Transgenerational inheritance of paternal DNA damage by linker histone H1-mediated DNA repair restriction

doi:10.21203/rs.3.rs-1602124/v1

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Transgenerational inheritance of paternal DNA damage by linker histone H1-mediated DNA repair restriction

https://doi.org/10.21203/rs.3.rs-1602124/v1

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published 21 Dec, 2022

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How paternal exposure to ionizing radiation (IR) affects genetic inheritance and disease risk in the offspring has been one of the most long-standing questions in radiation biology. While in humans, 80% of transmitted mutations arise in the paternal germline, the transgenerational effects of ionizing radiation (IR)-exposure has remained controversial and the mechanisms elusive. Here, we show that in sex-separated C. elegans strains paternal, but not maternal, IR-exposure leads to a transgenerational embryonic lethality. Offspring of irradiated males displayed various genome instability phenotypes, including DNA fragmentation, chromosomal rearrangement, and aneuploidy. We determined that paternal DNA double strand breaks (DSBs) are repaired by maternally provided error-prone polymerase-theta mediated end joining (TMEJ). Mechanistically, we show that depletion of a human histone H1.0 ortholog, HIS-24, or the heterochromatin protein, HPL-1, could significantly reverse the transgenerational embryonic lethality. Removal of HIS-24 or HPL-1 reduced the heterochromatin marker histone 3 lysine 9 di-methylation (H3K9me2) and allowed the employment of the error-free Homologous Recombination Repair (HRR) in the germline of the F1 of IR-treated P0 males, consequently improving the viability of the F2 generation. Our work establishes the mechanistic underpinnings of transgenerational consequences of paternal radiation exposure on the health of offspring, which may lead to congenital disorders and cancer in humans.

The effect of parental exposure to ionizing radiation (IR) on the health of the offspring has been controversially debated over decades. IR induces DNA double-strand breaks (DSBs) in the germline of males and is associated with elevated sperm DNA fragmentation index and reduced fertility in humans^1,2. Concerns about the transgenerational effect of paternal IR exposure were raised in response to the epidemiologic studies in the last century. In the 1980s, the UK government had noticed a high incidence of leukemia and lymphoma in the children living in the vicinity of the Sellafield nuclear plant compared to the children who moved to the place after birth and the children living in the surrounding areas³. A case-control study revealed that the high risk of developing malignant diseases in offspring is correlated with paternal radiation exposure that had occurred around the time of conception⁴. These reports eventually led to the “Gardner’s hypothesis”, which proposed a relationship between the paternal exposure to IR and the cancer risk in the offspring^4–6. However, the “Gardner’s hypothesis” has been widely criticized and rejected latterly, as no supporting evidence was found from the studies focusing on the survivors and clean-up workers from the atomic bomb in Japan, the Chernobyl accident in the Soviet Union, and the workers in other nuclear plants^7–11. Moreover, no biological explanation was proposed for the transgenerational cancer risk of paternal radiation exposure. Thus, it was concluded that there is “no indication” for the risk of childhood cancer in the offspring of the irradiated fathers⁸. However, the epidemiologic studies in humans are typically limited by the sample size, the mixed population, and records of radiation doses. Thus, the transgenerational effect of paternal IR exposure in humans remains an open question.

IR is a major source of mutagenesis in the germline, thus human and animal studies have also focused on the de novo mutations (DNMs) in the offspring of the irradiated parents^12,13. Although some studies reported an increase in minisatellite and microsatellite mutations in the children born to parents living in the highly exposed areas^14–16, two recent studies investigating the survivors and the cleanup workers of the Chernobyl accident reported no evident increase in DNMs in the offspring, regardless of the maternal or paternal exposure to IR^11,17. Thus, previous studies have not yet generated a conclusive result in the transgenerational genetic effect of parent radiation exposure. Considering sperm is responsible for the vast majority of the mutation rate in offspring ^18–22, it is especially interesting to evaluate the genome stability in the offspring of the exposed fathers, rather than mothers.

Apart from the genetic effect, several studies have focused on the transgenerational epigenetic effect of parental exposure to IR. For instance, a correlation between parentally inherited DNMs and the heterochromatin mark, histone 3 lysine 9 tri-methylation (H3K9me3) was noticed ²³, suggesting that epigenetic modifications might be involved in the parentally inherited genome instability. Studies in rats and mice revealed additional evidence of transgenerational epigenetic changes caused by paternal radiation exposure. These changes include loss of DNA methylation, and altered miRNA expression in the offspring of the animals exposed to a high dose of IR^24,25. Therefore, these studies indicate that paternal radiation exposure could potentially reshape the epigenetic landscape in the offspring.

