C. elegans strains
All strains were maintained based on standard conditions at 20 °C. Strains used were N2 (Bristol; wild type), CB4108 fog-2(q71), BC784 spe-8(hc50), RB1067 his-24(ok1024), MT13971 hpl-1(n4317), DW102 brc-1(tm1145), FX1524 cku-70(tm1524), FX2026 polq-1(tm2026), BJS1017 his-24(ok1024); fog-2(q71), BJS1018 hpl-1(n4317); fog-2(q71), BJS1019 brc-1(tm1145); fog-2(q71), BJS1020 cku-70(tm1524); fog-2(q71), BJS1021 polq-1(tm2026); fog-2(q71).
Measurement of ionizing radiation-induced progeny lethality
For the feminized mutants, synchronized L4 females and males were separated and maintained overnight. On the second day, the adult females or males either remained untreated or were irradiated with the indicated dose of ionizing radiation inflicted by a caesium 137 irradiation source (Biobeam GM 8000, Eckert & Ziegler, Gamma-Service Medical). Afterwards, ≥3 irradiated adult worms and ≥3 non-irradiated opposite-sex adults were immediately transferred to 3 new plates served as 3 biological replicates and allowed to lay eggs for 2 h. Then the males were removed and left the females to continue egg-laying for another 4 h. The females were then removed and the number of eggs was counted. The number of surviving progeny was characterized 24 h later to examine the progeny lethality of the P0 generation. Then, we transferred ≥3 surviving male progeny (F1) or ≥3 surviving female progeny (F1) to new plates, serving as one biological replicate, and placed them with the three untreated opposite-sex adults and allowed them to lay eggs for one day. At least three biological replicates were included in each experiment. The adults were then removed and the number of laid eggs was counted. Twenty-four hours later, the surviving progenies were counted to characterize the progeny lethality of the F1 generation.
For the hermaphrodite worms, synchronized hermaphrodite late L4 were separated from the rest of the worms by picking, and ≥3 late L4 hermaphrodites were either untreated or irradiated with the indicated dose of ionizing radiation. Three irradiated hermaphrodites were transferred to 3 separate plates as 3 biological replicates and allowed to lay eggs for 6 h. The adults were then removed and the number of eggs was counted 24 h later, the hatched progeny were quantified as the progeny lethality of the P0 generation. The surviving worms were transferred to three plates and allowed to lay eggs for one day. The adults were removed and the progeny lethality of the consequent generations was quantified 24 h later.
Developmental assay
Synchronized L1 worms of control and paternally treated F1 were generated via standard hypochlorite treatment. Arrested L1 worms were placed on NGM agar plates with OP50, fed with bacteria and incubated at 20 °C for 48 h. Then the larval stage of worms was characterized under a Zeiss discovery.V8 microscope. For each experiment, >30 L1 larvae were included for each replicate, n = 3 biological replicates were used.
Quantification of germ cell apoptotic corpses
Day-1 adult female worms were immobilized using 5 mM levamisole (AppliChem A431005) and mounted on a 2% agarose pad on a microscope slide. The number of apoptotic corpses was scored via Nomarski DIC microscopy on a Zeiss Axio Imager M1/2 based on the refractive morphological changes occurring in apoptotic germ cells within the gonad loop50.
RNAi treatment
RNAi feeding clones were obtained from the library of J. Ahringer. The E. coli feeding strain HT115 (DE3) with RNAi clones were cultured with LB medium containing ampicillin (100 μg ml−1) overnight. IPTG (1 mM) was added to the culture for the induction of RNAi product before seeded on RNAi agar plates (NGM agar with ampicillin and IPTG). For the RNAi feeding assay, >30 synchronized L1 larvae as P0 generation were placed on the RNAi agar plates seeded with E. coli feeding strain HT115 (DE3) containing specific RNAi or empty vector control. Three days later, adult males and females were separated and transferred to fresh RNAi plates for maintaining the RNAi efficiency until further experiments were performed. The subsequent experiments were performed as described in ‘Measurement of ionizing radiation-induced progeny lethality’.
