FANCM limits ALT activity by restricting telomeric replication stress induced by deregulated BLM and R-loops

ARTICLE FANCM limits ALT activity by restricting telomeric replication stress induced by deregulated BLM and R-loops Bruno Silva1, Richard Pentz1, Ana Margarida Figueira1, Rajika Arora1, Yong Woo Lee1, Charlotte Hodson2, Harry Wischnewski3, Andrew J. Deans 2,4 & Claus M. Azzalin 1 Telomerase negative immortal cancer cells elongate telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. While sustained telomeric replicative stress is required to maintain ALT, it might also lead to cell death when excessive. Here, we show that the ATPase/translocase activity of FANCM keeps telomeric replicative stress in check specifically in ALT cells. When FANCM is depleted in ALT cells, telomeres become dysfunctional, and cells stop proliferating and die. FANCM depletion also increases ALTassociated marks and de novo synthesis of telomeric DNA. Depletion of the BLM helicase reduces the telomeric replication stress and cell proliferation defects induced by FANCM inactivation. Finally, FANCM unwinds telomeric R-loops in vitro and suppresses their accumulation in cells. Overexpression of RNaseH1 completely abolishes the replication stress remaining in cells codepleted for FANCM and BLM. Thus, FANCM allows controlled ALT activity and ALT cell proliferation by limiting the toxicity of uncontrolled BLM and telomeric R-loops. https://doi.org/10.1038/s41467-019-10179-z OPEN 1Instituto de Medicina Molecular João Lobo Antunes (iMM), Faculdade de Medicina da Universidade de Lisboa, Lisbon 1649-028, Portugal. 2Genome Stability Unit, St. Vincent’s Institute of Medical Research, Fitzroy 3065 VIC, Australia. 3Institute of Biochemistry (IBC), Eidgenössische Technische Hochschule Zürich (ETHZ), Zürich 8093, Switzerland. 4Department of Medicine (St Vincent’s Hospital), University of Melbourne, Fitzroy 3065 VIC, Australia. Correspondence and requests for materials should be addressed to C.M.A. (email: [email protected]a.pt) NATURE COMMUNICATIONS | (2019) 10:2253 | https://doi.org/10.1038/s41467-019-10179-z | www.nature.com/naturecommunications 1 1234567890():,; Telomere shortening must be counteracted in immortal cells, including the large majority of cancer cells, to avoid senescence or death1. Approximately 90% of human cancers have reactivated the reverse transcriptase telomerase, which adds newly synthesized telomeric repeats to the 3′end of linear chromosomes2,3. About 10% of immortal cancer cells are telomerase-negative and replenish telomeres using the so-called Alternative Lengthening of Telomeres (ALT) pathway4.In humans, ALT was reported in tumors of mesenchymal or epithelial origin, including osteosarcomas, liposarcomas, glioblastomas, astrocytomas, and bladder carcinomas as well as in in vitro immortalized cell lines4–8. Molecular features considered markers for ALT comprise: (i) telomeres of heterogeneous lengths at different chromosome ends, including telomeres much longer than average telomeres in telomerase-positive cells8; (ii) elevated levels of the telomeric long noncoding RNA (lncRNA) TERRA9–13; (iii) clustering of multiple telomeres into ALT-associated PML bodies (APBs), nuclear structures containing promyelocytic leukemia protein (PML), telomeric factors such as TRF1, TRF2 and RAP1, TERRA, and DNA repair factors such as RAD51, RAD52, Replication Protein A (RPA), Brca1, and Bloom (BLM) and Werner helicases11,14–19; (iv) abundant extrachromosomal telomeric repeats (ECTRs) comprising double-stranded (ds) circles (t-circles), partially single-stranded (ss) circles (Cand G-circles) and linear dsDNA20–23; (v) recurrent mutations of the Alpha Thalassemia/ Mental Retardation Syndrome X-Linked (ATRX) gene12. Multiple DNA metabolism pathways collaborate to maintain telomeres in ALT cells. Break-induced replication (BIR) is active at ALT telomeres in the G2 phase of the cell cycle, and is stimulated by DSBs experimentally induced using the telomeretethered DNA endonuclease TRF1-FokI24,25. ALT BIR requires POLD3 and POLD4, two regulatory subunits of DNA polymerase delta24,25. Conservative mitotic DNA synthesis (MiDAS) was also documented in human ALT cells26. ALT MiDAS is stimulated by replication stress and requires RAD52 26. Finally, clustering of ALT telomeres within APBs is promoted by RAD51-dependent long-range movements, which are also stimulated by TRF1-FokIinduced DSBs27. Telomere movements may promote efficient homology searches and telomere synthesis, although both ALT BIR and MiDAS are independent of RAD51 25,26. A common notion deriving from all this work is that a sustained physiological damage must be maintained at ALT telomeres to promote telomere elongation. This is consistent with the presence of replication stress and DNA damage markers in APBs11,14–18. The triggers of this damage remain unclear, although RNA:DNA hybrids (R-loops), G-quadruplexes and oncogene expression were proposed as candidates11,26. This scenario implies that telomeric damage levels be maintained within a specific threshold that is high enough to trigger DNA synthesisbased repair, yet not too high to induce cell death. Consistently, telomeric R-loops (telR-loops) formed by TERRA and telomeric DNA activate replication stress at ALT telomeres, and their levels are tightly controlled by the endoribonuclease RNaseH1 11,28. When RNaseH1 is depleted, excessive replication stress rapidly leads to abundant telomere free chromosome ends (TFEs) and