Using sex-separated mutants of C. elegans as a model, we here report a previously unknown mechanism underlying the transgenerational effect of paternal exposure to IR. We show that paternal exposure to IR results in genome instability in the F1 generation and a transgenerational embryonic lethality. We determined that the paternal DNA damage is mainly repaired in the zygote through the maternally provided error-prone DNA repair machineries, the polymerase-theta mediated endjoining (TMEJ), which results in an elevated level of chromosomal aberrations. Removing the histone H1, HIS-24, or the heterochromatin protein, HPL-1, could reverse the transgenerational embryonic lethality of paternal IR exposure, through activating the error-free homologous recombination repair (HRR) pathway. Our work provides the mechanistic underpinnings of the transgenerational genetic and epigenetic effect of paternal exposure to IR.

Exposure of mature sperm to ionizing radiation (IR) leads to a transgenerational embryonic lethality.

To investigate transgenerational consequences of genotoxic stress, we exposed C. elegans to ionizing radiation (IR) and assessed the consequences in the subsequent generations. Unlike vertebrates, which are mainly dioecious, wild-type C. elegans has two sex forms: the self-fertilizing hermaphrodites and males. In order to distinguish the effect of DNA damage in male and female gametes, we used C. elegans sex determination mutants with a dioecious reproductive system: somatic hermaphrodites of the feminized mutant, fog-2, are defective in spermatogenesis and only generate a female germline, while fog-2 males generate a normal male germline. Using fog-2 mutant animals of either sex, we first addressed a question: which germ cell type might carry IR-induced DNA damage to the following generations. We irradiated adult female worms with low (30 Gy), medium (60 Gy) and high (90 Gy) doses of IR, and immediately transferred the irradiated females (∆f) onto a small plate in order to mate with un-irradiated males. After successful mating, the viability of the progeny laid by ∆f was characterized 24 h later. As expected, IR treatment led to a dose-dependent embryonic lethality (43.48%, 91.11%, and 95.24% lethality in 30 Gy, 60 Gy and 90 Gy treated groups, respectively) (Figure 1A), which is attributed to the excessive unrepaired DNA DSBs in oocytes. Nevertheless, when we crossed the survived F1 generation (∆ff and ∆fm) with the healthy opposite sex and examined the embryonic viability of the F2 generation, we found that the F2 generation showed nearly no embryonic lethality (Figure 1A). The observation indicates that the oocyte-carried DNA DSBs can lead to genome instability during embryogenesis of F1, and further result in embryonic lethality, but the maternal DNA damage will be eventually repaired before the gametogenesis of the F1 generation.

We next asked whether IR treatment in males (∆m) could affect subsequent generations. Immediately after IR exposure, males were placed with untreated females. Unlike the maternally irradiated group, the progeny lethality of ∆m was relatively low, as it led to 8.01 %, 14.29 %, and 8.33 % of progeny lethality in low, medium, and high doses groups, respectively (Figure 1A). This indicates that the paternal DNA damage has either been repaired or escaped from the check-point surveillance during the embryogenesis of the F1 generation. We then measured the embryonic viability of the F2 generation, after crossing the F1 generation animals with healthy animals of the opposite sex. Unexpectedly, we noticed that the embryonic lethality of the F2 generation was dramatically increased (Figure 1A). Noteworthy, this increase of embryonic lethality is significantly different between the progeny of female F1 (∆mf) and the progeny of male F1 (∆mm). The female F1 (∆mf) lead to 86.9 % and 96.35 % of progeny lethality, while the male F1 (∆mm) showed 35.58 % and 68.40 % of progeny lethality in the 60 Gy and 90 Gy treated groups, respectively (Figure 1A). Although we did not detect a significant increase of progeny lethality of the F1 generation in the 30 Gy irradiated group, there was a trend of increase observed (Figure 1A). Taken together, this observation suggests that male exposure to IR can lead to transgenerational lethality in the F2 generation.

We were next wondering how many generations this transgenerational lethality may last. To address this, we maintained the surviving progeny from F2 generations and mated them with the healthy opposite sex. We found that the progeny viability of F2 was restored (Figure 1B), indicating that the sperm-inherited detrimental effect can be majorly removed within two generations of intercrossing with healthy animals. Apart from using fog-2 mutant, we also verified this observation with an independent feminized mutant, spe-8 (Figure 1B).

In a case-control study of humans, an excess risk of childhood leukemia was associated with paternal IR exposure that occurred during the time of conception, but not three to six months before conception ²⁶. Like humans, spermatogenesis in male C. elegans is continuously ongoing in adults and in one nematode mating about two-thirds of the mature sperm are ejaculated. We hypothesized that mature sperm might be particularly vulnerable to DNA damage and might thus lead to transgenerational lethality. To test this hypothesis, we first collected the progeny derived from freshly irradiated fog-2 or spe-8 males to assess the effect of irradiation on mature sperm. On the second day, we transferred those irradiated males to new plates with untreated females and collected their progeny again to test the effect of irradiation on spermatogenesis prior to maturation (Figure 1C). Our data show that only the progeny derived from the freshly irradiated males could lead to the transgenerational lethality (Figure 1C), indicating that only the mature spermatozoa are responsible for the IR-induced transgenerational lethality.