Immunofluorescence staining
Adult worms were picked from plates and transferred to a drop of M9 buffer onto a 0.3% polylysine-treated three-well slide (3 × 14 mm printed wells slides from Fisher Scientific). Germline dissection was carried out with two syringe needles, followed by fixation with 3.7% formaldehyde for 1 h. Then, a 24 × 24 mm coverslip was placed onto the drop, and the slide was left in a −80 °C freezer for 10 min to perform the freeze-cracking procedure. Then the slide was quickly transferred to −20 °C methanol for less than 1 min. For visualizing the nuclei, slides were washed once with PBS and once with PBST (0.2% Tween in PBS) and mounted with DAPI Fluoromount-G mounting medium (Southern Biotech) and sealed with nail polish. For the other staining, after fixation, slides were washed 1 time with 1× PBS and 2 times with 1× PBT (0.5% Triton X-100 in PBS). To improve the signal quality, slides were first blocked for 20 min with Image-iT FX signal enhancer (Thermo Fisher) before blocking with 1× PBT containing 10% donkey serum for another 20 min. Afterwards, primary antibodies diluted with 1× PBT containing 5% donkey serum were applied to the slides and incubated at 4 °C overnight. After washing 3 times with 1× PBT, the slides were incubated with secondary antibodies diluted with 1× PBT at 37 °C for 30 min. Then slides were washed with 1× PBT 3 times, mounted with DAPI Fluoromount-G mounting medium (Southern Biotech) and sealed with nail polish. Slides were stored at 4 °C in the dark before imaging.
Primary antibodies used for immunofluorescence staining were rabbit polyclonal anti-phospho-RNAPII (Ser2) antibody (Thermo Fisher, A300-654A; dilution 1:500 in PBT); mouse monoclonal anti-H3K9me2 antibody (Abcam, ab1220; dilution 1:100 in PBT); rabbit polyclonal anti-HIM-8 (Novus Biologicals, 41980002; dilution 1:100 in PBT); rabbit anti-RAD-51 antibody (a gift from the laboratory of A. Gartner; dilution 1:2,000 in PBT). Secondary antibodies used were AlexaFluor 488 donkey anti-mouse IgG (Thermo Fisher, A21202; dilution 1:500 in PBT) and AlexaFluor 594 donkey anti-rabbit IgG (Thermo Fisher A21207; dilution 1:500 in PBT).
Fluorescence images for quantification were taken with a Zeiss Meta 710 confocal laser scanning microscope. For quantification, fixed exposure time was set for different treatments and strains. For H3K9me2, RNAPII p-Ser2 and RAD-51 staining, z-stack images were acquired with Zeiss Meta 710 confocal microscope, and the H3K9me2 and RNAPII p-Ser2 signal intensity and the foci number of RAD-51 foci per nucleus were quantified with Imaris x64 9.1.2 software. Fluorescence intensities were normalized to DAPI signal.
SILAC assay
The stable isotope labelling procedure was described in a previous study51. In brief, ET505 E. coli (lysine auxotrophy, from Coli Genetic Stock Center) were grown in M9 minimal medium (Na2HPO4 5.8 g l−1, KH2PO4 3 g l−1, NaCl 0.5 g l−1, NH4Cl2 1 g l−1, glucose 0.2% (w/v), MgSO4 1 mM, thiamine 0.01% (w/v) and 40 µg ml−1 13C6-labelled lysine (Cambridge isotope laboratory) or 40 µg ml−1 normal l-lysine, and incubated at 37 °C overnight to reach A600 = 1. Bacteria were concentrated to A600 = 50 and seeded to NGM-N plates (3 g of NaCl and 12 g of agarose dissolved in 970 ml deionised water).
Synchronized embryos generated by hypochlorite treatment were hatched and arrested in M9 buffer, and then L1 worms were transferred to NGM-N plates seeded with heavy isotope labelled lysine (heavy lysine)- or normal lysine (light lysine)-labelled ET505 E. coli. Worms were fed with labelled bacteria for two generations to reach the incorporation rate >97%, and then picked the late L4 stage worms to irradiate with ionizing radiation or mock-treated. Four replicates were included in this experiment, as two of them were the ionizing radiation-treated heavy lysine group and mock-treated light lysine group, whereas the other two replicates were the mock-treated heavy lysine group and ionizing radiation-treated light lysine group.
