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Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells

BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to PARP inhibitors. To understand resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show C20orf196 and FAM35A form a complex, “Shieldin” (SHLD1/2), with FAM35A interacting with single-stranded DNA via its C-terminal OB fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining through restricting DSB resection and counteract homologous recombination by antagonising BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitises BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.


Introduction
DNA double-strand breaks (DSBs) are highly cytotoxic cellular lesions that must be effectively and accurately repaired to maintain genome stability and prevent premature aging, neurodegeneration, immunodeficiency, cancer and other diseases1-3. In response to DSB detection, the apical kinases ATM, ATR and PRKDC (DNA-PKcs) become activated and phosphorylate numerous substrates to initiate the cellular DNA damage response (DDR)4. The ensuing cascade of molecular DDR events, which are promoted by various post-translational modifications including protein phosphorylation, ubiquitylation, sumoylation and poly (ADP-ribosyl)ation, impacts on a myriad of cellular components, amongst other things leading to assembly of DDR factors at DNA-damage sites, arrest or slowing of cell-cycle progression, and activation of DNA repair mechanisms4, 5. The two main types of DSB-repair pathway are non-homologous end-joining (NHEJ) which is active throughout the cell cycle, and homologous recombination (HR), which normally requires a sister chromatid as a template and hence only operates in S and G2 phases of the cell cycle. DSB-repair pathway choice is partly determined by functional antagonism between the HRpromoting factor BRCA1 and NHEJ-promoting proteins such as TP53BP1 (53BP1), RIF1 and MAD2L2 (REV7) 6-13. tumours hypersensitive to PARP-inhibitor drugs such as olaparib14-17. Unfortunately, intrinsic or acquired PARP-inhibitor resistance frequently leads to lack-of-response or to patient relapse and tumour regrowth15, 18. In the clinic, the most common PARP-inhibitor resistance mechanisms reported to date are restoration of BRCA1/2 expression or function. Notably, 53BP1 expression is lost in various triple-negative breast cancers7, which may account for certain clinically relevant examples of PARP-inhibitor resistance. Nevertheless, the mechanisms driving PARP-inhibitor resistance in a large proportion of BRCA1/2deficient tumours remain unexplained18, 19.
To systematically survey for genetic mechanisms of PARP-inhibitor resistance, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in human BRCA1-deficient breast cancer cells treated with PARP inhibitors. In addition to identifying known resistance factors such as 53BP1, RIF1 and MAD2L2 loss6-13, we identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers PARP-inhibitor resistance to BRCA1-deficient cells. Our ensuing work lead us to define the "Shieldin" (SHLD1 C20orf196 /SHLD2 FAM35A ) complex that promotes NHEJ by serving as the downstream effector of 53BP1, RIF1 and MAD2L2, restricts DSB resection, and counteracts HR in BRCA1-deficient cells by antagonising replacement of replication protein A (RPA) with BRCA2 and RAD51 on resected single-stranded DNA (ssDNA). Finally, we report that SHLD1 C20orf196 /SHLD2 FAM35A loss confers hypersensitivity to the DNAcrosslinking agent cisplatin, and that reduced SHLD1 C20orf196 or SHLD2 FAM35A expression is associated with evolution of PARP-inhibitor resistance in a patient-derived BRCA1deficient breast cancer xenograft model and in BRCA1-mutant cancers displaying intrinsic PARP-inhibitor resistance.

FAM35A or C20orf196 loss suppresses PARP-inhibitor sensitivity of BRCA1-mutant cells
To systematically explore genetic mechanisms imparting PARP-inhibitor resistance, we carried out genome-wide CRISPR-Cas9 gene-inactivation screens with the GeCKO library20 in the BRCA1-mutant breast cancer cell line SUM149PT treated in parallel with the PARP inhibitors olaparib, talazoparib (BMN673) or AZD2461 (Fig 1a, b, Supplementary  Fig 1a-c). In addition to identifying the known resistance genes TP53BP1, RIF1 and MAD2L2 whose products form a complex21, we identified several new suppressor candidates (Supplementary Table 1, Supplementary Fig 1d-e). These included DYNLL1, a known 53BP1 interaction partner22, and TEN1, a component of the CST telomere-capping complex (CTC1/STN1/TEN1) that also promotes telomere DNA replication23. In our ensuing studies, however, we focused on the uncharacterised proteins FAM35A and C20orf196 that collectively received the highest scores from our screens (Fig 1b and  Supplementary Table 1). Thus, by carrying out short-interfering RNA (siRNA) mediated mRNA silencing in non-transformed, hTERT immortalized human RPE1 cells ( Supplementary Fig 1f), we established that, as for 53BP1 loss7, depletion of FAM35A or C20orf196 markedly suppressed PARP-inhibitor sensitivity caused by BRCA1 inactivation while having no discernible effect in BRCA1-proficient cells (Fig 1c, Supplementary Fig  1g). This conclusion was independently confirmed by de novo CRISPR-Cas9 gene editing, with FAM35A or C20orf196 inactivation alleviating the olaparib hypersensitivity of BRCA1-deficient cells in a manner counteracted by re-introduction of wild-type FAM35A or C20orf196 (Fig 1d-

