells (Fig. 2f–i). This result was represent TERRA interacting proteins that localize to telomere re-capitulated in RNAseH1 knockdown cells that have been repeat sequences. recently reported to show increased abundance of telomeric RNA: NONO and SFPQ cover multiple biological functions that DNA hybrids (Fig. 2f–i, Supplementary Figure 2K)17. RNAi- include transcriptional regulation, the formation of subnuclear mediated depletion of TRF1 from U-2 OS cells resulted in a structures such as paraspeckles, but also DNA damage repair, compete loss of immunostaining for TRF1, thus excluding cross gene expression, cell cycle control, and circadian rhythm43,44.In reactivity of the anti-TRF1 antibody in our experimental setup this study, we exclusively focus on the role of NONO and SFPQ (Supplementary Fig. 2L-M). Together, this indicates that NONO in controlling telomere function in human cancer cells. and SFPQ have a role in suppressing TERRA:telomere RNA: DNA-hybrid formation in telomerase-positive and negative can- cer cells. NONO and SFPQ suppress telomere RNA:DNA hybrids and damage. We next wished to understand whether NONO and SFPQ impact on TERRA expression in U-2 OS cells that produce NONO and SFPQ suppress replication defects at telomeres. TERRA at levels readily detectable by RT-PCR, Northern blotting RNA:DNA hybrids trigger the formation of R-loop structures that and RNA-FISH. Northern blotting and quantitative RT-PCR represent obstacles during DNA replication, leading to replicative using primers that amplify subtelomeric portions of TERRA stress, DNA damage, and increased recombination frequencies at originating from 1 and 21 or chromosomes 2, 10, telomeres. Induction of replication defects at telomeres is linked and 13 revealed that RNAi-mediated depletion of NONO or with the phosphorylation of ATR and the phosphorylation of SFPQ does not impact on total TERRA levels (Supplementary Serine 33 of the 32 kDa subunit of the Replication protein A Figure 2A-C; Supplementary Table 4). However, RNA-FISH (RPA32pSer33), both surrogate markers for replication stress17. revealed that knockdown of NONO or SFPQ in U-2 OS ALT cells As expected, induction of replicative stress by treatment of U-2 significantly increased the number of TERRA foci per nucleus OS with hydroxyurea results in increased global (Fig. 2a, b). In line with this, the proportion of cells with high RPA32pSer33 staining (Fig. 3c). In line with increased RNA: TERRA foci number ( > 12) was increased in experimental cells DNA-hybrid formation, we found that depletion of NONO or (Supplementary Figure 2D). Finally, depletion of SFPQ resulted SFPQ from U-2 OS cells is paralleled by an increased co- in a significant increase in TERRA RNA-FISH signal intensity localization of TERRA with phosphorylated RPA32 (Fig. 3a, b). (Fig. 2a–c). These data suggest that NONO and SFPQ do not This effect is paralleled by a significantly increased co-localization impact on total TERRA levels but rather alter TERRA home- of TRF2 with RPA32pSer33 (Fig. 3c, d). Telomerase-positive ostasis at telomeres. Importantly, in NONO- or SFPQ-depleted H1299 cells recapitulate increased RPA32Ser33 phosphorylation interphase U-2 OS cells TERRA foci showed increased co- at telomere in the absence of SFPQ. However, in loss of NONO localization frequency with the DNA damage marker γH2AX conditions the increase in TRF2/RPA32Ser33 co-localization rates (Fig. 2d, e). Analysis of metaphase chromosomes by immuno does not reach significance in H1299 cells (Supplementary Fig- DNA-FISH showed increased localization of γH2AX at telomere ure 3A, B). We propose that this is due to the increased resistance sequences at chromosome ends in experimental H1299 and U-2 of telomerase-positive cancer cells to replication stress at telo- OS cells (Supplementary Figure 2E-H). Activation of a DNA meres, as previously shown for HeLa and H1299 cells17,45. damage response was validated by western blotting as shown by Replicative stress is reported to trigger the phosphorylation of increased γH2AX levels and stabilization of p53 in U-2 OS cells Ataxia telangiectasia and Rad3-related (ATR). In line with this we (Supplementary Figure 2I, J). Together, this indicates that loss of found an efficient phosphorylation of ATR at telomeres in SFPQ NONO and SFPQ leads to altered TERRA homeostasis and knockdown U-2 OS or H1299 cells (Fig. 3e, f; Supplementary promotes the formation of DNA damage at telomeres. Recent Figure 3C, D). Remarkably, although loss of NONO triggers studies showed that RNA:DNA hybrid and R-loop formation at RPA32Ser33 phosphorylation at telomeres in U-2 OS cells, we telomeres is linked with increased abundance of TERRA at were not able to observe increased telomeric ATR phosphoryla- telomeric chromatin, without altering total TERRA lncRNA tion under these conditions. The role of NONO in enhancing levels17,36. To test whether NONO or SFPQ depletion enhances ATR-mediated DNA damage response signaling may provide an RNA:DNA-hybrid formation between TERRA and the telomeric explanation for this observation43,46. C-strand, we performed co-immunocytochemistry using specific We next wished to test whether increased RNA:DNA-hybrid anti-TRF1 antibodies and affinity purified S9.6 monoclonal formation in NONO/SFPQ-depleted cells is directly linked to antibodies that detect RNA:DNA hybrids in a sequence inde- phosphorylation of RPA32Ser33. To address this issue, we aimed pendent manner. Confocal microscopy revealed that RNAi- to rescue RPA32Ser33 phosphorylation levels in NONO- and mediated depletion of NONO or SFPQ significantly increased co- SFPQ-depleted cells by ectopically expressing a mCherry-tagged localization frequencies between RNA:DNA hybrid and TRF1 version of human RNaseH1. As expected, co-depletion of NONO

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a RPA32 b TERRA pSer33 Merge

p = 0.011

p = 0.038

1 μm 60.87 ± 5.3 n = 97 ± 80 46.9 3.1 N = 3 32.5 ± 3.5 n = 97 60 n = 97 N = 3

si-NONO si-control N = 3 40

20 co-localizing with Fraction of TERRA foci 0 RPA32pSer33 (%) per nucleus si-SFPQ

si-control si-NONO si-SFPQ U-2 OS

c RPA32 d TRF2 pSer33 Merge

p = 0.003

1 μm p = 0.001 3.0 ± 0.3 n = 100 ± 4 2.5 0.1 N = 3 n = 101 3 N = 3 si-NONO 1.0 ± 0.21 2 n = 103 N = 3 1

0 si-SFPQ si-control Number of TRF2/RPA32pSer33 co-localization events per nucleus

si-control si-NONO si-SFPQ HU

U-2 OS e f TRF2 p-ATR Merge p = 0.012

p = 0.448 2.6 ± 0.5 n = 105 4

si-control N = 3

μ 1 m 3

2 0.2 ± 0.1 0.3 ± 0.1 n = 104 n = 103 per nucleus 1 si-NONO N = 3 N = 3 co-localization events Number of TRF2/pATR 0

si-control si-NONO si-SFPQ si-SFPQ

U-2 OS

g RPA32 h mCherry TRF2 pSer33 Merge p = 0.003 p = 0.03

si-control 2.6 ± 0.2 4 + mCherry n = 90 N = 3 1 μm 3 1.4 ± 0.3 n = 90 0.8 ± 0.2 N = 3 si-NONO/ 2 n = 90 si-SFPQ N = 3 + mCherry 1

