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Transmembrane nuclease NUMEN/ENDOD1 regulates DNA repair pathway choice at the nuclear periphery

Nature Cell Biology volume 25, pages 1004–1016 (2023)Cite this article

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An Author Correction to this article was published on 01 August 2023

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Abstract

Proper repair of DNA damage lesions is essential to maintaining genome integrity and preventing the development of human diseases, including cancer. Increasing evidence suggests the importance of the nuclear envelope in the spatial regulation of DNA repair, although the mechanisms of such regulatory processes remain poorly defined. Through a genome-wide synthetic viability screen for PARP-inhibitor resistance using an inducible CRISPR–Cas9 platform and BRCA1-deficient breast cancer cells, we identified a transmembrane nuclease (renamed NUMEN) that could facilitate compartmentalized and non-homologous end joining-dependent repair of double-stranded DNA breaks at the nuclear periphery. Collectively, our data demonstrate that NUMEN generates short 5′ overhangs through its endonuclease and 3′→5′ exonuclease activities, promotes the repair of DNA lesions—including heterochromatic lamina-associated domain breaks as well as deprotected telomeres—and functions as a downstream effector of DNA-dependent protein kinase catalytic subunit. These findings underline the role of NUMEN as a key player in DNA repair pathway choice and genome-stability maintenance, and have implications for ongoing research into the development and treatment of genome instability disorders.

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Fig. 1: A genome-wide inducible CRISPR screen identifies NUMEN as a regulator of PARPi resistance in BRCA1-deficient tumour cells.
Fig. 2: NUMEN exhibits endonuclease as well as 3′→5′ exonuclease activities and generates DNA termini preferred by NHEJ repair.
Fig. 3: NUMEN antagonizes the formation of 3′ overhang in cells and regulates NHEJ/HR repair pathway choice.
Fig. 4: NUMEN is an important regulator of NHEJ and functions downstream of DNA-PKcs.
Fig. 5: NUMEN localizes to the nuclear membrane and spatially controls DNA damage response.
Fig. 6: NUMEN could help resolve perinuclear heterochromatic DSBs.
Fig. 7: NUMEN contributes to the maintenance of genomic stability.

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Data availability

Mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD041562. Sequencing data from the CRISPR screen have been deposited in the NCBI Sequence Read Archive under the accession number PRJNA933943. The human breast cancer and pan-cancer data were derived from the TCGA Research Network (http://cancergenome.nih.gov/). The dataset derived from this resource that supports the findings of this study is available at https://github.com/Chenlt5/paper. The next-generation sequencing and mass spectrometric data have been deposited to the Figshare database and are available at https://doi.org/10.6084/m9.figshare.21592878. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

Custom codes are available at https://github.com/Chenlt5/paper.

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Acknowledgements

We thank J. Huang for providing the DR-GFP and EJ5-GFP U2OS cell lines. We also thank H. Hu for providing SUM149 cells. Our work was supported by grants from the National Key Research and Development Program of China (grant no. 2018YFA0107003), the National Natural Science Foundation of China (grant nos 31930058, 32170757, 31871479 and 92249304) and the Guangdong Basic and Applied Basic Research Foundation (grant no. 2020B1515020044).

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Author notes

  1. These authors contributed equally: Bohong Chen, Tianyu Ge.

Authors and Affiliations

  1. MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou, China

    Bohong Chen, Tianyu Ge, Meiqi Jian, Liutao Chen, Zhengwen Fang, Zibin He, Chengjing Huang, Yan An, Shanshan Yin, Yuanyan Xiong, JingKai Zhang, Ruofei Li, Miaoman Ye, Yubing Li, Feng Liu, Wenbin Ma & Zhou Songyang

  2. Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China

    Zhou Songyang

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Contributions

B.C. and Z.S. conceptualized, designed and oversaw the project. B.C. performed the CRISPR–Cas9 screen. B.C. and T.G. performed all cell biology and microscopy experiments, with help from M.J., C.H., Y.A., S.Y., M.Y. and Y.L. B.C., L.C. and Z.H. performed protein purification and in vitro nucleolytic activity assays. R.L. and F.L. conducted the BioID experiments. Z.F., Y.X. and J.Z. conducted the bioinformatic analysis. B.C. and Z.S. wrote the manuscript, and W.M. helped with proofreading and editing.

Corresponding author

Correspondence to Zhou Songyang.