The FAM35A/C20orf196 complex interacts with and acts downstream of 53BP1/RIF1/ MAD2L2
Sequence analyses indicated that FAM35A and C20orf196 are well conserved in vertebrates. Moreover, structure prediction modelling (RaptorX; http://raptorx.uchicago.edu/) revealed that FAM35A harbours a disordered N-terminus and an ordered C-terminal region containing three OB folds, with the last C-terminal OB fold/FAM domain containing a CXXC-type zinc finger motif (Fig 2a). Notably, this organization is highly similar to those of the RPA1 subunit of ssDNA binding protein RPA and the CTC1 subunit of the CST complex that also binds ssDNA23. In this regard, we noted that while the C20orf196 Nterminus (residues 1-70) is predicted to be intrinsically disordered, its C-terminal part is more structured and may harbour one-or two-winged helix (WH) domains (Fig 2a) similar to those in the yeast CST subunit Stn123, 24, suggesting that C20orf196 and Stn1 might play analogous or complementary roles.
Through combining cellular co-localization and co-immunoprecipitation experiments, we established that FAM35A and C20orf196 directly interact in a manner that is mainly, but not exclusively, mediated by the FAM35A OB3/FAM domain (Fig 2b-c, Supplementary Fig 2ab). Because loss of FAM35A or C20orf196 had similar effects to loss of 53BP1/RIF1/ MAD2L2 in BRCA1-deficient cells, we tested for possible interactions between these factors. Thus, via co-immunoprecipitation and mass spectrometry (MS) studies, we found that both C20orf196 and FAM35A interact with MAD2L2, the most distal factor of the 53BP1/RIF1/MAD2L2 axis mediating PARP-inhibitor sensitivity in BRCA1-deficient cells6-13 (Fig 2d, Supplementary Fig 2c).
Many DDR proteins accumulate at DSB sites within ionizing radiation (IR)-induced nuclear foci (IRIF)5. We established that both FAM35A and C20orf196 formed IRIF, and by livecell imaging studies found that the proteins were also recruited to localised DNA-damage sites induced by laser micro-irradiation ( Supplementary Fig 2d). Furthermore, we determined by confocal and super-resolution microscopy that FAM35A co-localised with the established DSB markers phosphorylated histone H2AFX25 (γH2AX) and 53BP126 (Fig  2e-f, Supplementary Fig 2e). Notably, siRNA/shRNA-depletion experiments established that while 53BP1 IRIF and MAD2L2 levels and IRIF were not significantly impaired by FAM35A or C20orf196 depletion ( Supplementary Fig 2f-h), IRIF formation by FAM35A and C20orf196 required 53BP1, RIF1 and MAD2L2 but not PTIP (Fig 2e and  Supplementary Fig 3a-c; note that total levels of GFP-tagged FAM35A/C20orf196 were minimally affected by 53BP1/RIF1/MAD2L2 depletion). We also established that C20orf196 IRIF were almost totally abrogated by FAM35A depletion, while C20orf196 depletion reduced but did not abolish FAM35A IRIF (Fig 2e). In addition, FAM35A formed nuclear foci when cells were treated with the DNA topoisomerase I inhibitor camptothecin (CPT; Supplementary Fig 3d). Significantly, the FAM35A N-terminus was necessary and Europe PMC Funders Author Manuscripts sufficient for its IRIF formation, these IRIF depended on 53BP1, RIF1, MAD2L2 and C20orf196, and this region could be co-immunoprecipitated with MAD2L2 (Fig 2f-g, Supplementary Fig 3e-f). Collectively, these findings indicated that FAM35A and C20orf196 act as downstream components of the 53BP1/RIF1/MAD2L2 molecular assembly21 at DSB sites.

