Inheritance of paternal DNA harm by histone-mediated restore restriction

C. elegans strains

All strains had been maintained based mostly on normal situations at 20 °C. Strains used had been N2 (Bristol; wild kind), 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 had been separated and maintained in a single day. On the second day, the grownup females or males both remained untreated or had been irradiated with the indicated dose of ionizing radiation inflicted by a caesium 137 irradiation supply (Biobeam GM 8000, Eckert & Ziegler, Gamma-Service Medical). Afterwards, ≥3 irradiated grownup worms and ≥3 non-irradiated opposite-sex adults had been instantly transferred to three new plates served as 3 organic replicates and allowed to put eggs for two h. Then the males had been eliminated and left the females to proceed egg-laying for an additional 4 h. The females had been then eliminated and the variety of eggs was counted. The variety of surviving progeny was characterised 24 h later to look at the progeny lethality of the P0 technology. Then, we transferred ≥3 surviving male progeny (F1) or ≥3 surviving feminine progeny (F1) to new plates, serving as one organic replicate, and positioned them with the three untreated opposite-sex adults and allowed them to put eggs for someday. No less than three organic replicates had been included in every experiment. The adults had been then eliminated and the variety of laid eggs was counted. Twenty-four hours later, the surviving progenies had been counted to characterize the progeny lethality of the F1 technology.

For the hermaphrodite worms, synchronized hermaphrodite late L4 had been separated from the remainder of the worms by selecting, and ≥3 late L4 hermaphrodites had been both untreated or irradiated with the indicated dose of ionizing radiation. Three irradiated hermaphrodites had been transferred to three separate plates as 3 organic replicates and allowed to put eggs for six h. The adults had been then eliminated and the variety of eggs was counted 24 h later, the hatched progeny had been quantified because the progeny lethality of the P0 technology. The surviving worms had been transferred to 3 plates and allowed to put eggs for someday. The adults had been eliminated and the progeny lethality of the resultant generations was quantified 24 h later.

Developmental assay

Synchronized L1 worms of management and paternally handled F1 had been generated through normal hypochlorite remedy. Arrested L1 worms had been positioned on NGM agar plates with OP50, fed with micro organism and incubated at 20 °C for 48 h. Then the larval stage of worms was characterised beneath a Zeiss discovery.V8 microscope. For every experiment, >30 L1 larvae had been included for every replicate, n = 3 organic replicates had been used.

Quantification of germ cell apoptotic corpses

Day-1 grownup feminine worms had been immobilized utilizing 5 mM levamisole (AppliChem A431005) and mounted on a 2% agarose pad on a microscope slide. The variety of apoptotic corpses was scored through Nomarski DIC microscopy on a Zeiss Axio Imager M1/2 based mostly on the refractive morphological adjustments occurring in apoptotic germ cells throughout the gonad loop50.

RNAi remedy

RNAi feeding clones had been obtained from the library of J. Ahringer. The E. coli feeding pressure HT115 (DE3) with RNAi clones had been cultured with LB medium containing ampicillin (100 μg ml−1) in a single day. IPTG (1 mM) was added to the tradition for the induction of RNAi product earlier than seeded on RNAi agar plates (NGM agar with ampicillin and IPTG). For the RNAi feeding assay, >30 synchronized L1 larvae as P0 technology had been positioned on the RNAi agar plates seeded with E. coli feeding pressure HT115 (DE3) containing particular RNAi or empty vector management. Three days later, grownup men and women had been separated and transferred to contemporary RNAi plates for sustaining the RNAi effectivity till additional experiments had been carried out. The next experiments had been carried out as described in ‘Measurement of ionizing radiation-induced progeny lethality’.

Immunofluorescence staining

Grownup worms had been 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, adopted by fixation with 3.7% formaldehyde for 1 h. Then, a 24 × 24 mm coverslip was positioned onto the drop, and the slide was left in a −80 °C freezer for 10 min to carry out the freeze-cracking process. Then the slide was shortly transferred to −20 °C methanol for lower than 1 min. For visualizing the nuclei, slides had been washed as soon as with PBS and as soon as with PBST (0.2% Tween in PBS) and mounted with DAPI Fluoromount-G mounting medium (Southern Biotech) and sealed with nail polish. For the opposite staining, after fixation, slides had been washed 1 time with 1× PBS and a pair of occasions with 1× PBT (0.5% Triton X-100 in PBS). To enhance the sign high quality, slides had been first blocked for 20 min with Picture-iT FX sign enhancer (Thermo Fisher) earlier than blocking with 1× PBT containing 10% donkey serum for an additional 20 min. Afterwards, major antibodies diluted with 1× PBT containing 5% donkey serum had been utilized to the slides and incubated at 4 °C in a single day. After washing 3 occasions with 1× PBT, the slides had been incubated with secondary antibodies diluted with 1× PBT at 37 °C for 30 min. Then slides had been washed with 1× PBT 3 occasions, mounted with DAPI Fluoromount-G mounting medium (Southern Biotech) and sealed with nail polish. Slides had been saved at 4 °C at midnight earlier than imaging.