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Extended data

Extended Data Fig. 1 Identification of PARPi resistance genes through a genome-wide CRISPR–Cas9 screen.

a, iCas9 MDA-MB-231 cells were cultured in doxycycline (1 μg ml−1) for 3 d before single clone isolation. Individual clones were harvested for immunoblotting using an anti-Flag to detect Flag-tagged Cas9. Uninduced cells were used as a control. GAPDH served as the loading control. Clone 1 was chosen for subsequent experiments. b, iCas9 MDA-MB-231 cells (clone 1) were transduced with lentiviruses encoding paired BRCA1-specific gRNAs and then induced with doxycycline (1 μg ml−1) for 7 d before single clone isolation. Immunofluorescence analysis (left) and western blotting (right) of parental (iCas9 MDA-MB-231) and KO clone 7 (iCas9 MDA-MB-231 BRCA1−/−) cells with anti-BRCA1 are shown here. Clone 7 was used for subsequent experiments. Scale bar, 20 μm. c, Parental (WT) and BRCA1-KO (BRCA1−/−) iCas9 MDA-MB-231 cells were cultured in olaparib for 14 d at the indicated concentrations before being examined in clonogenic survival assays. d, BRCA1−/− iCas9 MDA-MB-231 cells stably expressing control (sgVector) or sgRNAs targeting REV7 or 53BP1 were cultured in doxycycline (1 μg ml−1) for 7 d. The cells were then treated with olaparib for 14 d at the indicated concentrations before clonogenic survival assays. e, Density plots showing the distribution of sgRNAs with (Day 30) and without (Day 0) olaparib treatment from the inducible CRISPR screen. f, A sample list of DDR factors and PARPi-resistance genes among high-confidence hits. Interactions from BioGRID are shown on the right. Source numerical data and unprocessed blots are provided.

Source data

Extended Data Fig. 2 Loss of NUMEN results in PARPi resistance and sensitivity to different DNA lesions.

a, Phylogenetic tree derived from nucleotide sequences encoding NUMEN in metazoan. Phylogeny was inferred using the randomized axelerated maximum likelihood (RAxML) method with the GTRGAMMA model. Circle sizes correspond to bootstrap values from 500 replications. Evolutionary analyses were conducted in MegAlign and the tree was edited with iTOL. The scale bar represents the number of nucleotide substitutions per site. b, BRCA1−/− iCas9 MDA-MB-231 cells (Parental) were used to KO NUMEN (NUMEN−/−). GFP-tagged NUMEN (GFP–NUMEN) was then stably expressed in NUMEN and BRCA1-DKO cells. The expression of endogenous and exogenous NUMEN was confirmed by western blotting as indicated. c, A BRCA1-KO (BRCA1−/−) iCas9 HeLa cell line was similarly isolated as described above, and examined by immunofluorescence (left) and western blotting (right) for BRCA1 expression. Parental iCas9 HeLa cells served as controls. Scale bar, 20 μm. d,e, The BRCA1−/− HeLa cells from above (Parental) were used to KO NUMEN. GFP–NUMEN was then expressed in these NUMEN and BRCA1-DKO cells. NUMEN KO and rescue expression was confirmed by western blotting as indicated (d). The cells were then treated with the indicated concentrations of olaparib for 14 d before clonogenic survival assays (e). f, The BRCA1-mutant cell lines SUM149, MDA-MB-436 and HCC1937 were knocked out for NUMEN and validated by western blotting as indicated. GAPDH was used as a loading control. g,h, Parental (WT) and NUMEN-KO U2OS (g) and HeLa (h) cells were western blotted as indicated. GAPDH was used as a loading control. i–k, WT and NUMEN−/− HeLa cells were subjected to the indicated dosages of IR (i), zeocin (j) and cisplatin (k) treatments before survival assessment. The cells were assessed 7 d after the treatments. Data are the mean ± s.d. of three independent experiments. Statistical analysis was performed using a two-way ANOVA. Source numerical data and unprocessed blots are provided.

Source data

Extended Data Fig. 3 NUMEN functions as a structure-specific endo/exonuclease through its catalytic NUC domain.