increased C-circles. Conversely, RNaseH1 overexpression causes progressive TFE accumulation, likely due to inefficient de novo synthesis of telomeric DNA11. The DNA damage signaling kinase ATMand Rad3-Related (ATR) and the annealing helicase SWI/ SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein 1 (SMARCAL1) were also reported to restrict replicative stress at ALT telomeres29,30. The Fanconi anemia, complementation group M (FANCM) ATPase/translocase is a component of the Fanconi Anemia (FA) complex, where it supports efficient FANCD2 ubiquitination upon stalling of replication forks by physical impediments including DNA crosslinks31. Independently of the FA complex, FANCM remodels replication forks, recruits DNA repair factors at damage sites, suppresses meiotic crossovers and facilitates ATR checkpoint activation32–35. Moreover, the ATPase/translocase activity of FANCM resolves RNA:DNA hybrids in vitro and that R-loops accumulate genomewide in FANCM-deficient cells36.We hypothesized that FANCM suppresses replication stress at ALT telomeres, and while this work was in progress, a report from Pan et al.17 confirmed our hypothesis. The authors showed that, in ALT cells, FANCM allows efficient progression of the replication fork through the telomeric tract, and depletion of FANCM induces telomeric replication stress17. The same study also reported that FANCM depletion leads to accumulation of BLM and Brca1 at ALT telomeres and that codepletion of FANCM with Brca1 or BLM is lethal17. Here we show that FANCM depletion in ALT cells causes robust telomere replication stress and damage, activation of ATR signaling, nearly complete abrogation of proliferation, and cell death. In FANCM-depleted ALT cells telomeric ssDNA, ECTRs and mitotic DNA threads accumulate. Moreover, features of ALT activity including APBs and DNA synthesis in G2/M augment when FANCM is depleted. An ATPase/translocase inactive variant of FANCM fails to revert telomeric replication stress and APB accumulation in cells depleted for endogenous FANCM. Finally, FANCM resolves telR-loops in vitro and restricts them in cells, and the replicative stress induced by FANCM depletion is completely averted by simultaneous codepletion of BLM and overexpression of RNaseH1. We propose that FANCM keeps replicative stress and ALT in check by assuring regulated BLM activity and resolving telR-loops. Results FANCM supports viability of ALT cells. We depleted FANCM in several ALT (U2OS, HuO9, Saos2 and WI-38 VA13) and telomerase-positive (Tel+; HeLa, HOS, HT1080 and SKNAS) cells using short interference RNAs (siRNAs) against two sequences from FANCM coding region (siFa and siFb). Nontargeting siRNAs were used as controls (siCt). Two days after transfection, nearly complete depletion of FANCM protein was detected by western blot in Faand Fb-transfected cells, with the exception of siFb-transfected SKNAS cells, where about 10% of the protein remained (Fig. 1a). Fluorescence-activated cell sorting (FACS) of ethanol-fixed, propidium iodide (PI)-stained cells revealed that FANCM-depleted ALT cells, but not Tel+, accumulated in G2/M phase (Fig. 1b, c; Supplementary Fig. 1A). The clonogenic potential of ALT cells was largely abolished upon transfection of FANCM siRNAs, while the one of Tel+cells remained essentially unaffected (Fig. 1d, e). For colony formation experiments, cells were transfected only once with siRNAs before seeding and colonies were counted at least 8 days later. Hence, the antiproliferative effects exerted by FANCM depletion on ALT cells are fast and irreversible. Cell growth analysis upon prolonged siRNA treatment showed that FANCM-depleted U2OS cells were quickly eliminated from the population, while HeLa cells continued to grow although at lower rates (Fig. 1f). Finally, FANCM-depleted U2OS cells, but not HeLa cells, started to be permeable to PI already after 3 days of siRNA treatment denoting cell death (Supplementary Fig. 2A). No major changes in PARP1 cleavage were detected in the same cells (Supplementary Fig. 2B). Thus, FANCM depletion causes aberrant accumulation of ALT cells in G2/M phase, followed by PARP1independent cell death. Our data indicate that FANCM is essential for cell-cycle progression and viability in ALT cells. This is different to what ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10179-z 2NATURE COMMUNICATIONS | (2019) 10:2253 | https://doi.org/10.1038/s41467-019-10179-z | www.nature.com/naturecommunications was observed previously17. It is possible that less efficient protein depletion obtained by Pan and colleagues or retained expression of crucial FANCM splice variants (possibly including cell-type specific ones that are not reported in public databases) left residual amounts of FANCM protein sufficient to sustain cell proliferation. Less sensitive cell viability assays might also have underestimated the effects of FANCM depletion in the previous study. Telomeric replication stress sensitizes ALT cells to FANCM depletion. Several features of ALT cells could explain their sensitivity to FANCM depletion: absence of telomerase activity, very long telomeres, ATRX inactivation, and sustained telomeric replication stress. We ectopically expressed the catalytic (hTERT) and RNA (hTR) subunits of telomerase in U2OS and HeLa cells to generate supertelomerase cells37. Overexpression of hTERT and hTR was confirmed by quantitative RT-PCR (Supplementary Fig. 3A). As expected, HeLa supertelomerase cells had much longer telomeres