0 si-NONO/ Number of TRF2/RPA32pSer33 si-SFPQ co-localization events per nucleus

+ mCherry- si-control RnaseH1 mCherry mCherry

si-NONO/si-SFPQsi-NONO/si-SFPQ U-2 OS mCherry-RNaseH1

and SFPQ resulted in recruitment of RPA32Ser33 to telomeres of accumulation and R-loop-related replication defects at telomere U-2 OS cells, indicative for replicative stress (Fig. 3g, h). repeat sequences. Importantly, this effect is eliminated upon transient expression of RNaseH1 in NONO/SFPQ-depleted cells (Fig. 3g, h). Together, these data indicate that NONO and SFPQ are TERRA interacting NONO and SFPQ regulate telomere leading strand fragility. proteins that have a crucial role in preventing RNA:DNA-hybrid Increased RNA:DNA-hybrid levels at telomeres were shown to

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Fig. 3 Depletion of NONO and SFPQ cause replication defects at telomeres in U-2 OS cells. a Representative images of TERRA RNA-FISH combined with anti-RPA32pSer33 immunostaining performed in U-2 OS cells transfected with indicated siRNAs. Arrowheads indicate co-localization events. b Quantification of a; percentage of TERRA foci co-localizing with RPA32pSer33 per nucleus is indicated. c Representative images of combined immunofluorescence with anti-TRF2 and anti-RPA32pSer33 antibodies in U-2 OS cells transfected with indicated siRNAs. Arrowheads indicate co- localization events. Induction of replicative stress by treating cells with hydroxyurea (5 mM) for 6 h was used as control for RPA32pSer33 staining. d Quantification of TRF2/RPApSer33 co-localization events per nucleus. e Representative images of combined immunofluorescence using anti-TRF2 and anti-pATR antibodies (S428) in U-2 OS cells transfected with indicated siRNAs. Arrowheads indicate co-localization events. f Quantification of TRF2/pATR co-localization events per nucleus. g Representative images of combined immunofluorescence using anti-TRF2 and anti-RPA32pSer33 antibodies on U-2 OS cells transfected with indicated siRNAs and transiently expressing mCherry-RNase H1 or mCherry empty vector. Arrowheads indicate co-localization events. h Quantification of the number of TRF2/RPA32pSer33 co-localizations per nucleus. For quantifications in b, d, f, h, mean values and standard deviations are reported, error bars indicated standard deviation. N = number of independent experiments. n = number of analyzed nuclei. A two-tailed Student’s t-test was used to calculate statistical significance; p-values are shown. Source data is provided as a Supplementary Information File. Scale bar, 1 μm. siRNAs listed in Supplementary Table 1

fuel R-loop formation and fragility of the telomeric leading (C- SFPQ and NONO suppress recombination in cancer cells.To rich) strand in vertebrate cells17,36. Telomeric R-loop structures better understand the different function of NONO and SFPQ in are prone to breakage, thus providing DNA substrates for the control of telomere stability we used telomere CO-FISH to homologous recombination at yeast and human telomeres. In study recombination-based telomere sister chromatid exchange order to understand the direct importance of NONO and SFPQ (T-SCE), a reported downstream result of RNA:DNA-hybrid- for telomere integrity, we performed short term loss of func- related R-loop formation at eukaryotic telomeres (Fig. 5a)15,17. tion experiments using telomere chromosome orientation DNA- We found that NONO depletion does not significantly impact on FISH (CO-FISH). In this method BrdU incorporation during S- T-SCE in U-2 OS and H1299 cancer cells (Fig. 5b, c). In contrast, Phase and subsequent enzymatic digestion of the neo-synthesized SFPQ knockdown results in a significant increase in T-SCE fre- DNA strand after UV treatment allows the selective detection of quency in both, U-2 OS ALT and telomerase-positive H1299 cells the TTAGGG containing lagging telomeric strand or the (Fig. 5b, c). This identifies SFPQ as a novel repressor of telomere CCCTAA-repeat containing leading telomeric strand using dif- recombination in both, telomerase-positive and telomerase- ferentially labeled, telomere-strand-specific DNA-FISH probes negative human cancer cells. Activation of ATR was recently (Fig. 4a). This method allows to detect telomere-strand-specific shown to promote homologous recombination, providing an aberrations but also telomere recombination events (Figs. 4a, 5a). explanation for ATR phosphorylation in SFPQ, but not NONO RNAi-mediated depletion of NONO in U-2 OS cells significantly knockdown cells (Fig. 3e, f; Supplementary Figure 3C, D)47.We increased the appearance of aberrantly shaped or multi-dotted next wished to better understand a potential contribution of telomere signals the CCCTAA-repeat containing telomeric strand telomere fragility in NONO-depleted cells to the regulation of T- in U-2 OS cells ( + 60%; Fig. 4b). Interestingly, loss of NONO SCE events. We found that in the context of increased telomere does not have an impact on telomere fragility at the TTAGGG fragility caused by NONO knockdown, co-depletion of SFPQ repeat containing lagging strand (Fig. 4b). This data is in line with resulted in a dramatic 18-fold increase of T-SCE that involves leading strand fragility triggered by increased RNA:DNA-hybrid 35% of detectable telomeres (Fig. 5d, e). This effect was paralleled abundance in RNaseH1 or Flap endonuclease loss of function by a rescue of telomere fragility in NONO/SFPQ double knock- cells17,36. As expected, ectopic expression of NONO reduced basal down cells (Fig. 5d–f). This suggests that telomere sister chro- levels of telomere lagging strand fragility (Fig. 4c). Remarkably, matid recombination may represent a mechanism to resolve loss of NONO in telomerase-positive cells results in telomeric telomere fragility triggered by NONO depletion. This mechanism leading and lagging strand fragility; accordingly, NONO over- can also explain reduced basal levels of telomeric leading strand expression reduces basic telomere fragility levels on both telomere fragility in SFPQ-depleted H1299 and U-2 OS cells (Fig. 4d; strands (Supplementary Figure 4A, B). Together, these data Supplementary Fig. 4C). In line with increased T-SCE frequency identify NONO as novel suppressor of telomere fragility that has in NONO/SFPQ-depleted cells we observed an increased fre- a particular relevance in suppressing fragility at the telomeric quency of co-localization of TRF2 with Promyelocytic Leukemia leading strand in ALT cells that are reported to be prone to (PML) nuclear bodies and co-localization of RAD51 with TRF2 in exhibit telomeric RNA:DNA hybrids and leading strand fragility. U-2 OS cells that were depleted for SFPQ (Fig. 5g–j). Accordingly, NONO and SFPQ preferentially form heterodimers and have a we found significantly elevated TERRA–PML co-localization in common function in suppressing TERRA:telomere-hybrid for- SFPQ knockdown cells, a feature reported for ALT cells (ref. 17 mation. However, RNAi-mediated depletion of SFPQ had an Supplementary Figure 5A, B). This is indicative for an enhanced advert effect on telomere fragility in U-2 OS and H1299 cells generation of alternative lengthening of telomeres-associated when compared to loss of NONO expression conditions. In fact, PML nuclear bodies (APBs) and increased homologous recom- loss of SFPQ selectively reduces leading strand fragility in both, bination at telomeres under SFPQ loss of function conditions. As U-2 OS ALT and telomerase-positive H1299 cells (Fig. 4d; Sup- expected, depletion of NONO does not impact on APB frequency plementary Figure 4C). SFPQ overexpression does not impact on in U-2 OS cells. Remarkably, although siRNA-mediated depletion telomere fragility, suggesting that endogenous SFPQ expression of SFPQ in telomerase-positive H1299 cells triggers T-SCE we did levels are sufficient to keep telomere fragility at sustainable levels not observe significantly increased APB numbers (Fig. 6e, f). This (Fig. 4e; Supplementary Figure 4D). Our data shows that SFPQ suggests that loss of SFPQ can trigger telomere recombination in and NONO, although reported to preferentially act as hetero- telomerase-positive cancer cells; however, without efficiently dimer43, have different impact on telomere fragility. In particular, engaging APBs. NONO appears to be important to suppress telomere fragility; in Together, our data indicate that NONO and SFPQ have contrast, loss of SFPQ appears to activate molecular processes distinct roles in ensuring telomere stability. NONO suppresses that suppress telomere fragility. the generation of R-loop-related leading telomere fragility; in