a, Human NUMEN contains a putative signal peptide (SP) within the leader sequence, a conserved DNA/RNA endonuclease domain (NUC), and a C-terminus with multiple transmembrane regions (TMs). EXOG and ENDOG from the DNA/RNA non-specific endonuclease family are also shown. The RGQ/RGH motifs are marked in red. MLS indicates mitochondrial localization sequence. b,c, GFP-tagged full-length NUMEN (+GFP–NUMEN) or the NUC-deletion mutant (+GFP–NUMEN-ΔNUC) was stably expressed in BRCA1 and NUMEN-DKO (BRCA1−/−NUMEN−/−) MDA-MB-231 cells. b, Exogenous NUMEN expression was confirmed by western blotting using anti-GFP or anti-α-tubulin. c, The cells were cultured for 14 d in olaparib as indicated before clonogenic survival assays. Parental BRCA1−/− MDA-MB-231 cells served as controls. d, Recombinant full-length and mutant NUMEN NUC domain proteins were purified from E. coli using nickel-agarose columns and analysed by SDS–PAGE and Coomassie blue staining. e, 32P-labelled DNA substrates (40 nM) were incubated with (+) or without (−) NUMEN NUC proteins (50 nM) for 50 min at 37 °C. Stars indicate 5′-end labelling with 32P. The red arrowheads indicate how NUMEN processes the substrates from blunt-ended termini and/or past ds/ss junctions. The red arrows indicate the direction of processing. f, Alignment of the NUC domain sequences of NUMEN, ENDOG and EXOG from human and other species. The secondary structure of human NUMEN was obtained from AlphaFold 2. Multiple sequence alignments were performed using CLUSTALW and displayed by ESPript 3.0. The red and green stars underneath the sequences denote residue Gln143 and evolutionarily conserved cysteine residues, respectively. The β strands are marked by arrows and helices by spirals. Identical amino acids are shaded in red and conserved residues are boxed. g, ss- and dsDNA substrates were incubated with 50 nM recombinant WT or RGQ point mutants of NUMEN NUC domains for 40 min at 37 °C. Reactions with no NUC proteins (Mock) served as controls. Cy5 labelling is indicated by asterisks and biotin-TEG modifications by ellipses. h, GFP–NUMEN was stably expressed in MDA-MB-231 cells. Endogenous and exogenous NUMEN expression in WT, NUMEN−/− and NUMEN overexpression (GFP–NUMEN) cells was confirmed by western blotting as indicated. Source unprocessed blots are provided.

Source data

Extended Data Fig. 4 NUMEN deficiency impairs NHEJ and promotes HR.

a, HA-tagged ER-AsiSI was stably expressed in U2OS cells and the the indicated antibodies were used in western blots. b, Cells from a were cultured in 300 nM 4-OHT for 4 h and then immunostained using antibodies to the HA epitope (red). Induced translocation of ER-AsisI into the nucleus could be observed. DSBs generated by the restriction enzyme AsisI were visualized using anti-γH2AX (green). Representative images from confocal microscopy are shown. DNA was counterstained with DAPI (blue). Scale bars, 5 μm. c) DR-GFP and EJ5-GFP U2OS cell lines were transiently transfected with siRNAs targeting NUMEN. The knockdown efficiency was confirmed by western blotting, as indicated. d, Gating strategy used for Fig. 3d. The first gating was performed using FSC-A and SSC-A, the second gating was performed using FITC-A and PE-A. e, WT and NUMEN−/− MDA-MB-231 cells were treated with or without IR (2 Gy) and harvested 2 h later for immunostaining experiments using anti-RAD51. Data are the mean ± s.d. of three independent experiments. Circles are the mean values from each experiment; 30 nuclei were analysed in each group per experiment. Significance was calculated using a two-way ANOVA. NS, non-significant. Scale bar, 5 μm. f, Representative images of immunostaining analysis of WT and NUMEN−/− MDA-MB-231 cells that were harvested at the indicated time points after zeocin addition (500 μg ml−1), using antibodies to 53BP1 (left) or γH2AX (right). The cells were maintained without zeocin after 4 h. Scale bar, 5 μm. g, WT, TRF2−/− and TRF2 and NUMEN-DKO (NUMEN−/−TRF2−/−) HeLa cells were immunoblotted using anti-TRF2 and anti-GAPDH. h, BRCA1−/− and BRCA1 and NUMEN-DKO (BRCA1−/−NUMEN−/−) HeLa cells were treated with DMSO or zeocin (100 μg ml−1) for 4 h before being analysed for RAD51 foci formation. Data represent the mean ± s.d. of three independent experiments (n = 50 nuclei per condition per experiment). Significance was calculated using a two-way ANOVA. NS, non-significant. Scale bar, 5 μm. Source numerical data and unprocessed blots are provided.