than HeLa control cells; U2OS supertelomerase cells had reduced TFE frequencies, while the incidence of underreplicated, fragile telomeres (TFs) remained unchanged (Supplementary Fig. 3B)11,37. FANCM depletion inhibited cell proliferation and led to G2/M accumulation in U2OS supertelomerase cells, but not in HeLa supertelomerase cells (Fig. 1b–e). Moreover, HeLa cells treated with the telomerase inhibitor BIBR 1532 38 did not accumulate in G2/M when depleted for FANCM (Supplementary Fig. 4A). We then codepleted FANCM and ATRX in HeLa cells and did not observe the accumulation of G2/M cells (Supplementary Fig. 4B). Thus, the presence of ultra-long telomeres or the absence of active telomerase or ATRX alone do not explain the sensitivity of ALT cells to FANCM depletion. We then overexpressed the shelterin factor TRF1 in U2OS cells by retroviral infection, as this treatment halves the incidence of Ct Fa Fb FANCM LMB1 FANCM LMB1 FANCM Golgin FANCM Golgin FANCM Golgin FANCM Golgin FANCM Golgin FANCM Golgin FANCM FANCM KAP1 Golgin U2OS U2OSHeLa HeLa Supertelomerase U2OS U2OS HeLa U2OS HeLa HeLa Supertelomerase Supertelomerase G1 S G2/M 100 50 0 100 150 ** ** ** ** ** ** ** *** *** 50 0 siRNA: Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa U2OS HuO9 Saos2 VA13 HeLa HeLa HOS HT10 SK U2OS Supertelomerase U2OS HuO9 Saos2 VA13 HeLa HeLa HOS HT10 SK U2OS siCt siFa siFb siCt siFa siFb ev + ev + ev + :FL-TRF1 FANCM FL-TRF1 TRF1 LMB1 pS33 pRPA32 RPA32 Golgin DNA content (PI) ev FL-TRF1 G1 S G2/M 100 50 0 100 50 0FL-TRF1: ev + ev + ev + siCt siCt siFa siFa siFb siFb siCtsiFasiFb siCtsiFasiFb siCtsiFasiFb DNA content (PI) H-ST U-ST SK HT10 HOS HeLa VA13 Saos2 HuO9 U2OS Relative colony number (%) Relative cell count (%) Relative cell count (%) Day: 1 4 7 10 1 4 7 10 Normalized cell number (%) : siRNA ab c de fgh Fig. 1 FANCM supports normal cell-cycle progression and proliferation of ALT cells. aWestern blot analysis of FANCM protein levels in ALT and Tel+cells transfected with anti-FANCM siRNAs (siFa and siFb) or with control siRNAs (siCt). ALT cells (gray background) are: U2OS, HuO9, Saos2 and WI-38 VA13 (VA13); Tel+cells are: HeLa, HOS, HT1080 (HT10) and SKNAS (SK). U-ST and H-ST are supertelomerase U2OS and HeLa cells, respectively. Proteins were extracted 48 h after transfection. Lamin B1 (LMB1), Golgin 97 and KAP1 serve as loading controls. bExamples of FACS profiles of the indicated siRNAtransfected cells stained with propidium iodide (PI). Cell counts (yaxis) are plotted against PI intensity (xaxis). Cells were harvested 48 h after transfection. cQuantifications of experiments as in (b). The graph shows the percentage of cells in G1, S and G2/M phases from one representative experiment. dExamples of colony formation assays with the indicated siRNA-transfected cells. eQuantifications of experiments as in (d). The graph shows colony numbers relative to siCt-transfected samples. Bars and error bars are means and SDs from three independent experiments. Pvalues were calculated with a two-tailed Student’sttest. *P< 0.05, **P< 0.005, ***P< 0.001. fGrowth curves of U2OS and HeLa cells transfected with the indicated siRNAs every 3 days. Cell numbers are expressed relative to siCt-transfected cells. Data points and error bars are means and SDs from three independent experiments. gWestern blot analysis of U2OS cells infected with retroviruses expressing Flag-tagged TRF1 (FL-TRF1) or with empty vector (ev) control retroviruses. Five days after infections cells were transfected with the indicated siRNAs and harvested 48 h later. pS33: RPA32 phosphorylated at serine 33, pRPA32: phosphorylated RPA32. LMB1 and Golgin serve as loading controls. hExamples of FACS profiles of cells as in (g). The graph on the left shows the percentage of cells in G1, S and G2/M phases from one representative experiment. Source data are provided as a Source Data file NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10179-z ARTICLE NATURE COMMUNICATIONS | (2019) 10:2253 | https://doi.org/10.1038/s41467-019-10179-z | www.nature.com/naturecommunications 3 FTs39. FANCM depletion in TRF1 overexpressing cells still led to G2/M accumulation, yet less severely than in cells infected with empty vector (ev) retroviruses (Fig. 1g, h). However, HeLa cells depleted for TRF1 using an siRNA previously shown to induce telomere fragility39 did not accumulate in G2/M when codepleted for FANCM (Supplementary Fig. 4C). Similarly, FANCMdepleted HeLa cells did not show an altered cell-cycle distribution when treated with the replication stress inducer hydroxyurea (HU) followed by block release (Supplementary Fig. 4D). Hence, telomeric replication stress contributes to the sensitivity of ALT cells to FANCM depletion; nevertheless, telomeric or generalized replication stress alone are not sufficient to sensitize non-ALT cells to FANCM depletion. FANCM suppresses telomeric replication stress in ALT cells. To test the involvement of FANCM in telomere stability, we performed indirect immunofluorescence (IF) using antibodies against TRF2 combined with antibodies against RPA32 phosphorylated at Serine 33 (pS33) or p53 binding protein 1 (53BP1). RPA32 is phosphorylated at serine 33 during S phase by ATR upon replication fork stalling40; 53BP1 forms foci at dysfunctional telomeres that have activated either ATR, or the other DNA damage signaling kinase ataxia-telangiectasia mutated (ATM), or both41,42. Within 48 h of transfection, pS33 and 53BP1 accumulated at telomeres