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Fig. 4 NONO and SFPQ regulate telomeric leading strand fragility of U-2 OS a Leading (CCCTAA) Lagging (TTAGGG) strand fragility strand fragility cells. a Representative images and explanative models of leading strand and lagging strand telomeric fragility as detected by Chromosome Orientation FISH (CO-FISH). Fragile telomeres appear as multiple telomere signals. b Quantification of leading strand fragility (left panel) and lagging strand fragility (central panel) in U-2 OS cells transfected with NONO-specific siRNA or a non-targeting control siRNA. Right panel, western blotting showing NONO knockdown efficiency. c Quantification of leading strand Telomere CO-FISH fragility (left panel) and lagging strand fragility (central panel) in U-2 OS cells that stably overexpress FLAG-NONO or a control vector. Right panel, b p < 0.0001 p = 0.26 western blotting showing ectopic expression of FLAG-tagged NONO. 6.9 ± 0.4 ± 4.3 ± 0.3 3.6 ± 0.3 4.1 0.3 d Quantification of leading strand fragility (left panel) and lagging strand 20 N = 75 N = 75 N = 75 20 N = 75 n = 4415 n = 4415 fragility (central panel) in U-2 OS cells transfected with SFPQ-specific si-control n = 5113 n = 5113 si-NONO 15 15 50 kDa NONO siRNA or a non-targeting control siRNA. Right panel, western blotting fi fi 10 10 37 kDa Actin showing SFPQ knockdown ef ciency. e Quanti cation of leading strand fragility (left panel) and lagging strand fragility (central panel) in U-2 OS fragility (%) 5 (%) fragility 5 Leading strand Lagging strand cells that stably overexpress myc-tagged SFPQ or an empty vector. Right 0 0 panel, western blotting showing expression of ectopic myc-SFPQ. For quantifications in b–e, data points represent the percentage of fragile si-control si-NONO si-control si-NONO telomeres per metaphase spread. Only chromosome ends with detectable c p = 0.433 telomeres were considered for analysis. Metaphases from three p < 0.0001 independent experiments were analyzed. Box plot diagrams (b–e): middle line represents median; boxes extend from the 25th to 75th percentiles. 15 5.5 ± 0.2 15 ± ± 4.1 ± 0.2 3.9 0.2 3.7 0.2 N = 75 N = 75 N = 75 The whiskers mark the 10th and 90th percentiles. p-values were calculated n = 3341 N = 75 pLPC n = 3341 n = 3312 FLAG-NONO 10 n = 3312 10 using a two-tailed Mann–Whitney test. Median fragility values and 50 kDa FLAG standard deviation are indicated; n = number of analyzed telomere repeat 5 5 37 kDa Actin N = fragility (%)

fragility (%) signals, number of analyzed metaphase spreads. siRNAs listed in Lagging strand Leading strand Supplementary Table 1. Source blots are available in Supplementary 0 0 Figure 8 pLPC pLPC

FLAG-NONO FLAG-NONO d p = 0.76 p = 0.02 SFPQ and NONO control telomere length in cancer cells. ± 3.7 ± 0.3 5.5 0.4 20 3.8 ± 0.3 20 4.1 ± 0.3 N = 75 Recombination-based ALT ensures telomere maintenance in N = 75 N = 75

N = 75 si-SFPQ n = 4941 n = 4941 si-control 37,38 15 n = 4536 15 n = 4536 telomerase negative cancer cells . Accordingly, the impair- 100 kDa SFPQ 10 ment of pathways related to homologous recombination results in 10 Actin 37 kDa alterations in telomere length homeostasis in ALT cells48,49.

fragility (%) 5 fragility (%) 5 Lagging strand

Leading strand Therefore, we were interested to study the role of SFPQ and 0 0 NONO in telomere length homeostasis in telomerase negative U- 2 OS ALT cells but also telomerase-positive H1299 cells. Quan- si-SFPQ si-control si-SFPQ si-control titative telomere DNA-FISH revealed that siRNA mediated, e transient depletion of NONO or SFPQ in U-2 OS ALT cells for p = 0.305 p = 0.941 3 days induces a 8% or 17% increase in telomere length, 6.6 ± 0.5 4.5 ± 0.4 respectively (Fig. 6a, b). This telomere elongation phenotype is 7.1 ± 0.5 N = 75 N = 75 25 N = 75 n = 4054 25 exacerbated upon combined depletion of NONO and SFPQ ( + n = 3447 n = 3447 4.4 ± 0.3 pLPC MYC-SFPQ 20 20 N = 75 35%) (Fig. 6a, b). This is in line with the dramatically increased T- 15 15 n = 4054 100 kDa MYC SCE frequency under these conditions (Fig. 5d, e). In contrast to Actin 10 10 37 kDa telomere elongation in U-2 OS cells, we found that unleashing fragility (%) fragility (%)