Equal numbers of the F1 adult worms were washed off from heavy lysine plates and light lysine plates with M9 buffer and combined. After removing the M9 buffer, lysis buffer was added to the worm pellet (6 M guanidinium chloride (GuCl), 10 mM TCEP, 40 mM CAA, 100 mM Tris-HCl). Heat the sample at 95 °C for 10 min and sonicate the sample with Bioruptor (30 s sonication, 30 s break, 10 cycles, high performance). Heating and sonication were repeated once more. Then samples were centrifuged at 20,000g for 20 min, and the supernatant was collected. Five microlitres of protein solution was diluted with 20 mM Tris to reduce the concentration of guanidinium chloride to below 0.6 M. 50 mM TEAB was added to dilute the samples to 100 µl and then 1 µg Lys-C was added for incubation at 37 °C for 4 h. Samples were further diluted with 180 µl TEAB and treated with 2 µg Lys-C at 37 °C overnight. Enzyme digestion was stopped by adding formic acid to 1%, and the sample purification by StageTip was carried out according to CECAD/CMMC Proteomics Core facility’s standard protocol (https://www.proteomics-cologne.com/protocols). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD031873.
Single-worm WGS
Single L4 male and female fog-2, and hermaphrodite wild-type with indicated treatment were transferred to plates with UV-killed OP50 bacteria, in order to reduce the contamination of bacterial DNA. On the second day, a single adult worm was picked to 10 µl of M9 buffer, then the samples were frozen at −80 °C. DNA was extracted following the standard Illumina DNA preparation protocol. Libraries were prepared for sequencing using the standard Illumina protocols. In brief, 120 ng of genomic DNA was tagmented with adaptor sequence using bead-linked transposomes. Tagmented DNA was amplified by PCR for 5 cycles. Libraries were sequenced on the Illumina HiSeq 2500 following the manufacturer’s protocols. The data have been deposited with links to BioProject accession number PRJNA826255 in the BioProject database.
Telomere FISH
Telomere FISH was carried out by a modified protocol52. Twenty adult females or males were transferred to a drop of M9 buffer onto a 0.3% polylysine-treated 3-well slide (3 × 14 mm printed well slides; Fisher Scientific). Germline dissection was carried out with two syringe needles, followed by fixation with 3.7% formaldehyde for 1 h. Then a 24 × 24 mm coverslip was placed onto the drop, and the slide was left in a −80 °C freezer for 10 min to perform the freeze-cracking procedure. Then the slide was quickly transferred to −20 °C methanol for less than 1 min. The slides were washed once with 1× PBS and incubated in permeabilization buffer (0.5% Triton X-100 in 1× PBS) for 1 h at room temperature followed by a wash in 1× PBS. Then slides were quickly washed with 0.01 N HCL followed by a wash with 0.1 N HCL for 2 min. To prevent unspecific binding of the FISH probe, 50 µg ml−1 RNase A solution (10 µg ml−1 RNase A in 1× PBS) was added to the slide and incubated at 37 °C for 45 min. Afterwards, slides were washed 2 times with 2× SSC. For pre-hybridization, 50 µl of pre-hybridization solution (2× SSC, 50% formamide) was added on the slides and incubated in a humid chamber for 2 h at room temperature. Then the FISH probe (PNA-FISH TTAGGC telomeric probe, Panagene, resuspended to 100 µM, fluorophore: Cy3) was diluted as 1:500 in hybridization buffer (2× SSC, 50% formamide, 10% (w/v) dextran sulfate, 50 µg ml−1 heparin, 100 µg ml−1 sheared salmon sperm DNA). After pre-hybridization, the solution was removed from the slide as much as possible, then 50 µl of FISH probes were added to the sample and covered with Frame-Seal in situ PCR and Hybridization Slide Chamber (Bio-Rad SLF0601), then the slides were incubated over-night at 37 °C. On the second day, the samples were denatured for 5 min at 80 °C and continued incubating at 37 °C for 2 days. Afterwards, the hybridization chambers were removed, and slides were washed in 2× SSC for 5 min at room temperature. To fixate the staining, the slides were washed 3 times in preheated 2× SSC at 37 °C, and twice in preheated 0.2× SSC at 55 °C. As the last step, slides were washed in 1× PBT for 10 min at room temperature and mounted by 7 µl Vectashield mounting medium containing DAPI (Vector Laboratories H-1200-10).
Staining images were taken with a Zeiss Meta 710 confocal laser scanning microscope was used. To visualize the telomeric signal, z-stack images were acquired with a Zeiss Meta 710 confocal microscope, and the z-stack pictures were processed and the number of DNA fragments was counted with Image J/Fiji v2.3.0/1.53f.