Source data

Extended Data Fig. 5 Identification of the interaction network of NUMEN through BioID proximity labelling strategy.

a,b, BioID proximity screen. a, HEK293T cells were transduced with lentiviruses encoding Flag–BioID-tagged NUMEN and cultured in biotin-containing media before being harvested for large-scale affinity purification and identification of biotinylated proteins by mass spectrometry. Flag–BioID fused to an NLS served as the control. Two repeat control experiments were performed. b, Flag–BioID was inserted internally either N-terminal (BioID-NUMEN) or C-terminal (NUMEN–BioID) of the NUC domain. c, Enrichment patterns of the proteins identified from the two control repeat experiments (Ctrl-1 and Ctrl-2) versus the two NUMEN baits. d, Number of overlapping candidate proteins between different sample groups. e, The degree of correlation between control repeats and the two NUMEN baits is shown using log2-transformed FOT values. f, Gene Ontology enrichment analysis of cellular component for the preys identified from both NUMEN baits. The ER and nuclear membrane-associated terms are marked in blue and red, respectively. The top 20 enriched Gene Ontology terms under the ‘cellular component’ category for the baits are listed. Orange and grey bars represent respectively fold enrichments and −log10-transformed P values. g, Nuclear membrane proteins identified from our BioID screens are plotted as shown. The nuclear envelope LINC complex and LAD proteins are illustrated (top). The top-right quadrant of NUMEN–BioID scatter plot is shown (bottom), with enriched proteins (log2-transformed ratio) over controls. Significantly enriched nuclear membrane proteins (LINC complex in orange and LAD proteins in blue) and DDR factors (red) are highlighted. Putative NUMEN-interacting partners based on the BioGRID database are marked in black. Dashed lines indicate 1.5-fold change of FOT values.

Extended Data Fig. 6 Epistasis analysis of NUMEN on the NHEJ repair pathway.

a, siRNAs taregting the indicated genes were transiently transfected into U2OS EJ5-GFP and HeLa cells. The knockdown efficiencies were determined by RT–qPCR. A non-silencing siRNA (siNC) served as the control. Data are the mean ± s.d. of three independent experiments. Significance was calculated using a two-tailed unpaired t-test. b, siRNAs targeting the indicated gene products were introduced into U2OS EJ5-GFP cells before the NHEJ efficiencies were measured. All results were normalized to the control sample. Data are the mean ± s.d. of three different experiments. Statistical analysis was performed using a one-way ANOVA. NS, not significant. c, WT and NUMEN−/− MDA-MB-231 cells were treated with the DNA-PK inhibitor NU7441 (1 μM for 24 h) or knocked out for Artemis. The cells were then treated with zeocin (100 μg ml−1) for 4 h before immunostaining analysis using anti-RAD51. Data are the mean ± s.d. (n = 3 independent experiments). Quantification was carried out on 30 nuclei in each group per experiment. Statistical analysis was performed using a one-way ANOVA. NS, not significant. d, WT and NUMEN−/− MDA-MB-231 cells were treated with DMSO or 100 µg ml−1 zeocin for 4 h before immunofluorescence analysis and quantification. An antibody to phospho-DNA-PK S2056 (p-DNA-PK) was used. Data are the mean ± s.d. of three independent experiments (n = 30 nuclei per condition per experiment). Statistical analysis was performed using a one-way ANOVA. NS, not significant. e, 53BP1 was knocked down in WT and NUMEN−/− HeLa cells before treatment with the indicated concentrations of zeocin for 4 h. The cells were then maintained in normal culture medium for 7 d and then assessed for survival. f, WT and NUMEN−/− HeLa cells were knocked out for 53BP1 and subjected to zeocin (100 μg ml−1) treatment for 4 h before immunostaining using anti-RAD51. Data represent the mean ± s.d. (n = 3 independent experiments, 30 nuclei were analysed per experiment). Significance was calculated using two-tailed unpaired t-tests. NS, not significant. Source numerical data are provided.

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Extended Data Fig. 7 NUMEN is anchored to the nuclear membrane through its SP and TM domains.

a, MDA-MB-231, HeLa and HEK293T cells were co-stained with antibodies to NUMEN (green) and Lamin A/C (red). Representative images from confocal microscopy are shown. DNA was counterstained with DAPI (blue). b, HEK293T cells transiently expressing Flag-tagged full-length or truncation mutants of NUMEN were co-stained with antibodies to Lamin A/C (red) and Flag (green). Representative images from confocal microscopy are shown. The Flag epitope was inserted C-terminal of the SP sequence whenever possible to avoid disrupting NUMEN localization. c, Representative images of HEK293T cells stably expressing BioID-Flag-tagged NUMEN that were immunostained with antibodies to Flag (green) and Lamin A/C (red). d, Different NUMEN mutants were stably expressed in MDA-MB-231 cells and their expression confirmed by western blotting with antibodies to the Flag epitope. GAPDH was used as a loading control. e, Cells from d were treated with zeocin (100 μg ml−1) for 4 h before immunostaining analysis using anti-RAD51. Data represent the mean ± s.d. (n = 3 independent experiments). Quantification was carried out on 30 nuclei in each group per experiment. Significance was calculated using two-tailed unpaired t-tests. a–d, Scale bars, 5 μm. Source numerical data are provided.