Apart from using feminized mutants, we also wondered whether we could recapitulate the transgenerational lethality with wildtype (WT) C. elegans hermaphrodites. The development of the hermaphroditic gonad is strictly regulated in a spatiotemporal order. Here, spermatogenesis is initiated at the late L3 larval stage and ceases by the late L4 larval stage, where the germline switches to oogenesis that is maintained during the adult stage. The mature sperm is stored in the spermatheca at late L4 stage until the oocytes subsequently pass through and are fertilized. As we established above that the sperm is responsible for the transgenerational lethality, we irradiated WT at late L3 (start of spermatogenesis), young L4 (ongoing spermatogenesis), and late L4 (completed spermatogenesis) stages and characterized the progeny lethality of the following generations. We observed that only irradiation of WT hermaphrodites at the late L4 stage led to the transgenerational lethality in the F2 and F3 generations (Supplementary Figure 1A). These results further validate that the mature sperm also of hermaphrodite WT animals could lead to transgenerational lethality upon IR exposure.

In the fog-2 mutant, we noticed that the transgenerational lethality of irradiated males can last up to two generations, while, in WT hermaphrodites, we observed transgenerational lethality also in the F3 generation. Thus, we assumed that, without outcrossing with un-irradiated animals, this paternally inherited transgenerational lethality cannot be eliminated by maintaining multiple generations. We treated late L4 hermaphrodites with different doses of IR and observed that the transgenerational lethality was maintained over at least six generations (Supplementary Figure 1B). Indeed, although the level of lethality reached a peak at the F2 generation in all groups, the transgenerational lethality can last more than six generations in the 30 Gy, 60 Gy, 90 Gy and 120 Gy treated hermaphrodites (Supplementary Figure 1B). These results suggest that the recovery of viability in the F3 generation of fog-2 mutant is mainly attributed to the dilution of the transgenerational effect by outcrossing the damaged genomes three times with the un-irradiated opposite sex.

Paternal exposure to IR leads to DNA fragmentation, chromosomal rearrangement, and aneuploidy in the F1 generation.

The high embryonic lethality that we observed in the F2 generation prompted us to ask whether the paternal DNA damage causes heritable changes on the DNA and/or chromatin. Therefore, we tracked the chromatin states from the irradiated sperm to the germ cells of the F1 generation. First, we noticed that IR treatment leads to morphological changes in the sperm of fog-2 ∆m, as the sperm show a “comet-like” conformation (Figure 2A). This change is attributable to the formation of DSBs in the sperm’s DNA, as mature sperm cannot detect and repair DSBs properly due to its highly compacted chromatin structure that prevents access of the repair machinery²⁷. After fertilization, these embryos show various chromosomal instability phenotypes during the early embryogenesis, including the formation of chromosomal bridges and anaphase lagging (Figure 2B). The formation of chromosomal bridges indicates that the dividing embryonic cells entered a “Breakage-Fusion-Bridge” (BFB) cycle, which is a common source of genome instability in malignant cancer cells²⁸. The BFB cycle can result in an accumulation of chromosomal rearrangements and fragmentations, while anaphase lagging is a cause of aneuploidy. Although these embryos show a series of chromosomal aberrations, the majority of them can develop into phenotypically healthy adults, indicating that the sperm-carried DSBs could bypass the surveillance of mitotic checkpoints during early embryogenesis and further leads to a mutational cascade through the formation of chromosomal rearrangement, fragmentation, and aneuploidy.

We next characterized the chromosomal states in the adult fog-2 F1 offspring with paternally inherited DNA damage. In the unirradiated control, worms underwent a set of programmed intestinal cell divisions during the late L1 stage that generates the invariant regular pattern of intestinal nuclei in the adult worms. However, in the fog-2 F1 offspring that originated from fog-2 ∆m, we noticed that the divided intestinal nuclei were separated but still connected with chromosomal bridges (Figure 2C). The occurrence of chromosomal bridges indicates the persistence of DNA breaks from embryogenesis until adulthood. In addition, we used the H2B::GFP stain to visualize the chromatin conformation in the somatic tissues of the hermaphrodite F1 offspring from irradiated L4, and observed similar chromosomal bridges in the hermaphrodite F1 worms (Supplementary Figure 2A).