in FANCM-depleted ALT cells (Fig. 2a, b) forming the so-called telomere dysfunction-induced foci (TIFs)41. FANCM depletion did not induce TIF formation in Tel+cells (Fig. 2a, b). Accumulation of pSer33 and 53BP1 outside of telomeres was negligible in all FANCM-depleted cell lines (Fig. 2a). FANCM-mediated suppression of telomere instability is likely to be direct, because the protein associated with telomeric DNA in chromatin immunoprecipitation (ChIP) experiments (Fig. 2c, d). FANCM also immunoprecipitated with the abundant, genomewide-spread Alu repeat DNA (Fig. 2c, d), indicating that the protein is not exclusively associated with telomeres. This is consistent with the reported localization of FANCM to cellular chromatin fractions43. Western blot analysis confirmed that FANCM depletion causes pS33 accumulation and revealed phosphorylation of the other ATR target checkpoint kinase 1 (CHK1) in U2OS but not HeLa cells (Fig. 2e). The ATM target KRAB domain-associated protein 1 (KAP1) was not phosphorylated in any of the tested cell lines (Fig. 2e). Moreover, pS33 accumulation was weakened in FANCM-depleted U2OS cells overexpressing TRF1 (Fig. 1g), while telomerase inhibition, ATRX or TRF1 depletion and HU treatment did not promote pS33 accumulation in FANCMdepleted HeLa cells (Supplementary Fig. 4A-D). Actually, pS33 failed to accumulate efficiently in HeLa cells depleted for FANCM and treated with HU, consistent with a role for FANCM in supporting activation of the canonical ATR-dependent intra Sphase checkpoint32. We propose that FANCM deficiency in ALT cells activates a specific ATR-dependent signaling cascade, which is not fully identical to the one triggered by generalized replication stress and stems at least partly from excessive telomeric replication stress. Such ATR response likely provokes the observed G2/M arrest and cell death. FANCM suppresses ALT features. In our IF images, TRF2 foci in FANCM-depleted ALT cells are both larger and brighter than in control cells (Fig. 2a). To confirm that this was not simply due to increased TRF2 at telomeres, we subjected siRNA-transfected U2OS interphase cells to DNA fluorescence in situ hybridization (FISH) using telomeric probes, and measured the number and area of telomeric foci. We controlled for possible secondary effects related to cell-cycle stage by arresting siCt-transfected cells at the G2/M border with the cyclin-dependent kinase 1 (CDK1) inhibitor RO-3306 44 (Fig. 3a, b; Supplementary Fig. 1B). The overall number of telomeric foci decreased upon FANCM depletion (Fig. 3c, d), while their area distribution was broader, with slightly increased frequencies of very small foci (S in Fig. 3c–e) and substantially increased frequencies of very large foci (L in Fig. 3c–e). RO-3306-treated cells also had less telomeric foci than control cells (Fig. 3d), likely due to clustering of ALT telomeres in G2 19,45. However, the increase in very small and very large foci was more pronounced upon FANCM depletion than RO-3306 treatment (Fig. 3c–e). Approximately 60% of FANCM-depleted cells had at least five large foci, vs. approximately 10 and 15% of untreated or RO-3306-treated siCttransfected cells, respectively (Fig. 3f). We then analyzed the localization of PML, RAD51 and POLD3 at telomeres by combining PML and RAD51 IF with telomere FISH, and double IF for POLD3 and RAP1. We observed increased telomeric localization of all three factors in FANCMdepleted U2OS cells, without obvious increase in PML, POLD3 and RAD51 total protein levels (Figs. 3a, 4a, b). RO-3306 treatment did not substantially affect the number of telomeric PML and RAD51 foci, while it increased the one of telomeric POLD3 foci yet less importantly than FANCM depletion (Fig. 4a, b). Moreover, we incubated cells treated as above with the thymidine analog 5-Ethynyl-2′-deoxyuridine (EdU) for 2.5 h, and performed telomere FISH combined with EdU detection to visualize newly synthesized telomeric DNA (Fig. 4a). To exclude S phase cells, we only scored cells showing a punctuate EdU staining and with not more than 25 EdU foci. FANCM depletion increased the incidence of telomeric EdU foci, as it did RO-3306 treatment albeit to lower extents (Fig. 4a, b). We conclude that FANCM depletion exacerbates ALT activity as shown by robust telomere clustering within large APBs containing PML, RAD51 and POLD3, and increased synthesis of telomeric DNA outside of S phase. FANCM depletion also generates short telomeric species, possibly representing ECTRs (see below). G2/M arrest alone cannot explain the aberrantly elevated ALT features observed in FANCM-depleted cells. FANCM suppresses telomeric ssDNA and ECTRs in ALT cells. We performed in-gel telomere restriction fragment (TRF) analysis of genomic DNA from ALT (U2OS and WI-38 VA13) and Tel+(HOS and HeLa) cells harvested 48 h after siRNA transfection. Blots were hybridized with telomeric oligonucleotides of either 5′-TTAGGG-3′or 5′-CCCTAA-3′repeats. When hybridization was performed under native (nondenatured) conditions, we observed increased C-rich telomeric ssDNA of very diverse lengths in FANCM-depleted ALT cells (Fig. 5a, upper panel). Conversely, a decrease of G-rich ssDNA was observed in correspondence of the bulk of telomeres, likely due to shortening of the G-overhang (Fig. 5a, lower panels). For both probes, a fraction of the signal was in the gel wells, possibly corresponding to ssDNA exposed from molecules with significant secondary structures (Fig. 5a). We did