Source data

Extended Data Fig. 8 NUMEN participates in compartmentalized NHEJ repair at the nuclear periphery.

a, HEK293T cells stably expressing the 53BP1trunc–mApple DSB reporter (red) together with NUMEN–GFP (green) were cultured with zeocin (100 μg ml−1) in a glass-bottomed dish before time-lapse live-cell imaging. 53BP1trunc–mApple is a fusion protein of truncated 53BP1 and the Apple fluorescent protein. Images were captured every 3 min during a 4-h window, and those from the indicated time points are shown. Each boxed region is enlarged in the zoom panel (bottom). Arrows point to 53BP1trunc–mApple signals that moved closer to NUMEN–GFP signals over time. b, The 53BP1trunc–mApple DSB reporter cells described above were treated with 2 Gy IR and visualized by time-lapse live-cell imaging. Images were captured every 3 min during a 2-h window. c, GFP-tagged m6A-Tracer and Dam-LaminB1–mAmetrine co-expression HEK293T cells were cultured with (+) or without (−) the Shield-1 ligand for 24 h. In the absence of Shield-1 ligand, only m6A-Tracer (green) was expressed and showed predominantly cytoplasmic localization. Following induction, Dam-Lamin B1 (blue) became stabilized and along with m6A exhibited ring-like localization patterns at the nuclear lamina. d, HEK293T cells stably co-expressing m6A-Tracer–GFP (green, to mark LADs) and Dam-Lamin B1–mAmetrine were cultured with the Shield-1 ligand and immunostained using anti-γH2AX (red) following 100 μg ml−1 zeocin treatment for 4 h. The boxed region is enlarged in the zoom panel. Scale bars, 5 μm, unless specified otherwise in the zoom panel. Source numerical data are provided.

Extended Data Fig. 9 NUMEN enables in situ NHEJ repair of heterochromatic breaks and maintaining of genome stability.

a, MDA-MB-231 cells were treated with DMSO, zeocin (100 μg ml−1 for 4 h), bleomycin (40 μg ml−1 for 4 h), IR (2 Gy and harvested 2 h later) or cisplatin (2.5 μM for 4 h) before immunofluorescence analysis using antibodies to γH2AX (green) and H3K9me2 (red). DAPI was used to stain the nuclei (blue). Scale bars, 5 μm. b, Analysis of CNV counts from four cancer types with significant negative correlations from a. LIHC (n = 337), COAD (n = 407), HNSC (n = 468), and BRCA (n = 1,027) samples with valid outcome data were grouped by the expression levels of BRCA1 and NUMEN (median cutoff) and are shown in violin plots. Each box outline shows the 25th and 75th percentiles, and the solid line indicates the median value. Whiskers extend to the most extreme data points that are no more than 1.5× the interquartile range. n, number of subgroup samples. Statistical analysis was performed using a two-tailed Mann–Whitney U-test. c, DSB repair pathway choice is tightly regulated to ensure the faithful repair of chromosome breaks. Nuclear compartmentation may help shape such decisions. We propose that nuclear membrane anchoring of NUMEN facilitates the establishment of an environment that favours NHEJ for the repair of repetitive DNA sequences at LADs as well as lesions dynamically moving from the nuclear interior to the periphery. NUMEN may thus antagonize HDR near the nuclear periphery and help minimize ectopic repair between heterochromatic repetitive sequences. Source numerical data are provided.

Supplementary information

Reporting Summary

Supplementary Table 1

Complete list of MAGeCK analysis of PARPi resistance CRISPR screen.

Supplementary Table 2

The proximity/interaction proteins of NUMEN identified by mass spectrometry from the BioID pulldown expriments.

Supplementary Tables 3 and 4

Supplementary Table 3. List of oligonucleotides used in the study. Supplementary Table 4. List of antibodies used in the study.

Source data

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Chen, B., Ge, T., Jian, M. et al. Transmembrane nuclease NUMEN/ENDOD1 regulates DNA repair pathway choice at the nuclear periphery. Nat Cell Biol 25, 1004–1016 (2023). https://doi.org/10.1038/s41556-023-01165-1

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