The unresolved DNA damage in germ cells of the F1 generation leads to the embryonic lethality we observed in the F2 generation. To characterize these germ cells, we next evaluated the chromosomal state of the germline of the fog-2 F1 generation that originated from fog-2 ∆m. Compared to control germlines, we observed DNA fragments in the pachytene zone of the fog-2 F1 germlines (Figure 2D; left panels). We further verified the DNA fragmentation by using telomere Fluorescence In Situ Hybridization (tel-FISH), and detected no telomere sequence on those DNA fragments (Figure 2D; left panels). In the diakinesis zone of fog-2 female germlines, the meiotic chromosomes form six highly condensed and discrete oocyte bivalents, which are visible as six DAPI-stained bodies. However, in the fog-2 F1 female germlines, we detected more than six DAPI-stained bodies in the diakinesis zone, indicating aneuploidy (Figure 2D; right panels and E). Importantly, the six oocyte bivalents in the control’s diakinesis-stage oocytes are similar in size, while the DAPI-stained bodies observed in the fog-2 ∆mf’s diakinesis-stage oocytes have irregular size (Figure 2D; right panels). With tel-FISH, we noticed that the telomere sequences are randomly allocated on these DAPI-stained bodies (Figure 2D; right panels and F), and some DAPI-stained bodies even do not have telomere sequence (Figure 2D; right panels and F). Taken together, these observations indicate that the germ cells of the F1 generation display chromosomal rearrangement, fragmentation, and aneuploidy.

To detect de novo genomic aberrations in the fog-2 F1 generation at the DNA sequence level, we next performed whole-genome sequencing (WGS) of single P0 and their direct F1 progeny. The number of mapped reads was comparable between all samples. We detected between 3 and 21 de novo translocations in each female (∆mf) and between 2 and 28 de novo translocations in each male (∆mm) F1 individual. In total, we mapped 154 chromosomal translocations, in the fog-2 ∆mf and fog-2 ∆mm’s genome compared to the parental generation (Figure 2G, Supplementary Table 2). Note, that due to the limitation of this technique, we were not able to detect the chromosomal fragmentations. In contrast to translocations, tandem duplications (n=1), and insertions (n=1) were rare (Supplementary Table 3). All the translocations detected in the F1 animals were heterozygous, consistent with only the paternally inherited chromosomes carrying genomic aberrations. Both female and male progeny genomes’ showed an overall similar number of autosomal translocations. In contrast, the paternal X chromosome is only inherited to fog-2 ∆mf but not to fog-2 ∆mm animals, which only carry a maternal X chromosome. Indeed, in contrast to the similar numbers of autosomal translocations, only the fog-2 ∆mf animals bear X-chromosomal translocations (Figure 2H, Supplement Figure 2B and C, Supplementary Table 3), indicating that the translocations only occur on the paternal chromosomes.

We were wondering whether the X-chromosomal translocations might be responsible for the particularly high progeny lethality in the fog-2 ∆mf. It was recently shown that the integrity of the X-chromosome is essential for the meiotic sex chromosomal inactivation (MSCI), an important process during meiosis conserved from worms to human. When the continuity of the X-chromosome is lost, X-chromosomes fail to undergo the MSCI transcriptional inactivation state resulting in a high progeny lethality²⁹. To verify if the embryonic lethality of female F1’s progeny is due to the disruption of MSCI, we stained a transcription active mark, Phospho-Ser-2 on the C-terminal domain of RNA Pol II (p-Ser2), which indicates the transcriptional state. In the pachytene region of the germline, the X-chromosomes in the untreated fog-2 female were devoid of RNAPII p-Ser2, while autosomes displayed the expected enriched RNAPII p-Ser2 levels. In contrast, in the fog-2 ∆mf, RNAPII p-Ser2 was evenly distributed on both autosomes and X-chromosomes (Figure 2I). Additionally, a gene set enrichment analysis (GSEA) for chromosomal gene distributions indicates a strong upregulation of X-chromosomal proteins in the F1 generation of irradiated WT worms (Figure 2J and Supplementary Table 4). No other chromosome was significantly enriched (Supplementary Figure 3 and Supplementary Table 4). This suggests indeed that the X-chromosome rearrangement in fog-2 ∆mf’s germline could disrupt the MSCI process, which together with chromosomal translocations and fragmentation on autosomes, results in significantly higher progeny lethality compared to the fog-2 ∆mm.