Public datasets
The following public datasets have been re-analysed: The deletions of the hard-filtered variants of the 20220216 CeNDR 29 release30 were downloaded from https://www.elegansvariation.org/data/release/20220216. The data for the C. elegans mutation accumulation experiment were downloaded from the supplementary data from Volkova et al.31. The filtered hg38 SNV_INDEL_SV_phased_panel.vcf files for all chromosomes from the 20220422 release of the 1000 Genomes Project34 were downloaded from http://ftp.1000genomes.ebi.ac.uk/vol1/ftp/data_collections/1000G_2504_high_coverage/working/20220422_3202_phased_SNV_INDEL_SV/. The hg38 illumina-polaris-v2.1-sv-truthset structural variants (https://github.com/Illumina/Polaris) were downloaded from https://s3-us-west-1.amazonaws.com/illumina-polaris-v2.1-sv-truthset/all_merge.vcf.gz. The processed hg38 variants of the 1,548 trios from Iceland including gamete-of-origin analysis were downloaded from the supplementary data from Jónsson et al.35.
Bioinformatics analysis
Gene set enrichment analysis
The enrichment analysis for chromosomal gene distributions was done in R v3.6.3 with the GSEA function of clusterProfiler v3.14.353 was used with maxGSSize = 20000 and nPerm = 20000.
WGS preprocessing
The fastq files were preprocessed with Fastp v0.20.054, and mapped with BWA-0.7.1755 with the parameters bwa mem -M -K 100000000, and the reference genome ce11. The mapped files were converted to BAM and sorted with samtools v1.656, and duplicated reads were removed with GATK v4.1.0.0 MarkDuplicates57.
Structural variant calling
Structural variants were called with Manta v1.6.058 and only structural variants that passed all of the Manta quality filters were used. To find structural variants that are newly induced in the F1 generation, structural variants that overlapped with any structural variant of any P0 sample were filtered out (full structural variation sites with or without filtering in Supplementary Table 3). Repeat regions are difficult to map and identify, we therefore deleted any insertion–deletion mutant within a repeat region, or any translocation for which both break points overlapped with the same repeat class. Manta calls translocations in both directions as two break points including a position confidence interval. To avoid duplicates, we filtered translocations that had the same start and end break point within the respective confidence interval in any combination.
Translocation types
A translocation is called as two break points and can appear in four different ways in a VCF file. The reference sequence s is replaced by the sequence t after the fusion to position p, respective before the fusion at position p. This can happen in four ways:
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Type 1: t[p[ The genomic location extending right from the position p is fused after t. In other words, these are fusions between the 3′ sense strand with the 5′ sense strand.
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Type 2: t]p] The reverse component of the genomic location left of the position p is fused after t. In other words, these are fusions between the 3′ sense strand and the 5′ anti-sense strand.
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Type 3:]p]t The genomic location extending left from the position p is fused before t. This is the same as Type 1.
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Type 4: [p[t The reverse component of the genomic location extending right from the position p is fused before t. In other words, these are fusions between the 3′ anti-sense strand and the 5′ sense strand.
See https://github.com/samtools/hts-specs/blob/master/VCFv4.1.pdf for further details.
Circos plots
The library circlize v0.4.1259 in R v3.6.3 was used to generate circos plots.
Templated insertions with distribution
The inserted sequence between the break points was searched within ±25 bp around the break points in both directions, in the normal orientation, as well as in the reverse, complement, and reverse complement orientation. Insertions ≥3 bp for which a template could be found within ±25 bp were called templated insertions, while any other insertion was classed as miscellaneous.
Microhomology with permutations
For each translocation 8 bp surrounding both break sites (that is, 4 bp upstream and 4 bp downstream of both break sites) were compared in an 8 × 8 grid (that is, each of the surrounding bases is compared to every other base). Matching bases were scored 1 and nonmatching bases were scored 0. One map therefore contains a 1 for each of the 64 combinations that have the same base, and 0 otherwise. The heat maps shown in the figure contain the sum of all such respective heatmaps divided by the total number of translocations. For each of the four translocation classes a separate microhomology was calculated. For type 1 translocations the left and right flank are both 5′ to 3′ on the sense strand. The left flank of type 2 translocations is the 5′ to 3′ sense strand, while the right flank is the reverse complement sequence. For type 4 translocations the left flank is the reverse complement sequence, while the right flank is the 5′ to 3′ sense strand sequence.