FAM35A and C20orf196 promote NHEJ
Since 53BP1, RIF1 and MAD2L2 promote NHEJ6-13, we tested whether FAM35A and C20orf196 fulfil a similar role. Indeed, as for depletion of the NHEJ factor XRCC4, siRNA depletion of 53BP1, FAM35A or C20orf196 impaired NHEJ as measured by random integration of plasmid DNA into chromosomes27 (Fig 3a). In addition, FAM35A or C20orf196 depletion conferred IR hypersensitivity to both human and mouse cells (Fig 3b  and Supplementary Fig 4a). 53BP1 and associated factors promote NHEJ-mediated classswitch recombination (CSR) at the immunoglobulin heavy-chain locus, a process that allows B-lymphocytes to change antibody production from one type to another28.

FAM35A and C20orf196 antagonise DNA-end resection
To explore FAM35A and C20orf196 function further, we carried out assays in mouse cells harbouring a temperature-sensitive allele of the telomere-associated factor TRF2 (TRF2ts). TRF2ts inactivation at higher temperatures results in de-protected chromosome ends and causes NHEJ-mediated telomere fusions (Fig 4a)6, 33. Strikingly, through use of shorthairpin RNA (shRNA) mediated mRNA silencing, we found that such chromosome fusions were diminished by FAM35A or C20orf196 depletion like that elicited by MAD2L2 depletion (Fig 4b, Supplementary Fig 5a-b).
The impacts of 53BP1, RIF1 or MAD2L2 depletion in the TRF2ts system are connected to these factors counteracting DSB resection6-13, 34. We thus explored whether FAM35A and C20orf196 might also have this function. Indeed, as for 53BP1/RIF1/MAD2L2 inactivation6-13, 35, loss of FAM35A or C20orf196 in human cells enhanced DSBresection as measured by RPA and ssDNA staining intensity in pre-extracted nuclei after treatment with camptothecin (Fig 4c-e; RPA1 kinetics at DNA damage sites induced by laser micro-irradiation were not altered by FAM35A or C20orf196 depletion, Supplementary Fig  5c). In line with this resection being mediated by canonical pathways, it was diminished by depletion of the resection promoting factors RBBP8 (CtIP) or BLM (Fig 4f). Furthermore, we established that recruitment of BLM to sites of laser micro-irradiation was enhanced by FAM35A or C20orf196 depletion (Fig 4g). Similarly, as shown for 53BP1 depletion in mouse cells6, FAM35A or C20orf196 depletion in such cells led to higher levels of the DNA-end resection marker, Ser4/8 phosphorylated RPA2, after IR treatment (Fig 4d).
Together with our other findings, these data established FAM35A and C20orf196 as crucial components of 53BP1/RIF1/MAD2L2-mediated chromosomal NHEJ, and suggested that their pro-NHEJ function is connected to limiting DSB resection.

The FAM35A OB fold region interacts with ssDNA and promotes IR survival
Consistent with our prediction of structural similarity between FAM35A and RPA1, the FAM35A C-terminus could be retrieved from cell extracts via interaction with a ssDNA oligonucleotide (Fig 5a). Sequence alignment to RPA1 and structural modelling of FAM35A identified two Trp (W) residues predicted to be at the protein-ssDNA interface, based on analogous residues critical for RPA binding to ssDNA (Fig 5b, Supplementary Fig 5d). In accord with this prediction, we found via electrophoretic gel-mobility shift assays (EMSAs) that the bacterially-expressed, purified FAM35A C-terminal region bound preferentially to ssDNA rather than double-stranded DNA (Fig 5c, Supplementary Fig 5e), and ssDNA binding was reduced when the two Trp residues were mutated to Ala (W489/W640A; Fig  5c). Furthermore, while full-length FAM35A bearing these mutations (FAM35A W489/W640A ) still interacted with C20orf196 ( Supplementary Fig 5f) and formed IRIF in cells, these IRIF were consistently less pronounced than those of the wild-type FAM35A protein (Fig 5d). This suggested that following IRIF recruitment via its N-terminal region, the FAM35A C-terminal ssDNA binding region may allow further FAM35A recruitment, retention and/or stabilization. In addition, unlike the wild-type protein, FAM35A W489/W640A did not confer significant IR resistance when reintroduced into FAM35A null cells (Fig 5e). In parallel studies, expression of the FAM35A C-terminus did not complement the IR hypersensitivity of FAM35A null cells. Moreover, expression of the FAM35A N-terminus rendered cells IR hypersensitive irrespective of whether they expressed endogenous FAM35A, implying that the N-terminal IRIF-forming domain of FAM35A may have a dominant-negative effect on NHEJ (Fig 5f; overexpression of these FAM35A derivatives did not affect olaparib sensitivity in a wild-type background, Supplementary Fig 5g).