not observe alteration of telomeric ssDNA in Tel +cells (Fig. 5a). Hybridization of the same gels in denatured conditions using a long telomeric probe (Telo2 probe) revealed no appreciable alteration of telomere length in FANCM-depleted cells (Fig. 5a). We then dot-blotted genomic DNA from cells as above and hybridized it under native conditions to telomeric oligonucleotides, followed by denaturation and hybridization with an Alu repeat, as a control for total DNA loaded. This experiment confirmed that FANCM-depleted ALT cells contain more telomeric C-rich ssDNA than siCt-transfected cells (Fig. 5b). As previously reported11, depletion of RNaseH1 in U2OS cells also ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10179-z 4NATURE COMMUNICATIONS | (2019) 10:2253 | https://doi.org/10.1038/s41467-019-10179-z | www.nature.com/naturecommunications siCt siFa siFb siCt siFa siFb siCt siFa siFb siCt siFa siFb siCt siFa siFb siCt siFa siFb Merge+DAPITRF2 pS33 TRF2 53BP1 Merge+DAPI [TTAGGG]5 AIu U2OS VA13 HOS HeLa HT10 Ct Fa Fb Ct Fa Fb – ––– – +– – – + :siRNA :CPT FANCM GoIgin pS824 KAP1 pS33 pS345 pRPA32 RPA32 Actin CHK1 LMB1 U2OS HeLa siCt siFa siFb U2OS HuO9 Saos2 VA13 HeLa HOS HT10 SK U2OS VA13 HOS HeLa HT10 U2OS VA13 HOS HeLa HT10 U2OS VA13 HOS HeLa HT10 [TTAGGG]5 [TTAGGG]5 high contrast Alu HeLa U2OS HeLa U2OS pS33 TIFs/nucleus 60 40 20 0 60 40 20 0 **** **** **** **** **** **** **** **** **** ** 5 4 3 2 1 0 Input DNA in IP (%) 53BP1 TIFs/nucleus In Bd Ip 100% 10% a b c d e Fig. 2 FANCM suppresses telomeric DNA damage and localizes to telomeres in ALT cells. aExamples of pS33 or 53BP1 immunostaining (red) combined with TRF2 immunostaining (green) on U2OS and HeLa cells transfected with the indicated siRNAs and harvested 48 h after transfection. In the merge panel, DAPI-stained DNA is also shown (blue). Arrowheads point to pS33 and 53BP1 TIFs. Scale bar: 10 μm. bQuantifications of numbers of TIFs per nucleus in experiments as in (a) performed on the indicated cell lines. ALT cells are on a grey background. Each dot represents an individual nucleus. A total of at least 196 nuclei from three independent experiments were analyzed for each sample. Bars and error bars are means and SDs. Pvalues were calculated with a Mann−Whitney Utest. **P< 0.005, ****P< 0.0001. cDot-blot hybridization of endogenous FANCM ChIPs in the indicated cell lines using radiolabeled oligonucleotides comprising telomeric G-rich repeats or Alu repeats. A high contrasted image is shown to facilitate visualization of the telomeric signal for Tel+cells. In Input, Bd only beads control, Ip anti-FANCM immunoprecipitation. dQuantifications of experiments as in (c). Signals are graphed as the fraction of In found in the corresponding Ip samples, after subtraction of Bd-associated signals. Bars and error bars are means and SDs from three independent experiments. eWestern blot analysis of DNA damage activation in the indicated siRNA-transfected cells. Proteins were extracted 48 h after transfection. Untransfected cells treated with camptothecin (CPT) were included to control for antibody specificity. pS824: KAP1 phosphorylated at serine 824, pS345: CHK1 phosphorylated at serine 345. Beta Actin and LMB1 serve as loading controls. Source data are provided as a Source Data file NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10179-z ARTICLE NATURE COMMUNICATIONS | (2019) 10:2253 | https://doi.org/10.1038/s41467-019-10179-z | www.nature.com/naturecommunications 5 increased telomeric C-rich ssDNA, albeit at lower levels than FANCM depletion (Fig. 5b). No major difference in total telomeric DNA was detected using dot-blot hybridization of denatured DNA for U2OS, HOS and HeLa cells (Fig. 5b). An increase in total C-rich telomeric DNA was observed in FANCMdepleted WI-38 VA13 cells (Fig. 5b). We then performed phi-29-mediated C-circle assays46 using DNA from ALT and Tel+cells and found a remarkable increase in C-circles in ALT cells depleted for FANCM (Fig. 5c). Accumulation of ECTRs, likely to correspond partly but not exclusively to C-circles, was also detected in FANCM-depleted U2OS cells using two-dimensional gel electrophoresis (Fig. 5d). Metaphase chromosome FISH of FANCM-depleted U2OS cells showed abundant extrachromosomal telomeric signals, probably corresponding to ECTRs, and DNA threads extending from the termini of single chromosomes, or bridging two independent a b c d e siCt: 25.49 ± 15.15 (n=21656) siCt+R: 26.48 ± 19.57 (n=18262) siFa: 35.08 ± 70.15 (n=18654) siFb: 34.99 ± 34.99 (n=18599) Ct – Fa – Fb – Ct + :siRNA :RO-3306 FANCM POLD3 LMB1 PML RAD51 Actin 100 50 0 siRNA: Ct Ct+R Fa Fb siRNA: Ct Ct+R Fa Fb siRNA: Ct Ct+R Fa Fb Relative cell count (%) G1SG2/M 0 50 100 Telom foci/nucleus **** **** **** SNL siCt siCt+R siFa siFb 0 3 6 9 12 15 18 Frequency (%) 0 5 10 15 20 25 30 35 40 45 50 55 60 Area of telomeric foci (pixels) Area of telomeric foci (pixels) 60 65 70 75 80 85 90 95 100 105 110 115 120 L siCt: 25.49 ± 15.15 (n = 21,656) siCt+R: 26.48 ± 19.57 (n = 18,262) siFa: 35.08 ± 70.15 (n = 18,654) siFb: 34.99 ± 34.99 (n = 18,599) 100 80 60 40 20 0 Cells with large foci ≥5 (%) ** * ***** 1 0.5 0 siCt siCt+RO–3306 siFb siFa S N f Fig. 3 FANCM depletion alters telomeric DNA in interphase U2OS cells. aWestern blot analysis of FANCM protein levels in U2OS cells transfected and treated with RO-3306 as indicated. Cells were harvested 48 h after transfection. POLD3, RAD51 and PML levels were also analyzed. Beta Actin and LMB1 serve as loading controls. bQuantifications of FACS profiles of cells as in (a) stained with PI. The graph shows the percentage of cells in G1, S and G2/M phases from one representative experiment. R: RO-3306. cExamples of telomeric FISH on interphase cells as in (a). Telomeric DNA is shown in green, nuclear outlines are shown by dotted lines. S small foci, N normal foci, L large foci. Scale bar: 20 μm. dQuantifications of numbers of telomeric foci per nucleus in experiments as in (c). Each dot represents an individual nucleus. A total of at least 300 nuclei from three independent experiments were analyzed for each sample. Bars and error bars are means and SDs. Pvalues were calculated with a Mann−Whitney Utest. eArea distribution of telomeric foci areas in experiments as in (c). 