In addition to the fog-2 mutant, we also performed WGS of single hermaphrodite WT F1 worms and their P0 parents. Unlike the dioecious fog-2 system, the self-fertilized hermaphrodites carried on average roughly twice the number of heterozygous translocations in their F1 generation’s genome (Supplementary Figure 2D and Supplementary Figure 4). This could be explained by two possibilities: 1. The hermaphrodites’ sperm is more vulnerable compared to the sperm produced in the male worms; 2. In contrast to self-fertilizing WT hermaphrodites, mating of the fog-2 males with healthy females providing a set of undamaged maternal chromosomes could greatly improve the genome stability. Similarly, to the above result, we also observed the de-silencing of the X-chromosome and the aneuploidy phenotypes in the F1 of irradiated hermaphrodite WT worms (Supplementary Figure 2E, F).

Sperm-inherited DNA damage is mainly repaired by the maternal TMEJ repair mechanism

IR-induced DNA DSBs are mainly repaired by two pathways: the error-free homologous recombination repair (HRR), which uses an undamaged DNA strand as a template for repair and the error-prone non-homologous end joining (NHEJ), which joins the two ends of a break with minimal processing. Apart from the two main repair pathways, there is an alternative repair pathway termed theta-mediated end joining (TMEJ), which is defined by the requirement of the DNA polymerase theta ³⁰. TMEJ was determined to involve a microhomology in DNA flanking the breaks and to use DNA close to the breakpoints as a template for de novo synthesis, which induces insertions in between the breakpoints in ~25 % of cases ³⁰, with 9 % classified as templated insertions. TMEJ is crucial for viability in cells deficient in NHEJ or HR, although it is an error-prone repair mechanism and highly associated with carcinogenesis in mammals ³¹.

To test which DNA repair machinery is involved in the translocation events, we analyzed the sequence context of the pooled 154 de novo translocations identified in the F1 generation of fog-2 animals, using a breakpoint analysis similar to Roerink et al ³⁰. While most translocations showed no insertions in between their breakpoints, ~23 % contained at least 1 inserted base pair, and 6 % had an insertion of ³3 bp whose template could be found within ±25 bp around the breakpoints, thereby matching the TMEJ pattern defined in C. elegans ³⁰. (Figure 3A, Supplement Figure 5A). WT samples showed a similar pattern with 11 % templated inserts (Supplement Figure 5B, C). This suggests that translocation-inducing error-prone DSB repair could use DNA close to the breakpoints as a template for de novo synthesis preceding end joining.

Since the template insertion at the DNA breakpoint is a signature of TMEJ activation, we next ask whether microhomology is present at the flanking regions of DNA breakpoints. To address this, we analyzed the sequence around the breakpoints of the de novo translocations in the F1 generation. We divided the translocations into 4 types based on their sequence orientation (see methods for details), and identified a microhomology in each of the types. For clarity, type 1&3 is displayed in Figure 3B (A translocation with the same strand orientation on both sides can be either annotated from the left or from the right breakpoint. Type 1 and type 3 annotate the same translocation, just once annotated from the 5’ and once from the 3’ direction and are therefore the same. For clarity we refer to both as Type 1 in the following text.) and type 2&4 in Supplement Figure 5D-F. We assessed the degree of sequence similarity in an 8 bp window around the breakpoints. The last base pair within the translocation, i.e., the breakpoint, denoted with 0, the three following bases denoted with positive numbers, and the last four bases that are not included in the translocation, denoted with negative numbers, are shown in the heatmap in Figure 3B (bottom) and Supplement Figure 5E. We identified the same patterns in the genomes of WT F1 animals (Supplement Figure 5F), suggesting that the paternally inherited DSBs use the microhomology-mediated end joining, TMEJ, to repair.

Lastly, we investigated the base composition around the breakpoints. TMEJ-repair outcomes were previously characterized in the experimental conditions when TMEJ is employed as a result of defects in the DNA translesion synthesis (TLS) polymerases eta and kappa ³⁰. Under those conditions, TMEJ-induced deletions show a base composition bias at the first deleted base. However, the breakpoints of the translocations we detected following paternal IR-induced DSBs did not result in the same pattern, which indicates that paternal DSBs-induced TMEJ is independent of the N2-dG and/or 8-oxo-dG adducts caused by deficiency of other TLS polymerases (Supplement Figure 5G). Taken together, the sequence context of DNA breakpoints is highly similar to the outcomes of error-prone TMEJ.

To corroborate the sequence context analysis and to verify which repair pathway is required for the paternally inherited DNA damage, we crossed the mutants of each repair pathway’s core component with fog-2. Intriguingly, upon paternal exposure to IR, the HRR mutant, brc-1; fog-2, did not further increase the progeny lethality of the P0 and F1 generations, compared to the fog-2 mutant (Figure 3C), indicating the paternally inherited DNA damage does not engage the error-free HRR repair pathway. In stark contrast, the TMEJ mutant, polq-1; fog-2, was hypersensitive to the paternal DNA damage, as paternal irradiation caused 89.6 % and 97.98 % progeny lethality of the P0 generation in 60 Gy and 90 Gy groups, respectively (Figure 3C), and the F1 survivors were sterile due to a germline developmental defect. In addition, depletion of NHEJ repair protein CKU-70 further exacerbated the progeny lethality in the cku-70; fog-2 ∆m and cku-70; fog-2 ∆mm. We further verified this observation by using RNAi to knockdown these repair proteins on fog-2 mutant, and our RNAi results were consistent with the finding in mutants (Supplementary Figure 5H). Taken together, these results indicate that the paternal DNA damage is predominantly repaired by the error-prone repair pathways, especially TMEJ, and thus providing a profound explanation for the chromosomal abnormalities observed in the F1 generation.