FAM35A and C20orf196 antagonise HR in BRCA1-deficient cells
PARP inhibitors generate replication-associated DNA lesions that require BRCA1-mediated HR for their effective repair19, and loss of 53BP1/RIF1/MAD2L2 partly restores the ability of BRCA1-deficient cells to repair such lesions6-13. This has led to a model in which BRCA1 and 53BP1/RIF1/MAD2L2 play antagonistic roles in channelling DSBs towards HR or NHEJ, respectively. We thus speculated that BRCA1 might antagonise FAM35A/ C20orf196 action. Accordingly, both FAM35A and C20orf196 IRIF, but not 53BP1 IRIF, were significantly enhanced in number and intensity upon BRCA1 but not BRCA2 depletion (Fig 6a- Supplementary Fig 6a-b).
Collectively, our results suggested that FAM35A/C20orf196 act at the interface between the opposing functions of BRCA1 and 53BP1/RIF1/MAD2L2 to regulate DSB-repair pathway choice. While this action could operate at least in part through control of DSB resection, which is misregulated and of slower kinetics in BRCA1-deficient cells11, 36, we reasoned that FAM35A/C20orf196 might also contribute to the severe defect in BRCA2-mediated RAD51 loading at DNA-damage sites in BRCA1-deficient cells11, 35, 37. Indeed, as for 53BP1 inactivation, loss of FAM35A or C20orf196 restored RAD51 IRIF formation in BRCA1-null cells (Fig 6c). While exploring the mechanism for this effect, we found elevated resection levels in FAM35A and C20orf196 knockout cells, as measured by RPA recruitment at DNA-damage sites, were still maintained in the absence of BRCA1 ( Supplementary Fig 6c-d). Furthermore, FAM35A/C20orf196 depletion also alleviated the impaired recruitment of BRCA2 to DNA-damage sites in BRCA1-deficient cells (Fig 6d,  Supplementary Fig 6e). Accordingly, studies with a cell-based chromosomal traffic light reporter (TLR) HR system 38, 39 established that FAM35A or C20orf196 depletion in BRCA1-deficient cells restored HR to levels similar to those acquired upon 53BP1 depletion in this setting (Fig 6e, Supplementary 6f). In addition, removing FAM35A or C20orf196 rescued the spontaneous genomic instability phenotype of BRCA1 knockout cells (Fig 6f).
Building on our findings that the FAM35A N-terminal region largely mediates its localisation to IRIF (Fig 2f, Supplementary Fig 3e-f), introducing the FAM35A N-terminus, but not the C-terminus, enhanced PARP-inhibitor sensitivity of BRCA1/FAM35A null cells (Fig 6g, Supplementary Fig 6g). Furthermore, FAM35A inactivation was epistatic to 53BP1 inactivation in relation to conferring PARP-inhibitor resistance in BRCA1 knockout cells ( Supplementary Fig 6h). Considering our findings together, we propose that C20orf196 and FAM35A be named SHLD1 and SHLD2, respectively, or collectively as the "Shieldin complex" because it shields DSBs from inappropriate activities and promotes appropriate modes of DSB repair.

FAM35A/C20orf196 loss correlates with PARP inhibitor resistance in cancers
Having identified SHLD1 C20orf196 and SHLD2 FAM35A as mediating the PARP-inhibitor sensitivity of a BRCA1-deficient breast cancer cell line, we speculated that this might also apply in more physiological settings. Consequently, we employed a patient-derived xenograft (PDX) model of BRCA1-deficient breast cancer propagated in mice in the presence (cohort 2) or absence (cohort 1) of olaparib (Fig 7a). The resistant tumour was further serially passaged into new hosts that were treated in the presence (cohort 4) or absence (cohort 3) of olaparib to confirm and sustain drug resistance (Fig 7a; see also Supplementary Fig 7a). The tumours were then harvested and subjected to whole-genome RNA-sequencing. Notably, our analyses revealed that in contrast to the other cohorts, nearly all resistant tumours after chronic olaparib treatment (cohort 4) correlated with reduced mRNA expression of SHLD1 C20orf196 , SHLD2 FAM35A , 53BP1 and/or PARP1 (Fig 7a; each heatmap column represents one tumour/mouse sample). Because this tumour model is polyclonal40, our data suggested that olaparib resistance mechanisms might arise through parallel evolutionary trajectories converging on loss of Shieldin activity. Furthermore, when we stratified a cohort of BRCA1-deficient PDX tumours by SHLD1/2 expression, ensuing analyses indicated that low SHLD1 C20orf196 transcript levels correlated with intrinsic olaparib resistance (Fig 7b). One of the olaparib resistant models (PDX127) demonstrated concomitant loss of both SHLD1 C20orf196 and SHLD2 FAM35A , while two other resistant models exhibiting normal SHLD1/2 transcript levels harboured deleterious 53BP1 mutations. Notably, several of these resistant PDX models also display BRCA1 nuclear foci41, suggesting the presence of multiple mechanisms of resistance, due to tumour heterogeneity and/or mechanistic cooperation.
Finally, we found that in contrast to 53BP1 deficiency42, SHLD1 C20orf196 or SHLD2 FAM35A loss increased the sensitivity of BRCA1-proficient and BRCA1-null cells to IR, and even more markedly enhanced their sensitivity to the DNA crosslinking agent cisplatin (Fig 7c-d, Supplementary Fig 7b-c). Furthermore, enhanced cisplatin sensitivity upon SHLD1 C20orf196 or SHLD2 FAM35A inactivation was associated with increased DNA-damage focus formation by the FANCD2 protein that is involved in detection and repair of DNA crosslinks (Fig 7e,  Supplementary Fig 7d). These findings therefore suggested that, if loss/reduced expression of SHLD1/2 occurs in patients, it may provide collateral therapeutic vulnerabilities that could be exploited clinically.