5 Lagging strand 5 Leading strand telomere recombination by single depletion of SFPQ or combined 0 0 depletion of NONO and SFPQ resulted a 21% reduction of tel-

pLPC pLPC omere length in telomerase-positive H1299 cells, respectively (Fig. 6c, d). We thus hypothesize that enhancing T-SCE rates in MYC-SFPQ MYC-SFPQ absence of functional APBs leads to an inefficient homologous recombination process at telomeres in SFPQ knockdown H1299 cells, resulting a net loss of telomeric repeats (Fig. 6c, d). To test contrast, SFPQ has a critical role in suppressing uncontrolled this hypothesis, we aimed to rescue the telomere shortening recombination events triggered by the accumulation of telomere phenotype by stimulating the efficiency of homologous recom- R-loops and replication stress. bination pathways in SFPQ-depleted H1299 cells. SFPQ was We further propose that increased telomere fragility in NONO- depleted from telomerase-positive H1299 cells that were co- depleted cells increases the availability of recombinogenic treated with 5-Aza-2′-deoxycytidine to unleash recombination- substrates at telomeres that efficiently engage in homologous based telomere elongation. 5-Aza-2′-deoxycytidine treatment has recombination in the absence of SFPQ. We thus propose that been reported to reduce subtelomeric DNA methylation, thus NONO and SFPQ collaborate to suppress RNA:DNA-hybrid driving recombination events in cancer cells, that are detectable triggered telomere recombination in both ALT- and telomerase- by telomere CO-FISH50. As expected, 5-Aza-2′-deoxycytidine positive cancer cells. treatment lead to increased APB frequency in H1299 cells

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p = 0.01 p = 0.06 p = 0.038 abp = 0.1 c

± 5.7 0.7 ± 1.64 ± 0.55 2.3 ± 0.4 3 0.06 0.03 ± ± N = 75 30 N = 75 3.0 0.4 N = 75 3.2 0.4 N = 75 0.09 ± 0.02 25 n = 4536 n = 6711 N = 75 n = 4415 30 N = 75 n = 7113 N =75 20 n = 5113 25 n = 4941 2 n = 6718 20 15 20 15 0.14 ± 0.05 10 1 10 N = 75 T-SCE T-SCE 10 n = 7030 5 5 (% of telomeres) Telomeric sister chromatid (% of telomeres)

0 0 T-SCE (% of telomeres) 0 0 exchange: T-SCE T-SCE (% of telomeres)

si-control si-NONO si-control si-SFPQ si-control si-NONO si-control si-SFPQ

defsi-control si-NONO/si-SFPQ p < 0.0001 p = 0.689

± 34.7 3.5 p = 0.045 p = 0.899 N = 75 11.0 ± 0.9 100 n = 2806 50 11.7 ± 0.7 N = 75 80 40 9.7 ± 0.5 N = 75 n = 2806 N = 75 n = 3757 60 ± 30 n = 4005 1.9 ± 0.2 1.9 0.3 40 N = 75 N = 75 20 n = 4005 n = 3757 20 10

0 Fragile telomeres(%) 0 T-SCE (% of telomeres) U-2 OS

si-control si-NONO si-control si-NONO

si-NONO/si-SFPQ si-NONO/si-SFPQ

g TRF2 PML Merge i TRF2 RAD51 Merge

1 μm 1 μm si-NONO si-NONO si-SFPQ si-control si-SFPQ si-control si-SFPQ si-SFPQ si-NONO/ si-NONO/ U-2 OS U-2 OS U-2 OS

hjp = 0.006 p = 0.009

p = 0.035 p = 0.015

2.2 ± 0.2 p = 0.356 ± p = 0.336 2.9 0.2 n = 91 2.4 ± 0.3 4 n = 94 3 1.8 ± 0.2 N = 3 n = 95 N = 3 n = 91 1.5 ± 0.3 N = 3 N = 3 3 1.0 ± 0.1 1.1 ± 0.3 n = 95 2 0.8 ± 0.2 n = 97 N = 3 n = 92 n = 90 2 N = 3 N = 3 N = 3 1

per nucleus 1 co-localization Numberof APBs events per nucleus 0 0 Number of TRF2/RAD51

si-control si-NONO si-SFPQ si-control si-NONO si-SFPQ

si-NONO/si-SFPQ si-NONO/si-SFPQ

(Fig. 6e, f). This effect was found to be enhanced in SFPQ- under these conditions, quantitative telomere DNA-FISH depleted and 5-Aza-2′-deoxycytidine-treated H1299 cells, sug- experiments revealed a reversion of the telomere shortening gesting the activation of a functionally relevant subnuclear phenotype of SFPQ-depleted H1299 cells, as demonstrated by a compartment for the ALT pathway (Fig. 6e, f). Importantly, 47% telomere elongation in 5-Aza-2′-deoxycytidine-treated SFPQ

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Fig. 5 NONO collaborates with SFPQ to suppress recombination at telomeres. a Representative images and explanative models of telomeric sister chromatid exchange (T-SCE) as detected by CO-FISH. b Quantification of T-SCEs in U-2 OS cells transfected with control siRNAs, NONO-specific siRNAs (left panel), or SFPQ-specific siRNAs (right panel). c Quantification of T-SCEs in H1299 cells transfected with control siRNAs, NONO-specific siRNAs (left panel), or SFPQ-specific siRNAs (right panel). d Representative images of CO-FISH experiments performed in U-2 OS cells transfected with indicated siRNAs. e Quantification of T-SCEs in U-2 OS cells transfected with NONO-specific siRNA, or with both NONO- and SFPQ-specific siRNAs compared to control cells. f Quantification of telomere fragility in U-2 OS cells transfected with NONO-specific siRNA, or with both NONO and SFPQ-specific siRNAs. g Representative images of anti-TRF2 and anti-PML co-immunofluorescence to detect APBs in U-2 OS cells transfected with indicated siRNAs. h Quantification of the number of APBs per nucleus of data shown in g. i Representative images of anti-TRF2 and anti-RAD51 co-immunofluorescence in U- 2 OS cells transfected with indicated siRNAs. j Quantification of RAD51-TRF2 co-localization events per nucleus of data shown in i. Box plot diagrams (b, c, e, f): middle line represents median; boxes extend from the 25th to 75th percentiles. The whiskers mark the 10th and 90th percentiles. p-values were calculated using a two-tailed Mann–Whitney test. Median T-SCE/fragility values and standard deviation are indicated; n = number of analyzed telomere repeat signals, N = number of analyzed metaphase spreads. Panels h, j means (bars) and standard deviation (error bars) are reported. N = number of independent experiments. n = number of analyzed nuclei. A two-tailed Student’s t-test was used to calculate statistical significance; p-values are shown. Source data is provided as a Supplementary Information File. Scale bar, 1 μm. siRNAs listed in Supplementary Table 1