We next tested whether TMEJ was acting in mature sperm or after fertilization with damaged sperm. To this end, we sex separated the polq-1 mutation and crossed each combination between male and female fog-2 mutants proficient or deficient (polq-1) for TMEJ. Strikingly, only when polq-1 females were mated with IR-treated males regardless of paternal polq-1 status, progeny lethality increased to nearly 100% (Figure 3D). These observations indicate that maternally provided TMEJ is the major route for repair of mature sperm DNA.

Removal of histone H1 and heterochromatin protein HPL-1 can alleviate the sperm-inherited lethality

To further elucidate the mechanisms underlying the paternally inherited DNA damage, we used stable isotope labelling with amino acids (SILAC)-based proteomics to identify the global changes in the proteome of the F1 worms of IR-treated parents. Instead of using fog-2 mutants, we quantified the proteome of F1 animals of hermaphrodites WT P0’s that were irradiated at the late L4 stage (Figure 4A). We chose the hermaphrodite treatment for feasibility as no mating was required and a large number of F1 worms with a homogenous phenotype could be obtained. The GO term analysis of the data revealed that the proteome changes are highly enriched in the biological processes of protein-DNA assembly, nucleosome assembly and nucleosome organization (Figure 4A). We noticed that the majority of the proteins in those categories were up-regulated in the IR F1 worms, especially the histone proteins (Figure 4B). The 3 most highly up-regulated proteins were the linker histone (H1) variants, HIL-3, HIS-24 and HIL-2. We thus wondered whether this increased histone H1 in the F1 generation was responsible for the embryonic lethality in the F2 generation. We, therefore, knocked down these histone H1 proteins in WT animals and examined the embryonic lethality in the F2 generation carrying paternally inherited DNA damage. Knock-down of hil-2 by RNAi led to larval arrest regardless of irradiation (data not shown), while hil-3 RNAi showed only a mild, but not significant, reduction of the F1 progeny’s lethality. Strikingly, RNAi knockdown of his-24 significantly restored the embryonic viability of the WT F2 generation (Supplementary Figure 6A).

We next generated his-24; fog-2 double mutants to examine the rescue effect in the sex-separated model. Indeed, removal of HIS-24 could significantly suppress the transgenerational embryonic lethality caused by paternal IR exposure (Figure 4C). However, the rescue effect of HIS-24 depletion was quickly reduced after maintaining this strain multiple generations, implying that the function of HIS-24 can be gradually compensated by other regulators. To circumvent such an adaptation, we knocked down his-24 in fog-2 mutants by using RNAi and detected a significant rescue effect in the F2 generation –albeit milder than the inactivation of his-24 by genetic mutation. In contrast to the significant restoration of ∆mm progeny’s viability, his-24 RNAi was insufficient to reduce the high lethality levels in progeny of ∆mf, likely because even one aberrant X-chromosome structure is sufficient to prevent the MSCI thus resulting in lethality (Supplementary Figure 6B). These data indicate that the inherited paternal DNA damage leads to an increase in the level of histone H1, which is responsible for the embryonic lethality of the F2 generation.

Histone H1 is a conserved chromosomal protein that is mainly involved in DNA packaging and chromosomal condensation. Previous studies have shown that the histone H1 and heterochromatin proteins function together to regulate chromatin mobility, higher-order chromatin structure, and transcription^32–35. Thus, the up-regulated histone H1 we observed in the F1 generation might be associated with an increase in heterochromatin regions. We thus evaluated the heterochromatin structure by staining the heterochromatin mark, di-methylation on histone 3 lysine 9 (H3K9me2). In the non-irradiated fog-2 mutants, H3K9me2 was weakly, but evenly, distributed on the chromatin of late pachytene cells of the females, while H3K9me2 was strongly enriched on the X-chromosome in the pachytene cells of the males, as it is essential for the X-chromosome silencing during meiosis in the male’s germline (Figure 4D). As predicted, in the germline of the F1 worms, we found a dramatic increase in the level of H3K9me2, and the increased H3K9me2 deposition was not specifically associated with the X chromosome, but randomly distributed (Figure 4D). We next asked if this increased H3K9me2 in the F1 progeny of IR-treated P0 males is due to the elevated histone H1. We thus knocked down his-24 in fog-2 mutants and measured the H3K9me2 level in the F1s’ germline. Indeed, depletion of HIS-24 significantly abolished the elevated H3K9me2 in the germlines of both fog-2 ∆mf and fog-2 ∆mm animals (Figure 4E).