Discussion
Over the past two decades, it has become evident that eukaryotic cells have evolved multiple mechanisms of DNA DSB repair that are regulated in complex and sophisticated ways to optimise genome stability. In particular, much attention has focused on how cells strategically employ the two prime modes of DSB repair -NHEJ and HR -which antagonise one another, operate optimally in different contexts and whose relative usage is regulated by factors such as chromatin structure and cell-cycle stage. In addition to being of academic interest, work on such subjects is also of clinical relevance, particularly in cancer therapy where DSB-inducing chemotherapeutic agents are frequently used, and molecularlytargeted drugs such as PARP inhibitors are being increasingly employed in specific settings. Intrinsic or arising PARP-inhibitor resistance in patients with BRCA1/2 mutations is nevertheless an increasing clinical problem. Using whole-genome CRISPR-Cas9 syntheticviability/resistance screens, we have uncovered two, previously uncharacterised proteins -SHLD1 C20orf196 and SHLD2 FAM35A -whose loss mediates PARP-inhibitor resistance and which we have shown act as the most distal factors of the 53BP1/RIF1/MAD2L2 molecular axis to promote NHEJ and restrict HR in BRCA1-deficient settings. Our screens have also identified additional candidate PARP-inhibitor resistance factors that await validation in future studies.
Mechanistically, we have shown that SHLD1 C20orf196 and SHLD2 FAM35A form a complex, termed Shieldin, with SHLD1 C20orf196 recruitment to DNA-damage sites via its interactions with SHLD2 FAM35A and other factors, and SHLD2 FAM35A interacting with single-stranded DNA via its C-terminal OB fold region. Moreover, we have established that SHLD1 C20orf196 and SHLD2 FAM35A promote NHEJ in a manner that may be mediated via their effects on restricting DNA-end resection, and serve as a barrier to HR by antagonising the replacement of RPA with BRCA2/RAD51 on resected ssDNA in a manner counteracted by BRCA1. Our work is in line with a recent report43, that independently identified SHLD1 C20orf196 and SHLD2 FAM35A as NHEJ-promoting factors and antagonists of HR in BRCA1-defective cells. This study also identified a third component, RINN1/SHLD3 CTC-534A2.2 that is proposed to serve as a molecular bridge from RIF1 to MAD2L2 and SHLD1/2.
While it seems possible that Shieldin loss contributes to HR restoration in BRCA1-deficient cells through effects on both resection and on BRCA2/RAD51 loading, the relative importance of these mechanisms needs further investigation. We note that more extensive and possibly faster resection in the absence of Shieldin might enhance BRCA2/RAD51 loading. Alternatively, or in addition, Shieldin might serve as a physical barrier to BRCA2/ RAD51 loading at dsDNA/ssDNA junctions in BRCA1-deficient cells -perhaps through it being tethered to DSB-flanking chromatin via the 53BP1/RIF1/MAD2L2 complex allowing interactions between the C-terminus of distal SHLD2 FAM35A and ssDNA (see Fig 7f for a model, and Supplementary Fig 7e for SHLD2 FAM35A domain function summary). Nevertheless, we found that overexpression of the SHLD2 FAM35A N-but not the C-terminus confers olaparib hypersensitivity to BRCA1/FAM35A double knockout cells, suggesting that at least in this context, chromatin binding by SHLD2 FAM35A plays a dominant role in restricting HR. By contrast, we found that both the SHLD2 FAM35A N-and C-termini are important for IR resistance (in BRCA1-proficient cells). As IR sensitivity in Shieldindeficient cells likely reflects impaired NHEJ, we speculate that Shieldin potentiates NHEJ by restricting DSB resection as well as by assembling with other NHEJ-promoting factors to tether DSB ends together to facilitate their juxtaposition and repair.
Notably, we have found that SHLD1 C20orf196 or SHLD2 FAM35A inactivation confers enhanced cisplatin sensitivity to BRCA1-null or BRCA1-proficient cells. Such sensitivity probably does not reflect the roles for Shieldin in promoting NHEJ, restricting DNA-end resection or antagonising BRCA1-mediated BRCA2/RAD51 loading because, in our hands, 53BP1 loss does not have pronounced effects on cisplatin sensitivity. Intra-strand DNA crosslinks (ICLs) generated by cisplatin and other compounds are detected and repaired by the Fanconi anemia (FA) pathway, with a key FA protein being FANCD2, which forms foci at sites of these lesions44. We have observed that following cisplatin treatment, FANCD2 foci were more pronounced in cells in which SHLD1 C20orf196 or SHLD2 FAM35A was inactivated. It will thus be of interest to determine if Shieldin -like MAD2L2, which (with REV3L) functions as a regulatory subunit of the trans-lesion DNA synthesis (TLS) polymerase Pol zeta, and whose biallelic inactivation has been associated with FA45-47might also promote ICL repair via TLS mechanisms.
Finally, based on our findings, it will be interesting to evaluate SHLD1/2 expression in tumour biopsies from patients, establish whether this information can be used in patient stratification for PARP-inhibitor therapies, and determine whether SHLD1/2 expression changes arise in patients whose BRCA1-deficient cancers develop resistance after PARPinhibitor treatment. In this regard, we note that if down-regulation of Shieldin components in BRCA1-deficient cancers does confer clinical resistance, this might allow alternative treatments, such as ones based on platinum compounds.