knockdown H1299 cells, when compared to control cells (Fig. 6g, at telomere repeats of NONO- or SFPQ-depleted cells. This is in h). This indicates that functional subnuclear APB compartments line with independent studies that show an involvement of NONO are necessary to convert enhanced telomere recombination of and SFPQ in promoting DNA damage pathways, including SFPQ-depleted telomerase-positive cells into a telomere elonga- homologous recombination, non-homologous end joining, tion phenotype. We thus conclude that NONO and SFPQ are nucleotide excision repair, and interstrand cross-link repair43,44. novel regulators of telomere length in both telomerase-positive The fact that NONO and SFPQ contain multiple RNA binding and negative cancer cells. domains suggests an “RNA-based” role of NONO and SFPQ in Here, we show that RNA binding proteins NONO and SFPQ preventing RNA:DNA-hybrid-related DNA damage. Remarkably, are novel component of telomeric chromatin that suppress although acting as heterodimers, NONO and SFPQ have appar- replicative stress at telomeres by antagonizing the formation of ently different roles in suppressing downstream events related to RNA:DNA hybrids. Remarkably, although preferentially acting in RNA:DNA hybrids at telomeres. NONO appears to play an heterodimers, NONO and SFPQ control different pathways that important role in preventing fragility of the leading, CCCTAA- result from the formation of RNA:DNA hybrids and R-loop repeat containing telomeric strand. In line with this, leading strand structures: NONO appears to suppress telomere fragility; in fragility was previously shown to be a direct consequence of contrast, SFPQ has a central role in suppressing homologous telomeric RNA:DNA hybrids in different model systems17,36,52.In recombination (Fig. 7). Combined loss of NONO and SFPQ contrast, CO-FISH analysis revealed that SFPQ acts as barrier to depletion results in a massive increase in homologous recombi- homologous recombination at telomeres. Loss of SFPQ drives nation and altered telomere length homeostasis. Altogether, our RNA:DNA-hybrid formation, impaired replication and results in data highlight a role for NONO and SFPQ in controlling RNA: dramatically increased recombination frequencies in the context of DNA-hybrid-related telomere instability and telomere length ATR phosphorylation at telomeres of telomerase-positive and homeostasis in both telomerase-positive and negative cancer cells. ALT cancer cells. This effect is paralleled by a reduction of telo- mere fragility, leading to the interesting speculation that homo- logous recombination may help to rescue telomere fragility. Our Discussion data show that SFPQ functions as barrier to homologous Here, we show that NONO and SFPQ are novel telomere repeat recombination at telomeres. Phosphorylation of ATR in SFPQ associated proteins that collaborate to suppress RNA:DNA- knockdown cells is in line with a recent report that demonstrated hybrid-related replicative stress, telomere fragility and telomere that activation of ATR promotes homologous recombination47. recombination in both U-2 OS ALT cells and H1299 telomerase- Remarkably, removing the SFPQ dependent recombination bar- positive human cancer cells. Loss of NONO or SFPQ results in an rier in the context of increased fragility mediated by NONO increased appearance of TERRA foci without affecting total depletion results in massive T-SCE events that involve 35% telo- TERRA expression levels suggesting that trapping TERRA in meres detectable by DNA-FISH in U-2 OS ALT cells. We propose RNA:DNA-hybrid structures may increase the amount of that increased chromosome fragility in the absence of NONO detectable TERRA transcripts at telomeres. In ALT cells, extra- leads to the generation of elevated numbers of recombinogenic chromosomal telomeric repeats (ECTR) can constitute a notable DNA products that efficiently engage in telomere recombination fraction of the total telomere repeat content, suggesting that a when removing the recombination barrier SFPQ. Thus, NONO fraction of NONO and SFPQ may also locate to this type of and SFPQ have a direct role in suppressing RNA:DNA-hybrid- telomere repeat sequences51. However, alterations at telomeres of related telomere fragility and recombination (Fig. 7). Given the metaphase chromosomes observed in loss of function experiments fact that NONO and SFPQ do not contain enzymatic activity and support a role for NONO and SFPQ at telomeres. We directly may rather function as scaffold we propose that localization of show that loss of NONO and SFPQ mediates enhanced formation NONO or SFPQ to telomeres is necessary to recruit specific—yet of telomeric RNA:DNA hybrids in both, telomerase-positive and to define—factors that suppress RNA:DNA-hybrid-related telo- negative human cancer cells. A common role of NONO and SFPQ mere fragility and recombination43,44. Homologous recombina- in suppressing RNA:DNA-hybrid abundance and replication tion represents a key pathway for the regulation of telomere length defects in human cancer cells is in accordance with several studies homeostasis. In line with this, we found that NONO/SFPQ that propose that NONO and SFPQ act on various aspects of RNA knockdown in U-2 OS triggers increased T-SCE frequencies, metabolism by preferentially forming heterodimers43. In line with increased localization of RAD51 at telomere repeats as well as the the increased abundance of RNA:DNA hybrids, we observed engagement of telomeres in APBs, thus resulting telomere elon- increased appearance of the classic DNA damage marker γH2AX gation. Under the same conditions, telomerase-positive H1299

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abTelomere cdTelomere p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p = 0.0532 9115 p = 0.0960 ± 143.9 9274 n = 4460 7936 80,000 si-control 80,000 ± N = 90 9932 121.1 ± 7893 125.7 ± 116.2 n = 4877 7862 1 μm ± n = 3584 1 μm ± n = 4952 N = 90 117.6 7322 115.3 n = 3872 N = 90 N = 90 ± 107.3 n = 5015 N = 90 6780 N = 90 60,000 ± 115.5 n = 4856 60,000 n = 3941 N = 90 N = 90 si-NONO si-NONO 40,000 40,000

20,000 20,000 si-SFPQ si-control si-SFPQ Telomere fluorescence units (a.f.u.) Telomere fluorescence units (a.f.u.)

0 0

si-control si-NONO si-SFPQ si-control si-NONO si-SFPQ si-SFPQ si-SFPQ si-NONO/ si-NONO/ si-NONO/si-SFPQ si-NONO/si-SFPQ U-2 OS H1299

ehTRF2 PML Merge g Telomere p < 0.0001

p < 0.0001 p < 0.0001

p < 0.0001 ± si-control 10,019 si-control 128.4 1 μm 1 μm N = 90 n = 5970 120,000 8349 ± 98.27 si-SFPQ si-SFPQ N = 90 n = 6923 6810 ± 5503 ± 80,000 86.74 87.14 N = 90 N = 90 n = 5417 n = 3823 5-Aza 5-Aza si-control si-control 40,000 Telomere fluorescence units (a.f.u.) 0 5-Aza 5-Aza si-SFPQ si-SFPQ

H1299 H1299 si-control si-SFPQ

si-control 5-Azasi-SFPQ 5-Aza f p = 0.0083

p = 0.0001 p = 0.002

p = 0.08 0.99 ± 0.09 N = 3 1.5 n = 90 0.69 ± 0.02 ± N = 3 1 0.5 0.04 0.4 ± 0.02 N = 3 n = 90 N = 3 n = 90 n = 90

Number of 0.5 APBs per nucleus 0

si-control si-SFPQ

si-control 5-Azasi-SFPQ 5-Aza cells, that lack functional APB compartments, show rapid telo- Together, this identifies NONO and SFPQ as regulators of mere shortening. Remarkably, telomere shortening can be rescued telomere length homeostasis by controlling RNA:DNA-hybrid by 5-Aza-2′deoxycytidine treatment that forces APB formation abundance at telomeres. Given the relevance of RNA:DNA and T-SCE in SFPQ loss of function cells50. This suggests that hybrids in recombination and telomere length homeostasis, functional APBs are important to convert increased T-SCE fre- NONO and SFPQ are expected to be of special relevance in quencies in SFPQ loss of function cells into an overall increase in human tumors that maintain telomeres length via the ALT telomere length. pathway. In line with this, SFPQ mutations have been recently