3D images were sum projected and areas of individual nuclear FISH signals were measured using DAPI staining to identify nuclei (not shown). A total of at least 300 nuclei from three independent experiments were analyzed for each sample. Areas of telomeric foci (in pixels) are binned into 25 intervals of 5-pixel width (xaxis; numbers indicate bin centers) and plotted against frequencies (yaxis; %). S small foci (0−2.5 pixels), N normal foci (2.5−57.5 pixels), L large foci (57.6−125.5 pixels). The distribution of large foci is represented in the right graph using a smaller y axis scale to facilitate visualization. fQuantification of cells with at least five Large (L) foci in experiments as in (c). Pvalues were calculated with a twotailed Student’sttest. *P< 0.05, **P< 0.005, ***P< 0.001, ****P< 0.0001. Source data are provided as a Source Data file ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10179-z 6NATURE COMMUNICATIONS | (2019) 10:2253 | https://doi.org/10.1038/s41467-019-10179-z | www.nature.com/naturecommunications chromosome ends (Supplementary Fig. 5A and B). C-rich ssDNA-containing ECTRs and DNA threads may explain the well-retained DNA molecules observed in our TRF analysis and the increased telomeric ssDNA observed in our dot-blot analysis (Fig. 5a, b). We did not observe an increase in the incidence of TFEs in FANCM-depleted U2OS cells (Supplementary Fig. 5A and B). FANCM regulates BLM in ALT cells. FANCM and BLM were reported to collaborate in maintaining ALT telomeres17.We depleted FANCM in U2OS and HeLa cells and performed indirect IF using anti-BLM and anti-TRF2 antibodies. Because FANCM is necessary for BLM recruitment to damage sites induced by stalled replication34, we included cells treated with the topoisomerase I inhibitor Camptothecin (CPT; Supplementary Fig. 6A). CPT induced robust formation of nuclear (nontelomeric) BLM foci in siCt-transfected U2OS and HeLa cells, but not in siFa-transfected cells (Supplementary Fig. 6B and C). On the other hand, BLM TIFs were already abundant in siCttransfected U2OS and only rarely observed in siCt-transfected HeLa cells, and FANCM depletion increased the number of BLM TIFs in U2OS cells (Supplementary Fig. 6B and C). CPT treatment marginally affected TIF frequencies in all samples (Supplementary Fig. 6B and C). BLM nuclear relocalization occurred without major changes in total protein levels (Supplementary Fig. 6A). Hence, we confirm that FANCM depletion causes BLM accumulation at ALT telomeres17, while it prevents it at nontelomeric sites of damage both in ALT and Tel+cells34. The telomeric accumulation of BLM upon FANCM depletion might involve reported interactions with TRF1 and TRF2 47. We then depleted FANCM and BLM simultaneously in U2OS cells (Fig. 6a). BLM depletion alone did not alter cell-cycle a b siCt siCt+RO–3306 siFa siFb siCt siCt+RO–3306 siFa siFb siCt siCt+RO–3306 siFa siFb siCt siCt+RO–3306 siFa siFb siCt siCt+R siFa siFb siCt siCt+R siFa siFb siCt siCt+R siFa siFb Merge+DAPI PML Telomeres Merge+DAPI RAD51 TelomeresMerge+DAPI EdU Telomeres Merge+DAPI POLD3 RAP1 30 25 20 15 10 5 0 APBs/nucleus RAD51+telom/nucleus 25 20 15 10 5 0 20 15 10 5 0 POLD3+RAP1/nucleus **** *** **** **** **** **** **** siCt siCt+R siFa siFb 25 20 15 10 5 0 EdU+telom/nucleus **** **** **** **** **** **** Fig. 4 FANCM depletion increases ALT features. aUpper panels: examples of PML or RAD51 immunostaining (red) combined with telomeric DNA FISH (green); lower left panel: examples of POLD3 immunostaining (red) combined with RAP1 immunostaining (green); lower right panel: examples of EdU detection (red) combined with and telomeric DNA FISH (green). Experiments were performed on U2OS cells transfected with the indicated siRNAs and harvested 48 h after transfection. SiCt-transfected cells treated with RO-3306 were included. In the merge panel, DAPI-stained DNA is also shown (blue). Arrowheads point to colocalization events. Scale bar: 10 μm. bQuantifications experiments as in (a). Each dot represents an individual nucleus. A total of at least 300 nuclei from three independent experiments were analyzed for each sample. Bars and error bars are means and SDs. Pvalues were calculated with a Mann−Whitney Utest. ***P< 0.001, ****P< 0.0001. Source data are provided as a Source Data file NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10179-z ARTICLE NATURE COMMUNICATIONS | (2019) 10:2253 | https://doi.org/10.1038/s41467-019-10179-z | www.nature.com/naturecommunications 7 distribution and number of colonies formed, while it decreased proliferation rates and only minimally augmented the fraction of PI-permeable cells (Fig. 6b–d; Supplementary Figs. 1C, 2A). Unexpectedly, FANCM and BLM codepletion resulted in