Major antibodies used for immunofluorescence staining had been 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 present from the laboratory of A. Gartner; dilution 1:2,000 in PBT). Secondary antibodies used had been 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 photographs for quantification had been taken with a Zeiss Meta 710 confocal laser scanning microscope. For quantification, fastened publicity time was set for various therapies and strains. For H3K9me2, RNAPII p-Ser2 and RAD-51 staining, z-stack photographs had been acquired with Zeiss Meta 710 confocal microscope, and the H3K9me2 and RNAPII p-Ser2 sign depth and the foci variety of RAD-51 foci per nucleus had been quantified with Imaris x64 9.1.2 software program. Fluorescence intensities had been normalized to DAPI sign.

SILAC assay

The steady isotope labelling process was described in a earlier research51. Briefly, ET505 E. coli (lysine auxotrophy, from Coli Genetic Inventory Middle) had been 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−113C6-labelled lysine (Cambridge isotope laboratory) or 40 µg ml−1 regular l-lysine, and incubated at 37 °C in a single day to achieve A600 = 1. Micro organism had been 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 remedy had been hatched and arrested in M9 buffer, after which L1 worms had been transferred to NGM-N plates seeded with heavy isotope labelled lysine (heavy lysine)- or regular lysine (mild lysine)-labelled ET505 E. coli. Worms had been fed with labelled micro organism for 2 generations to achieve the incorporation charge >97%, after which picked the late L4 stage worms to irradiate with ionizing radiation or mock-treated. 4 replicates had been included on this experiment, as two of them had been the ionizing radiation-treated heavy lysine group and mock-treated mild lysine group, whereas the opposite two replicates had been the mock-treated heavy lysine group and ionizing radiation-treated mild lysine group.

Equal numbers of the F1 grownup worms had been washed off from heavy lysine plates and lightweight lysine plates with M9 buffer and mixed. After eradicating 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). Warmth the pattern at 95 °C for 10 min and sonicate the pattern with Bioruptor (30 s sonication, 30 s break, 10 cycles, excessive efficiency). Heating and sonication had been repeated as soon as extra. Then samples had been centrifuged at 20,000g for 20 min, and the supernatant was collected. 5 microlitres of protein resolution was diluted with 20 mM Tris to scale back the focus of guanidinium chloride to beneath 0.6 M. 50 mM TEAB was added to dilute the samples to 100 µl after which 1 µg Lys-C was added for incubation at 37 °C for 4 h. Samples had been additional diluted with 180 µl TEAB and handled with 2 µg Lys-C at 37 °C in a single day. Enzyme digestion was stopped by including formic acid to 1%, and the pattern purification by StageTip was carried out in accordance with CECAD/CMMC Proteomics Core facility’s normal protocol (https://www.proteomics-cologne.com/protocols). The mass spectrometry proteomics information have been deposited to the ProteomeXchange Consortium through the PRIDE accomplice repository with the dataset identifier PXD031873.

Single-worm WGS

Single L4 female and male fog-2, and hermaphrodite wild-type with indicated remedy had been transferred to plates with UV-killed OP50 micro organism, with the intention to scale back the contamination of bacterial DNA. On the second day, a single grownup worm was picked to 10 µl of M9 buffer, then the samples had been frozen at −80 °C. DNA was extracted following the usual Illumina DNA preparation protocol. Libraries had been ready for sequencing utilizing the usual Illumina protocols. Briefly, 120 ng of genomic DNA was tagmented with adaptor sequence utilizing bead-linked transposomes. Tagmented DNA was amplified by PCR for five cycles. Libraries had been sequenced on the Illumina HiSeq 2500 following the producer’s protocols. The info have been deposited with hyperlinks to BioProject accession quantity PRJNA826255 within the BioProject database.