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Fig. 6 NONO and SFPQ regulate telomere length homeostasis in human cancer cells. a Telomere length measurements by quantitative telomere DNA-FISH on interphase U-2 OS cells after transient transfection with indicated siRNAs. Representative images are shown. b Telomere fluorescence intensity was analyzed for each telomere of a. c Telomere length measurements by quantitative telomere DNA-FISH on interphase H1299 cells after transient transfection with indicated siRNAs. Representative images are shown. d Telomere fluorescence intensity analyzed for each telomere of c. e Representative images of combined immunofluorescence using anti-TRF2 and anti-PML antibodies in H1299 cells transfected with SFPQ-specific siRNA or a non-targeting control siRNA. Cells were treated or not treated with 5-aza-2’-deoxycytidine (5-Aza). f Quantification of e; number of TRF2-PML co-localization events per nucleus. Mean values (bars) and standard deviations (error bars) are reported. N = number of independent experiments. n = number of analyzed nuclei. A two-tailed Student’s t-test was used to calculate statistical significance; p-values are shown. Source data is provided as Supplementary Information File. g Representative images of telomere length measurements by quantitative telomere DNA-FISH on interphase H1299 cells transfected with SFPQ-specific siRNA or a non-targeting control siRNA. Cells were treated or not treated with 5-aza-2’-deoxycytidine (5-Aza). h Telomere fluorescence intensity analyzed for each telomere of g. For telomere length measurements, Box plot diagrams (b, d, h): middle line represents median arbitrary fluorescence units (a.f.u.), boxes extend from the 25th to 75th percentiles. The whiskers mark the 10th and 90th percentiles. p-values were calculated using a two-tailed Mann–Whitney test. Median a.f.u. values with standard deviation are indicated; n = number of analyzed telomere repeat signals, N = number of analyzed interphase nuclei. Arbitrary fluorescence units (a.f.u.) are shown. Scale bar, 1 μm. siRNAs listed in Supplementary Table 1

Stable telomeres NONO and SFPQ bind TERRA and telomeres

- Suppression of RNA:DNA hybrids - Suppression of R-loop formation 5′ TERRA - Suppression of fragility 5′ RPA32 - Suppression of recombination NONO

SFPQ TTAGGG -3′ DNA RNA pol pol II AATCCC -5′

3′

Unstable telomeres NONO/SFPQ loss of function

NONO - Accumulation of telomeric R loops and RNA:DNA hybrids SFPQ - Telomere replication stress - Telomere fragility - Telomere damage 5′ TERRA - Telomere recombination - Altered telomere length homeostasis 5′ RPA32pSer33

RNA:DNA hybrids TTAGGG -3′ DNA pol AATCCC -5′ 5′ TTAGGG -3′ ′ 3′ AATCCC -5 3′

Fig. 7 A model for the role of NONO and SFPQ in controlling telomere stability. Top panel: NONO and SFPQ bind TERRA transcripts at telomeres and control mechanisms that antagonize the accumulation of RNA:DNA hybrids, thereby stabilizing telomeres. Bottom panel: Loss of NONO and SFPQ leads to the accumulation of RNA:DNA hybrids at telomeres, resulting in the displacement of the non-template telomere strand (G-strand) and R-loop formation. Increased R-loop formation triggers replication defects, fragility, telomere damage and the exposition of recombinogenic telomeric DNA that engage in telomere recombination events and result alterations in telomere length homeostasis. DNA pol, DNA polymerase; RNA pol II, RNA polymerase II discovered by exome sequencing in human osteosarcoma, a (Fig. 7). Understanding the function of the entire NONO/SFPQ tumor type that is reported to use recombination-based complex at telomeres is expected to open new avenues in ALT as predominant pathway for telomere maintenance53. understanding the suppression of the ALT pathway in telomerase- We hypothesize that NONO and SFPQ act as platforms to negative tumors and activation of the ALT pathway in telomerase recruit factors that resolve DNA:RNA-hybrid structures negative cells. These insights are expected to be highly relevant for thereby suppressing fragility and recombination at telomeres potential telomere-based anti-cancer therapies.