a partial rescue of the aberrant cell-cycle distribution and cell proliferation and viability deriving from depleting FANCM (Fig. 6b–d; Supplementary Figs. 1C, 2A). Moreover, BLM depletion halved the incidence of pS33 TIFs in cells depleted for FANCM (Fig. 6e, f). These results establish that BLM depletion alleviates the adverse effects exerted by FANCM deficiency on ALT cells. FANCM suppresses TERRA and telR-loops in ALT cells.To test whether FANCM suppresses telomere replication stress in ALT cells by regulating TERRA and/or telR-loops, we first performed TERRA northern blot and found that the levels of this lncRNA were 3.5 and 2.5 folds higher in siFaand siFbtransfected cells, respectively, than in siCt-transfected ones. TERRA species up to ~2 kb in length were the most affected (Fig. 7a). We then performed in vitro R-loop resolution assays using telR-loop-containing plasmids generated by T7 transcription of a telomeric tract of approximately 1 kb11. We used two U2OSVA13 HOS HeLa U2OSVA13 U2OS HOS HeLa HeLa HeLa U2OS U2OS U2OS U2OS siCt denat Telo2 U2OS siFa denat Telo2 HuO9 HOSHeLa 30 ****** *** ** *** Relative C-circle signal 20 10 0 HT10 SK siCt siFa siFb Saos2 VA13 U2OS HuO9 Saos2 VA13 HOS HOS HeLa siRNA: siRNA: Native [TTAGGG]5 [TTAGGG]5 siCt ss C-rich 1 ± 0.12 1 ± 0.05 1 ± 0.08 1 ± 0.04 1 ± 0.02 6.42 ± 0.24 8.56 ± 1.08 1.03 ± 0.07 1.83 ± 0.13 0.94 ± 0.06 1.23 ± 0.21 2.90 ± 0.06 1.04 ± 0.03 1.17 ± 0.02 1.02 ± 0.08 1.41 ± 0.14 1.59 ± 0.19 2.23 ± 0.11 1.59 ± 0.06 1.70 ± 0.01 1.15 ± 0.07 1 ± 0.16 1 ± 0.14 1 ± 0.07 1 ± 0.12 0.85 ± 0.07 1.06 ± 0.09 1.02 ± 0.21 1.57 ± 0.22 1.04 ± 0.13 0.86 ± 0.03 1.11 ± 0.13 0.73 ± 0.06 1 ± 0.06 1 ± 0.07 1 ± 0.04 1 ± 0.02 1.07 ± 0.03 0.95 ± 0.04 1.09 ± 0.02 0.84 ± 0.09 0.66 ± 0.02 1.09 ± 0.10 0.96 ± 0.07 1.13 ± 0.08 1 ± 0.02 1 ± 0.05 1 ± 0.09 Total C-rich Total G-rich ss G-rich siFa siFb siCt siFa siFb siCt siFa siFb siCt siFa siFb [CCCTAA]5Alu (loading) Alu (loading) Denat Telo2 Native [CCCTAA]5 Denat Telo2 NativeDenat NativeDenat Wells kb 10 8 6 5 4 3 2 1.5 1.2 1 Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa FbRHFbRHFbRHFbRH U2OSVA13 HOS HeLa U2OSVA13 HOS HeLa siRNA: Wells kb 10 8 6 5 4 3 2 1.5 1.2 1 Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa Ct Fa kb 23 9.4 3 2 1 0.5 kb 23 9.4 3 2 1 0.5 VA13 VA13 VA13 HOS siRNA: +φ29 –φ29 +φ29 –φ29 ab cd Fig. 5 FANCM restricts telomeric ssDNA and ECTRs in ALT cells. aTRF analysis of the indicated ALT (gray backgrounds) and Tel+siRNA-transfected cells. Genomic DNA was prepared 48 h after transfection, restriction digested and hybridized in-gel in native conditions to radiolabeled oligonucleotides comprising five telomeric G-rich or C-rich repeats ([TTAGGG]5 and [CCCTAA]5, respectively). After signal acquisition, gels were denatured and rehybridized to a long radiolabeled telomeric probe (Telo2). The position of the wells and the sizes in kb of a molecular weight marker are indicated on the left of the gels. bDot-blot hybridizations of digested genomic DNA from cells as in (a). Control transfections with siRNAs against RNaseH1 (siRH) were also included. Native or denatured DNA was first hybridized to radiolabeled telomeric oligonucleotides. After signal acquisition, membranes were denatured and rehybridized to radiolabeled Alu repeat oligonucleotides (loading). For quantifications (table below), telomeric signals were normalized through the corresponding Alu signal and expressed relative to siCt-transfected samples. Means and SDs from three technical replicates are indicated. Note the accumulation of C-rich ssDNA in FANCM-depleted ALT cells (thick borders). cC-circle assay analysis of genomic DNA from the indicated siRNA-transfected cells harvested 48 h after transfection. Products were dot-blotted and hybridized to a radiolabeled Telo2 probe. Control reactions were performed without phi29 polymerase (Φ29). Note that Tel+cells had no detectable signals. The graph at the bottom shows quantifications of C-circle signals relative to siCt-samples. Bars and error bars are means and SDs from three independent experiments. Pvalues were calculated with a two-tailed Student’sttest. **P< 0.005, ***P< 0.001. d2D gel electrophoresis of genomic DNA from siRNA-transfected U2OS cells as in a. DNA was denatured and hybridized to a radiolabeled Telo2 probe. Arrowheads point to arches corresponding to circular DNA. Source data are provided as a Source Data file ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10179-z 8NATURE COMMUNICATIONS | (2019) 10:2253 | https://doi.org/10.1038/s41467-019-10179-z | www.nature.com/naturecommunications plasmids with different insert orientations as to produce transcripts containing TERRA-like, G-rich RNA repeats, or complementary C-rich transcripts (Fig. 7b). As expected11, G-rich transcripts were less efficiently produced than C-rich ones (Fig. 7b). TelR-loop plasmids were incubated with recombinant FANCM in heterodimer with its stabilization partner FAAP24, with or without ATP and then resolved in agarose gels. FANCM promoted complete release of both G-rich and C-rich transcripts from R-loop-plasmids without RNA degradation and in an ATPdependent manner (Fig. 7b). Thus, FANCM efficiently unwinds the RNA moiety of telR-loops in vitro. To examine telR-loops in FANCM-depleted U2OS cells, we performed DNA:RNA immunoprecipitations (DRIP) using the monoclonal antibody S9.6 48. Dot-blot hybridization detected telomeric DNA in immunoprecipitated material from all samples, with a ~3-fold increase in siFa