The heterochromatin protein 1, HPL-1, is essential for the establishment and maintenance of the heterochromatin structure. We, therefore, knocked-down HPL-1 in the fog-2 mutant and observed the loss of H3K9me2 in the F1’s germline, similarly as we observed in the his-24 (RNAi); fog-2 (Figure 4E). These observations raise the next question, whether depletion of HPL-1 could mimic the rescue effect of his-24 RNAi. We characterized the progeny viability of the F1 generation of paternally irradiated hpl-1; fog-2 mutant worms, and detected a significant suppression of the progeny lethality (Figure 4F). We verified the restoration of viability by hpl-1 RNAi in fog-2 mutants (Supplementary Figure 6C). Taken together, our observations suggest that the paternal DNA damage causes histone H1 elevation that leads to an increase in heterochromatin formation, which in turn results in the embryonic lethality in the F2 generation.

Depletion of Histone H1 and heterochromatin protein HPL-1 can activate the error-free HRR machinery to repair the paternally inherited DNA damage

Elevated histone H1 and heterochromatization causes chromatin compaction, which is commonly proposed to represent a barrier for faithful DNA DSB repair such as HRR ^36–38. We have established above that the paternally inherited DNA damage does not engage HRR, but instead relies on the error-prone TMEJ and NHEJ repair machineries, which results in genomic aberrations. Therefore, we wondered whether the depletion of HIS-24 and HPL-1 could remove the heterochromatin barrier for the HRR machinery and facilitate the faithful repair. We monitored HRR engagement by foci formation of the RAD-51 protein that mediates the strand invasion reaction and is commonly used in C. elegans as a measure of HRR activation ³⁹. In the empty vector (EV) control, we noticed a modest increase in RAD-51 foci in the germline of F1 generation animals compared to offspring from non-irradiated animals. Strikingly, upon knocking-down the his-24 and hpl-1 in fog-2 mutants, we detected a significant elevation of RAD-51 foci on the germlines of the ∆mm, while the ∆mf’s germline also showed a mild, but significant, increase in RAD-51 foci in the hpl-1 (RNAi); fog-2. (Figure 5A and B). The increased RAD-51 foci are most obvious in the pachytene zone of the ∆mf’s germline and the transition zone of the ∆mm’s germline (Figure 5A and B), indicating an activation of the HRR pathway. Therefore, we hypothesized that the rescue effect we observed in the worms devoid of HIS-24 and HPL-1 is likely due to the activation of the HRR machinery. To verify our hypothesis, we knocked down his-24 and hpl-1 in the brc-1; fog-2 double mutant, which is HRR deficient. Compared to the fog-2 control, the rescue of progeny lethality upon his-24 and hpl-1 RNAi was completely abolished in fog-2; brc-1 mutants, indicating that HRR is mediating the restored viability of the F1 progeny upon H1 depletion (Figure 5C). Moreover, we also confirmed the requirement of HRR for the ability of HIS-24 and HPL-1 depletion to restore viability with hermaphrodite WT and brc-1 mutants (Supplementary Figure 7). Noteworthily, the removal of BRC-1 completely abolished the progeny viability of the H1-depleted F1 offspring but did not affect the progeny viability of the P0 generation, indicating that the H1-mediated DNA repair restriction is mainly involved in the gametogenesis in the F1 generation.

Taken together, we conclude that the paternally inherited DNA damage leads to elevated histone H1 and heterochromatin formation, which in turn prevents the F1 generation from using the faithful DSB repair machinery. Removal of the heterochromatin barrier facilitates the engagement of the error-free HRR and significantly rescues the transgenerational embryonic lethality caused by paternal IR irradiation.

The determination of transgenerational consequences of parental exposure to IR has been limited mainly because the mechanisms underlying this process have remained elusive. Here, we used sex-separated C. elegans strains as a model and establish that paternal exposure to radiation leads to transgenerational lethality in the F2 generation. In addition, we found that maternal POLQ-1 mediates the repair of paternal DNA through utilizing the error-prone TMEJ repair machinery, which ensures the viability of the F1 generation, albeit with severe chromosomal rearrangements in both somatic and germ cells. The embryonic lethality of the F2 generation could be alleviated by knocking down the linker histone H1, HIS-24, by disrupting the heterochromatin formation and activating the HRR machinery in the F1’s germlines. Our work provides insight into the transgenerational consequences of paternal exposure to radiation and uncovers a previously unknown mechanism underlying this phenomenon (Figure 6).