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Methods 2. Chan, S. R. W. L. & Blackburn, E. H. Telomeres and telomerase. Philos. Trans. Immunofluorescence. Cells were fixed in 4% paraformaldehyde (PFA) for 15 min, R. Soc. Lond. B. Biol. Sci. 359, 109–121 (2004). followed by treatment with citrate buffer [0.1% (w/v), 0.5% Triton X-100] for 5 min 3. Palm, W. & de Lange, T. How shelterin protects mammalian telomeres. Annu. at room temperature. Cells were blocked for 45 min in 3% BSA, 0.1% Tween-20 in Rev. Genet. 42, 301–334 (2008). 1x PBS and incubated with primary antibodies (as indicated in Supplementary 4. d’Adda di Fagagna, F. et al. A DNA damage checkpoint response in telomere- Table 2) diluted in blocking solution at room temperature for 2 h. Cells were initiated senescence. Nature 426, 194–198 (2003). washed in 0.3% BSA, 0.1% Tween-20 in 1x PBS and incubated with secondary 5. Schoeftner, S. & Blasco, M. A. Developmentally regulated transcription of antibodies (Supplementary Table 3) for one hour at room temperature. Slides were mammalian telomeres by DNA-dependent RNA polymerase II. Nat. Cell Biol. washed in 0.1% Tween-20 1XPBS, stained with DAPI (Sigma) and mounted in 10, 228–236 (2008). fl fi Vectashield (Vector laboratories). For S9.6 immuno uorescence, cells were xed 6. Azzalin, C. M., Reichenbach, P., Khoriauli, L., Giulotto, E. & Lingner, J. and permeabilized with ice-cold methanol for 10 min and acetone for 1 min on ice Telomeric repeat containing RNA and RNA surveillance factors at 23 as previously described . Blocking, antibody and washing solutions were per- mammalian chromosome ends. Science 318, 798–801 (2007). formed in 4x SSC. Cells with at least 5 nuclear extra-nucleolar S9.6 foci were 7. Ng, L. J., Cropley, J. E., Pickett, H. A., Reddel, R. R. & Suter, C. M. Telomerase analyzed. Co-localization events were quantified using ImageJ 1.46r or by visual ’ fi activity is associated with an increase in DNA methylation at the proximal inspection. The Student s t-test was used to calculate statistical signi cance. subtelomere and a reduction in telomeric transcription. Nucleic Acids Res. 37, 1152–1159 (2009). RNA FISH. Cells were permeabilized with three consecutive steps (30 sec each) of 8. Yehezkel, S., Segev, Y., Viegas-Péquignot, E., Skorecki, K. & Selig, S. cytobuffer (100 mM NaCl, 300 mM Sucrose, 3 mM MgCl2, 10 mM Pipes pH 6.8), Hypomethylation of subtelomeric regions in ICF syndrome is associated with cytobuffer/0.5% Triton-X, and cytobuffer. Cells were fixed for 10 min at room abnormally short telomeres and enhanced transcription from telomeric temperature in ice-cold 4% paraformaldehyde (PFA) in 1x PBS and then dehy- regions. Hum. Mol. Genet. 17, 2776–2789 (2008). drated twice in ice-cold 70% ethanol for 2 min, ice-cold 90% ethanol for 1 min, and 9. Nergadze, S. G. et al. CpG-island promoters drive transcription of human ice-cold 100% ethanol for 1 min. Dried slides were incubated overnight with a Cy3- telomeres. RNA 15, 2186–2194 (2009). labeled TERRA probe in a humid chamber (2x SSC, 50% formamide) at 37 °C. 10. Porro, A. et al. Functional characterization of the TERRA transcriptome at Slides were washed once in 2x SSC for 3 min at room temperature and three times damaged telomeres. Nat. Commun. 5, 5379 (2014). in 2x SSC for 5 min at 37 °C. Subsequently, slides were transferred to 4x SSC and 11. Porro, A., Feuerhahn, S., Reichenbach, P. & Lingner, J. Molecular dissection mounted in Vectashield (Vector laboratories). The TERRA probe was labeled with of telomeric repeat-containing RNA biogenesis unveils the presence of distinct the FISH Tag DNA kit (Invitrogen). and multiple regulatory pathways. Mol. Cell. Biol. 30, 4808–4817 (2010). 12. Azzalin, C. M. & Lingner, J. Telomere functions grounding on TERRA firma. Immunofluorescence combined with RNA FISH. Cell were permeabilized with Trends Cell Biol. 25,29–36 (2015). cytobuffer as described for RNA-FISH and then subjected to the immuno- 13. Cusanelli, E. & Chartrand, P. Telomeric repeat-containing RNA TERRA: A fluorescence protocol. Used primary and secondary antibodies are indicated in noncoding RNA connecting telomere biology to genome integrity. Front. Supplementary Table 2, 3. After the washing step for the secondary antibody, cells Genet. 6, 143 (2015). were fixed at room temperature for 2 min with 4% paraformaldehyde (PFA), 14. Scheibe, M. et al. Quantitative interaction screen of telomeric repeatcontaining dehydrated and RNA-FISH was performed. Co-localization of foci from FISH and RNA reveals novel TERRA regulators. Genome Res. 23, 2149–2157 (2013). immunostainings were quantified by visual inspection. 15. Balk, B. et al. Telomeric RNA-DNA hybrids affect telomere-length dynamics and senescence. Nat. Struct. Mol. Biol. 20, 1199–1205 (2013). Telomere DNA-FISH. DNA-FISH was carried out as previously described54. Cells 16. Pfeiffer, V., Crittin, J., Grolimund, L. & Lingner, J. The THO complex were fixed with 4% paraformaldehyde for 10 min at RT. After three washes with 1x component Thp2 counteracts telomeric R-loops and telomere shortening. – PBS for 5 min, slides were dehydrated by washing in 70%, 90%, and 100% ethanol EMBO J. 32, 2861 2871 (2013). for 5 min each. Slides were allowed to dry for 10 min at RT. A Cy3-labeled 17. Arora, R. et al. RNaseH1 regulates TERRA-telomeric DNA hybrids (CCCTAA)3 probe was added to sample and after denaturation at 80 °C for 3 min, and telomere maintenance in ALT tumour cells. Nat. Commun. 5, 5220 the slides were incubated for 2 h at room temperature in a humid chamber. Sub- (2014). sequently, the slides were washed under agitation, twice with FISH solution (70% 18. Yu, T. Y., Kao, Y. W. & Lin, J. J. Telomeric transcripts stimulate telomere formamide, 10 mM Tris pH 7.2, 0.1% BSA) for 15 min at room temperature and recombination to suppress senescence in cells lacking telomerase. Proc. Natl three times with 0.01% Tween-20 in 1x TBS at room temperature. Slides were then Acad. Sci. USA 111, 3377–3382 (2014). dehydrated with washes in 70%, 90%, and 100% ethanol. Samples where mounted 19. Sagie, S. et al. Telomeres in ICF syndrome cells are vulnerable to DNA damage with Vectashield (Vector laboratories). Interphase nuclei were analyzed using spot due to elevated DNA:RNA hybrids. Nat. Commun. 8, 14015 (2017). IOD analysis (TFL-TELO) software. A Mann–Whitney was used to calculate sta- 20. Ginno, P. A., Lott, P. L., Christensen, H. C., Korf, I. & Chédin, F. R-loop tistical significance. formation is a distinctive characteristic of unmethylated human CpG island promoters. Mol. Cell 45, 814–825 (2012). ′ Chromosome prientation FISH (CO-FISH). Confluent experimental cells were 21. Ginno, P. A., Lim, Y. W., Lott, P. L., Korf, I. & Chédin, F. GC skew at the 5 ′ subcultured in the presence of 5′-bromo-2′-deoxyuridine (BrdU, 10 µM; Sigma) and 3 ends of human links R-loop formation to epigenetic regulation – and cultivated for 18–20 h. Colcemid was added during the final period of BrdU and transcription termination. Genome Res. 23, 1590 1600 (2013). treatment (U-2 OS, 0.2 µg/m 3 h; H1299, 1 µg/ml for 2 h). Cells were recovered and 22. Sun, Q., Csorba, T., Skourti-Stathaki, K., Proudfoot, N. J. & Dean, C. R-loop metaphases prepared as previously described54. CO-FISH was performed as pre- stabilization represses antisense transcription at the arabidopsis FLC locus. 55,56 fi – viously described using rst a (CCCTAA)3 probe labeled with Cy3 and then a Science 340, 619 621 (2013). second (TTAGGG)3 probe labeled with FITC. Samples were processed as described 23. Bhatia, V. et al. BRCA2 prevents R-loop accumulation and associates for DNA-FISH. with TREX-2 mRNA export factor PCID2. Nature 511, 362–365 (2014). 24. Santos-Pereira, J. M. & Aguilera, A. R loops: new modulators of genome Reporting summary. Further information on experimental design is available in dynamics and function. Nat. Rev. Genet. 16, 583–597 (2015). the Nature Research Reporting Summary linked to this article. 25. Skourti-Stathaki, K., Proudfoot, N. J. & Gromak, N. Human senataxin resolves RNA/DNA hybrids formed at transcriptional pause sites to promote Xrn2- Data availability dependent termination. Mol. Cell 42, 794–805 (2011). The data that support the findings of this study are available from the corresponding 26. Skourti-Stathaki, K., Kamieniarz-Gdula, K. & Proudfoot, N. J. R-loops induce author upon reasonable request. Source data for western blot and ChIP experiments in repressive chromatin marks over mammalian gene terminators. Nature 516, Figs. 1 and 4 are available in the Supplementary Information File. 436–439 (2014). 27. Boque-Sastre, R. et al. Head-to-head antisense transcription and R-loop formation promotes transcriptional activation. Proc. Natl Acad. Sci. USA 112, Received: 29 January 2018 Accepted: 21 January 2019 5785–5790 (2015). 28. Skourti-Stathaki, K. & Proudfoot, N. J. A double-edged sword: R loops as threats to genome integrity and powerful regulators of gene expression. Genes Dev. 28, 1384–1396 (2014). 29. Yu, K., Chedin, F., Hsieh, C.-L., Wilson, T. E. & Lieber, M. R. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B References cells. Nat. Immunol. 4, 442–451 (2003). 1. O’Sullivan, R. J. & Karlseder, J. Telomeres: protecting chromosomes against 30. Aguilera, A. & García-Muse, T. R loops: from transcription byproducts to genome instability. Nat. Rev. Mol. Cell Biol. 11, 171–181 (2010). threats to genome stability. Mol. Cell 46, 115–124 (2012).