Telomere FISH

Telomere FISH was carried out by a modified protocol52. Twenty grownup females or males had been transferred to a drop of M9 buffer onto a 0.3% polylysine-treated 3-well slide (3 × 14 mm printed effectively slides; Fisher Scientific). Germline dissection was carried out with two syringe needles, adopted by fixation with 3.7% formaldehyde for 1 h. Then a 24 × 24 mm coverslip was positioned onto the drop, and the slide was left in a −80 °C freezer for 10 min to carry out the freeze-cracking process. Then the slide was shortly transferred to −20 °C methanol for lower than 1 min. The slides had been washed as soon as with 1× PBS and incubated in permeabilization buffer (0.5% Triton X-100 in 1× PBS) for 1 h at room temperature adopted by a wash in 1× PBS. Then slides had been shortly washed with 0.01 N HCL adopted by a wash with 0.1 N HCL for two min. To stop unspecific binding of the FISH probe, 50 µg ml−1 RNase An answer (10 µg ml−1 RNase A in 1× PBS) was added to the slide and incubated at 37 °C for 45 min. Afterwards, slides had been washed 2 occasions with 2× SSC. For pre-hybridization, 50 µl of pre-hybridization resolution (2× SSC, 50% formamide) was added on the slides and incubated in a damp chamber for two 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 answer was faraway from the slide as a lot as attainable, then 50 µl of FISH probes had been added to the pattern and coated with Body-Seal in situ PCR and Hybridization Slide Chamber (Bio-Rad SLF0601), then the slides had been incubated over-night at 37 °C. On the second day, the samples had been denatured for five min at 80 °C and continued incubating at 37 °C for two days. Afterwards, the hybridization chambers had been eliminated, and slides had been washed in 2× SSC for five min at room temperature. To fixate the staining, the slides had been washed 3 occasions in preheated 2× SSC at 37 °C, and twice in preheated 0.2× SSC at 55 °C. Because the final step, slides had been 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 photographs had been taken with a Zeiss Meta 710 confocal laser scanning microscope was used. To visualise the telomeric sign, z-stack photographs had been acquired with a Zeiss Meta 710 confocal microscope, and the z-stack photos had been processed and the variety of DNA fragments was counted with Picture J/Fiji v2.3.0/1.53f.

Public datasets

The next public datasets have been re-analysed: The deletions of the hard-filtered variants of the 20220216 CeNDR 29 launch30 had been downloaded from https://www.elegansvariation.org/information/launch/20220216. The info for the C. elegans mutation accumulation experiment had been downloaded from the supplementary information from Volkova et al.31. The filtered hg38 SNV_INDEL_SV_phased_panel.vcf information for all chromosomes from the 20220422 launch of the 1000 Genomes Challenge34 had been 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) had been 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 together with gamete-of-origin evaluation had been downloaded from the supplementary information from Jónsson et al.35.

Bioinformatics evaluation

Gene set enrichment evaluation

The enrichment evaluation for chromosomal gene distributions was performed in R v3.6.3 with the GSEA operate of clusterProfiler v3.14.353 was used with maxGSSize = 20000 and nPerm = 20000.

WGS preprocessing

The fastq information had been preprocessed with Fastp v0.20.054, and mapped with BWA-0.7.1755 with the parameters bwa mem -M -Ok 100000000, and the reference genome ce11. The mapped information had been transformed to BAM and sorted with samtools v1.656, and duplicated reads had been eliminated with GATK v4.1.0.0 MarkDuplicates57.

Structural variant calling

Structural variants had been referred to as with Manta v1.6.058 and solely structural variants that handed all the Manta high quality filters had been used. To seek out structural variants which might be newly induced within the F1 technology, structural variants that overlapped with any structural variant of any P0 pattern had been filtered out (full structural variation websites with or with out filtering in Supplementary Desk 3). Repeat areas are tough to map and establish, we subsequently deleted any insertion–deletion mutant inside a repeat area, or any translocation for which each break factors overlapped with the identical repeat class. Manta calls translocations in each instructions as two break factors together with a place confidence interval. To keep away from duplicates, we filtered translocations that had the identical begin and finish break level throughout the respective confidence interval in any mixture.

Translocation varieties

A translocation is named as two break factors and might seem in 4 alternative ways in a VCF file. The reference sequence s is changed by the sequence t after the fusion to place p, respective earlier than the fusion at place p. This may occur in 4 methods:

  • Kind 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.

  • Type 2: t]p] The reverse part of the genomic location left of the place p is fused after t. In different phrases, these are fusions between the three′ sense strand and the 5′ anti-sense strand.

  • Kind 3:]p]t The genomic location extending left from the place p is fused earlier than t. This is similar as Kind 1.

  • Kind 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.

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