To calculate the significance of individual bins a permutation test was done. For each permutation the same number of translocations (of the same type) as in the original heatmap was randomly distributed on the genome to calculate the microhomology ratios for each of the 64 bins. To calculate a P value a permutation test was calculated with 100,000 permutations. To calculate the adjusted P value for the 64 bins statsmodels v0.11.160 multipletests methods with the parameter method=’fdr_bh’ in Python 3.661 was used.
Base composition
For all translocations with a microhomology of length 1 (n = 35 for fog-2, and n = 58 for wild type) the base composition for 8 bp around the break points was calculated. For each of the 8 positions the percentage of A, C, T and G was calculated. To be able to compare it to a random background distribution we sampled the same number of positions—that is, 35 for fog-2 and 58 for wild type—and calculated the average percentage of each A, C, T, and G for 25.000 such permutations.
Analyses of public C. elegans datasets
CeNDR
The deletions of the hard-filtered variants of the 20220216 CeNDR30 release were downloaded and further filtered for deletions between 8 and 200 bp. Deletions for which both break sites were annotated within the same repeat class were removed. Each deletion got categorized into non-homology, that is, no matching base at the break sites, microhomology—that is, exactly one matching base at the break sites, and macro-homology—that is, more than one matching base. By chance we would expect 75% of break sites to be non-homologous, 16.66% microhomologous, and 8.33% macro-homologous. The over-representation of microhomologous deletions sites were calculated with the binomial test function binom_test in Python’s Scipy-v1.5.1 package. The over-represented microhomologous variants were used in the heat map as described above for a 16 × 16 grid.
Mutation accumulation
The data for the mutation accumulation experiment were downloaded from the supplementary data from Volkova et al.31. Deletions for which both break sites were annotated within the same repeat class were removed, and only deletions with a length between 8 and 200 bp were considered for the subsequent analysis. The statistics and heat map were calculated as described above.
Analyses of public human datasets
1000 Genomes Project
The filtered hg38 SNV_INDEL_SV_phased_panel.vcf files for all chromosomes from the 20220422 release of the 1000 Genomes Project34 were downloaded and further filtered for deletions between 8 and 200 bp. Since the microhomology footprint of humans is 2–6 bp, we defined the 3 categories different from C. elegans. 0–1 bp homology is expected by chance in 75% + 16.66% = 91.66% of break sites. Microhomology—that is, 2–6 bp homology, in 8.325% of break sites, and macrohomology in 1/12288 ≈ 0.00008% of break sites. The statistics and heat map were otherwise calculated as described above.
Polaris
The hg38 illumina-polaris-v2.1-sv-truthset structural variants (https://github.com/Illumina/Polaris) were downloaded and filtered for deletions that had a PASS in the quality column. To focus on de novo deletions, any deletion that overlapped with either parent got filtered out, and only deletions between 8 and 200 bp were considered for the subsequent analysis. The statistics and heat map were calculated as described above.
Iceland trios
The processed hg38 variants of the 1548 trios from Iceland including gamete-of-origin analysis were downloaded from the supplementary data from Jónsson et al.35. Only deletions between 8 and 200 bp for which the gamete of origin was available were considered. The statistics and heat map were calculated as described above separately for deletions coming from the mother and father.
Data presentation and statistical analysis
All the data and statistical significances were analysed using the GraphPad Prism 7 software package (GraphPad) and R studio. For the proportion data shown in this paper, GLM with logit link function (R v4.0.2 and emmeans v1.5.2 (https://cran.r-project.org/web/packages/emmeans/index.html)) and ordinary ANOVA with arcsine transformed value (arcsine transformation equation: Y = arcsin(√(Y/n)) × 180/π) were both applied to confirm the significance of the observations, and the full statistic results are shown in the Supplementary Table 1. In addition, a QQ plot was attached for the ANOVA analysis to assess the normal distribution of the transformed value. Only the P values calculated from the GLM method are shown in the figures. Statistical methods, P values, sample size information and error bar descriptions are reported in the figure legends. Randomization was not applied because the group allocation was guided based on the genotype of the respective mutant worms. Worms of a given genotype were nevertheless randomly selected from large strain populations for each experiment without any preconditioning. Blinding was not applied as the experiments were carried out under highly standardized and predefined conditions such that an investigator-induced bias can be excluded. For progeny lethality characterization and staining quantifications, median with 95% confidence interval was used as these data types contain outliers.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