and siFb samples (Fig. 7c). Treatment of nucleic acids with recombinant RNaseH prior to antibody incubation largely abolished hybridization signals, confirming that they emanated from DNA:RNA hybrids (Fig. 7c). We also performed native DNA FISH using G-rich telomeric probes on interphase nuclei treated or not with RNaseH11.A punctate staining corresponding to C-rich telomeric DNA was already visible in untreated siCt-transfected cells, and its intensity was higher in RNaseH-treated cells likely due to degradation of TERRA transcripts within telR-loops and consequent increased binding sites for the probe (Fig. 7d, e). In untreated siFatransfected cells, the C-rich ssDNA signal was more prominent than in control cells and it was further augmented by RNaseH treatment (Fig. 7d, e). The total number of foci per cell was higher in FANCM-depleted cells but was not affected by RNaseH treatment (Fig. 7d, e). We conclude that FANCM suppresses TERRA and TERRA-containing telR-loops in ALT cells. Considering the ability of FANCM to resolve telR-loops in vitro (Fig. 7b) and the localization of FANCM to telomeres (Fig. 2c, d), we propose that FANCM directly resolves telR-loops on telomeric chromatin. The more prominent C-rich ssDNA signal already present in FANCM-depleted cells not treated with RNaseH (Fig. 7d, e) might originate from gaps in DNA replication or cellular degradation of the RNA moiety of telR-loops. Also, although we refer to the telomeric RNA:DNA hybrid structures arising upon FANCM depletion as telR-loops, our experiments do not distinguish between conventional R-loops, three-stranded nucleic acids comprising an RNA:DNA hybrid and a displaced ssDNA, and ds RNA:DNA hybrids devoid of a displacement loop. FANCM averts telR-loop-induced telomeric replication stress. We speculated that FANCM suppresses telomeric replication 40 35 550 000 0 0100 G1 S G2/M siCt siBl siCt siBl 200 150 Rel colony number (%) 100 50 0 ciCtsiFa5siFa20 50 Relative cell count (%) 040 0 000 0 0 20 20 20 20 15 55 35 20 20 20 siCt [nM]: siFa [nM]: siBl [nM]: siCt siCt siBl 100 50 0 Day: siCt 40 pS33 TIFs/nucleus 20 0 siCt siCt TRF2 pS33 Merge+DAPI siFa5siFa20 siCt siCtsiBl siBl siCt siBl siBl ** **** **** **** **** **** * siFa5 siFa20 14 710 Normalized cell number (%) siFa siF/B siFa5 P = 0.08 P = 0.11 **** **** *** siFa20 :siCt [nM] :siFa [nM] :siBl [nM] *FANCM BLM LMB1 1520 20 20 20 20 20 20 abc d e f * Fig. 6 BLM depletion substantially averts the phenotypes associated with FANCM depletion. aWestern blot analysis of FANCM and BLM in U2OS cells transfected with siFa, anti-BLM siRNAs (siBl), and siCt. Two different concentrations (5 and 20 nM) of siFa were used. Cells were harvested 48 h after transfection. LMB1 serves as loading control. The asterisk indicates a band cross-reacting with the anti-FANCM antibody. bQuantifications of FACS profiles of cells as in (a) stained with PI. The graph shows the percentage of cells in G1, S and G2/M phases from one representative experiment. cExample of colony formation assays using cells as in (a). siFa5: 5 nM siRNA, siFa20: 20 nM siRNA. The graph on the right shows colony numbers relative to siCttransfected samples. Bars and error bars are means and SDs from four independent experiments. Pvalues were calculated with a two-way ANOVA followed by Tukey’s HSD. dGrowth curves of U2OS cells transfected with the indicated siRNAs (20 nM each) every 3 days. Cell numbers are expressed relative to siCt-transfected cells. Data points and error bars are means and SDs from three independent experiments. SiCt and siFa curves are the same as the ones shown in Fig. 1f. eExamples of pS33 immunostaining (red) combined with TRF2 immunostaining (green) on cells as in (a). In the merge panel, DAPI-stained DNA is also shown (blue). Arrowheads point to pS33 TIFs. Scale bar: 10 μm. fQuantifications of numbers of pS33 TIFs per nucleus in cells as in (a). Each dot represents an individual nucleus. A total of at least 300 nuclei from three independent experiments were analyzed for each sample. Bars and error bars are means and SDs. Pvalues were calculated with a two-way ANOVA followed by Tukey’s HSD. *P< 0.05, **P< 0.005, ***P< 0.001, ****P< 0.0001. Source data are provided as a Source Data file NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10179-z ARTICLE NATURE COMMUNICATIONS | (2019) 10:2253 | https://doi.org/10.1038/s41467-019-10179-z | www.nature.com/naturecommunications 9 (P1EZP3-168771). Research in the Deans laboratory was supported by the Cancer Council of Victoria, Australian National Health and Medical Research Council (APP1139099), Buxton trust and the Victorian Government’s OIS Program. A.J.D is a Victorian Cancer Agency fellow. Publication costs were supported by UID/BIM/50005/2019, project funded by the Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estado. Author contributions R.P. and C.M.A. conceived the original project. B.S., R.P., R.A. and C.M.A designed experiments. B.S., R.P., R.A., A.M.F., H.W. and C.M.A. performed the experiments and data analysis. Y.W.L. performed the DRIP experiments. C.H. and A.J.D. designed and performed the in vitro R-loop resolution assays. A.J.D. generated the anti-FANCM monoclonal antibody. C.M.A wrote the manuscript with inputs from A.J.D., B.S., R.P. and R.A. Additional information Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467019-10179-z. Competing interests: The authors declare no competing interests. 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