Generation of human stable cell lines and knockouts
U2OS Trex or RPE1 p53 null FRT-derived cells stably expressing inducible GFP-tagged constructs, were generated by transfection of pcDNA5/FRT/TO-neo containing the GFPtagged construct and pOG44 (1:4 respectively). Selection began at 48 h using 0.5 mg/ml G418 (Invitrogen). Knockouts were generated in RPE1 p53 null cells by transfecting an 'All-in-one' plasmid48. Single-cell sorting by GFP expression was done using MoFlo (Beckman Coulter). Single clones were expanded, genomic DNA extracted and screened by PCR, TOPO-cloning and sequencing. Validated mouse Embryonic Stem Cell (mESC) knockouts of Fam35a and C20orf196 were obtained from Haplobank (www.haplobank.at).

Plasmids and cloning
See Supplementary

Random plasmid integration assay
Performed as previously described27.

DNA-damage induction using chemical agents, ionizing radiation and laser microirradiation
Performed as previously described39.

FRAP and association kinetics
Performed as previously described50.

TLR assays
The Traffic Light Reporter (TLR) assay and the constructs used herein have been described in detail previously38, 39.

Cell-cycle profiling
Performed as previously described39.

Whole cell extracts and immunoblotting
Were performed as previously described39. For detection of phospho-RPA (pS4/S8-RPA2), lysates were prepared by scraping cells in 2xSDS buffer followed by SDS-PAGE using 4-12% Bis-Tris gel (Invitrogen), and immunoblotting was done using SuperSignal West Pico PLUS (Thermo Scientific). IRDye800CW-and IRDye680-labelled secondary antibody was used for detection on the Odyssey Infrared imager (LI-COR). Quantification of blots was performed using ImageJ. All protein concentrations were determined using a BCA assay (Pierce). All antibodies are listed in Supplementary Table 2.

DNA pulldown experiments
Procedures were described in detail in39 using oligos with the sequence:

Recombinant protein purifications and Electrophoretic Mobility Shift Assays (EMSAs)
Wild-type and mutant FAM35A C-terminal domains were purified using the same method. DNA oligo F was used as ssDNA for EMSA. Equal volumes of DNA oligo F and R were mixed and annealed (heated to 95°C for 2min and cooled to 25°C over 45min) to generate dsDNA. Each 20 μl of EMSA reaction contained 10 nM of ssDNA/dsDNA incubated with different concentrations of proteins in 20 mM Tris-HCl pH 7.5, 50 mM KCl, 5% (v/v) glycerol, 100 μM DTT, 10 μg/ml BSA. Samples were incubated at 37°C for 15min and applied onto a 5% polyacrylamide native gel in 0.5xTBE buffer for electrophoresis at 4°C. DNA was visualized by Typhoon 9000 (GE Healthcare).