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31. Alzu, A. et al. Senataxin associates with replication forks to protect fork integrity 54. Canela, A., Klatt, P. & Blasco, M. A. Telomere length analysis. Methods Mol. across RNA-polymerase-II-transcribed genes. Cell 151, 835–846 (2012). Biol. 371,45–72 (2007). 32. Santos-Pereira, J. M. et al. The Npl3 hnRNP prevents R-loopmediated 55. Benetti, R., García-Cao, M. & Blasco, M. A. Telomere length regulates the transcription-replication conflicts and genome instability. Genes Dev. 27, epigenetic status of mammalian telomeres and subtelomeres. Nat. Genet. 39, 2445–2458 (2013). 243–250 (2007). 33. Helmrich, A., Ballarino, M. & Tora, L. Collisions between replication and 56. Bailey, S. M., Cornforth, M. N., Kurimasa, A., Chen, D. J. & Goodwin, E. H. transcription complexes cause common fragile site instability at the longest Strand-specific postreplicative processing of mammalian telomeres. Science human genes. Mol. Cell 44, 966–977 (2011). 293, 2462–2465 (2001). 34. Tuduri, S. et al. Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription. Nat. Cell Biol. 11, 1315–1324 (2009). Acknowledgements 35. Luke, B. et al. The Rat1p 5′ to 3′ exonuclease degrades telomeric repeat- The laboratory of S.S. is supported by the Italian Association for Cancer Research (AIRC) containing RNA and promotes telomere elongation in Saccharomyces Start up grant 10299, the AIRC-IG 18381 grant, a FRA 2015 intramural grant from the cerevisiae. Mol. Cell 32, 465–477 (2008). University of Trieste, Italy, and a grant by the European Union, European Regional 36. Teasley, D. C. et al. Flap endonuclease 1 limits telomere fragility on the leading Development Fund, and Interreg V-A Italia-Austria 2014–2020 (ITAT1096-P). R.B is strand. J. Biol. Chem. 290, 15133–15145 (2015). supported by a AIRC grant Rif 42/08, 6352 and 17756. E.P. was supported by an AIRC 37. Conomos, D., Pickett, H. A. & Reddel, R. R. Alternative lengthening of fellowship. We thank Alessandro Marcello and Fabrizio D’Adda di Fagagna for providing telomeres: remodeling the telomere architecture. Front. Oncol. 3, 27 (2013). vectors and Maria Blasco for DNA-FISH probes. 38. Draskovic, I. & Vallejo, A. Telomere recombination and alternative telomere lengthening mechanisms. Front. Biosci. 18,1–20 (2013). Author contributions 39. Benetti, R. et al. A mammalian microRNA cluster controls DNA methylation E.P., V.B., R.B., and S.S. designed experiments; E.P., V.B., A.Z., O.S., P.V.B., and R.D. and telomere recombination via Rbl2-dependent regulation of DNA carried out experiments; S.M. performed mass spectrometry analysis; E.P., V.B., R.B., and methyltransferases. Nat. Struct. Mol. Biol. 15, 268–279 (2008). S.S. wrote the manuscript. 40. Flynn, R. L. et al. Alternative lengthening of telomeres renders cancer cells hypersensitive to ATR inhibitors. Science 347, 273–277 (2015). 41. López De Silanes, I., Stagno d’Alcontres, M. & Blasco, M. A. TERRA Additional information transcripts are bound by a complex array of RNA-binding proteins. Nat. Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- Commun. 1, 33 (2010). 019-08863-1. 42. Takahama, K. et al. Regulation of telomere length by G-quadruplex telomere DNA- and TERRA-binding protein TLS/FUS. Chem. Biol. 20, 341–350 (2013). Competing interests: The authors declare no competing interests. 43. Knott, G. J., Bond, C. S. & Fox, A. H. The DBHS proteins SFPQ, NONO and PSPC1: a multipurpose molecular scaffold. Nucleic Acids Res. 44, 3989–4004 Reprints and permission information is available online at http://npg.nature.com/ (2016). reprintsandpermissions/ 44. Yarosh, C. A., Iacona, J. R., Lutz, C. S. & Lynch, K. W. PSF: nuclear busy-body or nuclear facilitator? Wiley Interdiscip. Rev. RNA 6, 351–367 (2015). Journal peer review information: Nature Communications thanks the anonymous 45. Min, J., Wright, W. E. & Shay, J. W. Alternative lengthening of telomeres reviewers for their contribution to the peer review of this work. Peer reviewer reports are mediated by mitotic DNA synthesis engages break-induced replication available. processes. Mol. Cell. Biol. 37, e00226–17 (2017). 46. Alfano, L. et al. NONO regulates the intra-S-phase checkpoint in response to Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in UV radiation. Oncogene 35, 567–576 (2016). published maps and institutional affiliations. 47. Buisson, R. et al. Coupling of homologous recombination and the checkpoint by ATR. Mol. Cell 65, 336–346 (2017). 48. Clynes, D. et al. Suppression of the alternative lengthening of telomere pathway Open Access This article is licensed under a Creative Commons by the chromatin remodelling factor ATRX. Nat. Commun. 6, 7538 (2015). Attribution 4.0 International License, which permits use, sharing, 49. Fan, Q., Zhang, F., Barrett, B., Ren, K. & Andreassen, P. R. A role for adaptation, distribution and reproduction in any medium or format, as long as you give monoubiquitinated FANCD2 at telomeres in ALT cells. Nucleic Acids Res. 37, appropriate credit to the original author(s) and the source, provide a link to the Creative 1740–1754 (2009). Commons license, and indicate if changes were made. The images or other third party 50. Vera, E., Canela, A., Fraga, M. F., Esteller, M. & Blasco, M. A. Epigenetic material in this article are included in the article’s Creative Commons license, unless regulation of telomeres in human cancer. Oncogene 27, 6817–6833 (2008). indicated otherwise in a credit line to the material. If material is not included in the 51. Episkopou, H. et al. Alternative lengthening of telomeres is characterized by ’ reduced compaction of telomeric chromatin. Nucleic Acids Res. 42, 4391–4405 article s Creative Commons license and your intended use is not permitted by statutory (2014). regulation or exceeds the permitted use, you will need to obtain permission directly from 52. Chawla, R. et al. Human UPF1 interacts with TPP1 and telomerase and the copyright holder. To view a copy of this license, visit http://creativecommons.org/ sustains telomere leading-strand replication. EMBO J. 30, 4047–4058 (2011). licenses/by/4.0/. 53. Kovac, M. et al. Exome sequencing of osteosarcoma reveals mutation fi signatures reminiscent of BRCA de ciency. Nat. Commun. 6, 8940 (2015). © The Author(s) 2019

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