GFP-Trap pull down for mass spectrometry
HEK293T cells were cultured in SILAC media containing either L-arginine and L-lysine, or L-arginine [ 13 C 6 , 15 N4] and L-lysine [ 13 C 6 , 15 N 2 ] (Cambridge Isotope Laboratories) as described previously51. Cells were lysed 48h post-transfection in modified RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% sodium deoxycholate) supplemented with protease, phosphatase inhibitors and N-ethylmaleimide. Lysates were cleared by centrifugation at 16,000×g for 15min at 4°C and protein concentrations were estimated using QuickStart Bradford Protein assay (BioRad). Per SILAC condition, 20 µl of pre-equilibrated GFP-Trap-A beads (ChromoTek) were added to 2 mg of lysate and incubated 1h at 4°C rotating, followed by 3x washes with modified RIPA buffer. Bound proteins were eluted in NuPAGE® LDS Sample Buffer (Life Technologies) supplemented with 1 mM dithiothreitol, heated at 70 ºC for 10min and alkylated with 5.5 mM chloroacetamide at RT. Samples were loaded onto 4-12% gradient SDS-PAGE gels, proteins were stained using the Colloidal Blue Staining Kit (Life Technologies) and digested in-gel using trypsin. Peptides were extracted from the gel and desalted on reversed phase C18 StageTips52.

Mass spectrometry analysis
Peptide fractions were analyzed on a quadrupole Orbitrap mass spectrometer (Q Exactive Plus, Thermo Scientific) equipped with an EASY-nLC 1000 (Thermo Scientific) as described53. Peptide samples were loaded onto C18 reversed phase columns and eluted with a linear gradient from 8 to 40% acetonitrile containing 0.1% formic acid for 2h. The mass spectrometer was operated in data dependent mode, automatically switching between MS and MS2 acquisition. Survey full-scan MS spectra (m/z 300-1650) were acquired in the Orbitrap. The ten most intense ions were sequentially isolated and fragmented by HCD54. Fragment spectra were acquired in the Orbitrap mass analyzer. Raw data files were analyzed using MaxQuant (development version 1.5.2.8)55. Parent ion and MS2 spectra were searched against a database containing 92,578 human protein sequences obtained from the UniProtKB released in December 2016 using Andromeda search engine56. Spectra were searched with a mass tolerance of 6 ppm in MS mode, 20 ppm in HCD MS2 mode, strict trypsin specificity and allowing up to three miscleavages. Cysteine carbamidomethylation was searched as a fixed modification, whereas protein N-terminal acetylation, methionine oxidation, n-ethylmaleimide modification of cysteines were searched as variable modifications. The dataset was filtered based on posterior error probability to arrive at a false discovery rate below 1% estimated using a target-decoy approach57.

Immunofluorescence and microscopy imaging
Confocal imaging for γH2AX, RAD51, RPA, ssDNA (BrdU), BLM, BRCA2, FANCD2, Cyclin A and GFP (FAM35A and C20orf196) was performed as described in39, for RAD51 and Cyclin A the pre-extraction step was omitted and cells were permeabilised for 15min in Europe PMC Funders Author Manuscripts 0.2% Triton X-100 (Sigma) in PBS after fixation. Super-resolution images were acquired using a Deltavision OMX 3D-SIM System V3 BLAZE (Applied Precision, a GE Healthcare company) equipped with 3 sCMOS cameras, 405, 488, 592.5nm diode laser illumination, an Olympus Plan Apo N 60x 1.42NA oil objective, and standard excitation and emission filter sets. Imaging of each channel was done sequentially using three angles and five phase shifts of the illumination pattern as described58. Sections were acquired at 0.125 μm z steps. Raw OMX data was reconstructed and channel registered in SoftWoRx software version 6.5.2 (Applied Precision, a GE Healthcare company). Voxelwise nearest-neighbour distances were measured for GFP-FAM35A signal relative to 53BP1 signal using a custom script (Butler R) for Fiji (https://github.com/gurdon-institute/OMX-Spatial-Analysis). The script maps signal volumes using Kapur's maximum entropy thresholding method59 and measures distances using the exact signed 3D Euclidean distance transform with internal distances set to zero for display on the histogram. For all images, scale bars = 10μm.

Multiplex fluorescence in situ hybridisation (M-FISH)
Human 24-colour multiplex FISH (M-FISH) probe preparation and slides treatments followed60. For each human cell sample, 10-30 metaphases were karyotyped based on the M-FISH classification and DAPI-banding pattern. FISH on metaphases spreads using BAC probes was performed as previously described61 and counted manually. For class-switch recombination (CSR) assays, DNA FISH on metaphases spreads was performed as previously described61 and counted manually. At least 470 metaphases were evaluated per genotype, using at least 2 independent clones for each condition. For telomere uncapping, cell harvesting, preparation of metaphase spreads and telomere FISH with an Alexa488-(CCCTAA repeat) peptide nucleic acid custom probe (PN-TC060-005, Panagene/ Eurogentec), metaphase chromosome analysis was done as described previously6. These data represent 2 independent experiments, ≥1300 chromosomes for each condition, counted manually after blinding the genotypes.

Telomere fusion assays: MEFs viral transduction
Cells were transduced as before49 with pLKO-puro shRNA lentiviruses obtained from the MISSION shRNA library (Sigma), against mouse genes as described or left untransduced ('empty'). Assessment of telomere NHEJ. TRF2ts MEFs were grown for 24h at the nonpermissive temperature of 39°C to inactivate TRF2 and induce NHEJ-dependent chromosome end-to-end fusions because of telomere uncapping.

Patient-derived tumour xenografts
PDXs were generated and established from consented breast or ovarian cancer patients' samples as previously described40. Counts were assigned to genome features using featureCounts (v1.5.2), whereby the alignment score is used to discern accurately between reads sourced from human and mouse68. Counts from multiple sequencing runs were merged and then normalised using the edgeR package69, 70.

Code availability
A custom FIJI script used in OMX analysis can be found at https://github.com/gurdoninstitute/OMX-Spatial-Analysis.

Statistics and Reproducibility
Unless stated otherwise Prism v7.0b (GraphPad Software) was used to generate graphs, perform statistical tests and calculate p values. Error bars, statistical tests and number of independent repeats (n) are indicated in figure legends with statistical source data including the precise p values provided in Supplementary Table 5. Statistical tests included two-tailed Student t-tests, Fisher's Exact test, and one-way analysis of variance (ANOVA), the latter all being corrected as recommended for multiple comparisons. Microscopy image analyses were performed using ImageJ/FIJI or Volocity 6.3 (Perkin-Elmer). CRISPR screens were performed with three clones per drug treatment. Mass spectrometry of GFP-FAM35A and GFP-C20orf196 was performed in two independent experiments. RNA-sequencing was performed as three replicates for each trial arm, due to sequencing across multiple lanes (which were merged prior to any further analysis). This was performed for the following number of independent biological samples: six PDXs in cohort 1, five PDXs in cohort 2, seven PDXs in cohort 3 and eight PDXs in cohort 4. For the SHLD1 high and low expression cohorts 12 and 4 independent PDXs were evaluated respectively. All immunofluorescence assay quantification data represent means ±SEM's of 3 independent biological repeats and n≥30 cells per condition unless otherwise specified. All immunoblots are representative of two independent experiments with unprocessed scans of immunoblots shown in Supplementary Fig 8.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.  a, Random plasmid integration assay. b, FAM35Ako and C20orf196ko cells were treated with IR and analysed for clonogenic survival, right panel shows AUC. a-b, Bars represent mean ± SEM, one-way Anova; n=3 independent experiments, except C20orf196ko in b (n=4), with individual data points plotted over bars; statistical source data can be found in Supplementary Table 5. c, Schematic representation of class-switch recombination and chromosomal instability in murine IgM + B cells (germline configuration with C μ

Europe PMC Funders Author Manuscripts
Europe PMC Funders Author Manuscripts GFP-FAM35A W489/W640A fails to efficiently form IRIF (12 h after 5Gy of IR). Scale bar 10µm. Representative images from 3 independent experiments. e, FAM35Ako RPE1 cells complemented with FAM35A derivatives in clonogenic survival assays; right panel shows AUC. f, Overexpression of FAM35A N-terminus but not C-terminus or full-length FAM35A sensitises wild-type cells to IR in clonogenic assays; right panel shows AUC. e-f, Bars represent mean ± SEM, one-way Anova; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant (p≥0.05); n=3 independent experiments except group 2 and 4 in e (n=2), with individual data points plotted over bars; statistical source data including the precise p values can be found in Supplementary Table 5. All immunoblots are representative of two independent experiments; unprocessed scans of immunoblots are shown in Supplementary  Fig 8. Dev et al.