Although the studies on the transgenerational effect of parental irradiation in humans are highly controversial, studies in animals provided evidence that paternal exposure to IR could result in compromised viability, fertility and genome stability in the offspring ⁴⁰. Studies in bovine embryos have shown that exposing mature sperm to IR does not impair the fertilization of oocytes but instead leads to chromosomal fragmentation and translocations of the paternal chromosomes during early embryogenesis ¹. Due to the absence of genomic surveillance in early embryos, these paternal chromosomal fragments are randomly distributed during the first cell divisions, resulting in aneuploidy and unequal chromosome segregation ⁴¹. This phenomenon is called genetic mosaicism, which is the leading cause of the high miscarriage rate and failed implantation rate in in vitro fertilization (IVF) ^42–45. Although this embryonic selection force for genetic mosaicism can greatly eliminate the embryos carrying paternal DNA damage, other studies reported that the viable F1 offspring of irradiated male mice showed increased genomic instability and elevated incidence of cancers ^46,47. These works together suggest that the paternal DNA damage cannot be eliminated by embryonic surveillance and may lead to genome instability in the non-irradiated offspring. Unlike the mammalian system, the holocentric structure of C. elegans chromosomes can ensure equal segregation even of chromosome fragments and thus prevent genetic mosaicism during embryogenesis⁴⁸. Therefore, most embryos carrying fragmented paternal chromosomes can escape the developmental arrest, so that the paternal DNA damage is majorly preserved in the F1 generation. This feature makes C. elegans a powerful tool to investigate the transgenerational effect of paternal DNA damage.

We found that specifically the mature sperm is responsible for the transgenerational effect of IR. This is highly consistent with the correlation in humans between the elevated malignant disease risk in children and paternal radiation exposure that occurred around the time of conception, i.e. when mature sperm has been exposed to radiation ^4,5. Importantly, the specific vulnerability of mature sperm might explain the absence of a transgenerational effect on the survivors of the atomic bomb in Japan and the cleanup workers of the Chernobyl accident, as the radiation effect at the time of conception varied at those accidents. Our data urges that a further analysis in humans should be carried out, especially in the offspring of fathers working in an environment that might be exposed to radiation.

The transgenerational genomic variation originating from paternal exposure to IR has thus far been poorly recognized. Animal studies have revealed that a high dose of IR can lead to de novo mutations in offspring ^13–15,23, although recent studies in humans failed to support this finding ^11,17. Some studies reported that paternal irradiation leads to an elevated mutation rate at Expanded Tandem Repeat Loci (ESTRs) in the offspring, and this transgenerational instability was detected in both germline and somatic tissues in mice ^49–51. In addition, the frequency of chromosomal aberrations, including chromosomal bridges, is increased in the progeny of male mice exposed to IR ^46,47,52. In contrast, maternal irradiation does not increase the mutation rate in F1 offspring, in line with the observation that the paternal germline is responsible for the majority of transmitted mutations. Here, instead of focusing on the mutation rate at specific loci, we monitored the chromosomal aberrations from the mature sperm to the F1 adulthood, and we observed significantly elevated chromosomal rearrangement, chromosome fragmentation, and template insertion in the F1 generation of IR-exposed P0 males.

Interestingly, we noticed that there is a significant distinction of transgenerational effect between the female the male offspring of the irradiated male. This distinction can be explained by the fragmented X-chromosome the female progeny inherited from the father. We suggested that paternal hereditary sex-chromosome rearrangement in the female progeny is associated with the disruption of MSCI, which is consistent with a recent finding that the integrity of X-chromosome is essential for the maintenance of MSCI²⁹. However, our current data cannot rule out the possibility that paternal irradiation-induced specific mutations in the female offspring are responsible for the high progeny lethality.

Since the chromatin in mature sperm is highly compacted precluding the engagement of effective DSB machineries, it relies on the maternal factors to repair the paternal DNA damage upon fertilization ²⁷. Depletion of the HRR and NHEJ factors in mice could significantly increase the chromosomal structural aberrations in the early embryos carrying paternal DNA DSBs ²⁷. However, we found that in C. elegans, instead of HRR or NHEJ, the maternal TMEJ is the predominant repair machinery responsible for the paternal DNA repair after fertilization. TMEJ has thus far been considered as a “back-up” mechanism to canonical HRR and NHEJ pathways ^30,31. Unexpectedly, our data establish that TMEJ is the major DSB repair mechanism acting on the paternal genome upon fertilization. To illuminate how specifically TMEJ is engaged, it will be important to understand the recruitment mechanism of polymerase theta to paternal DSBs. Previous studies showed that the poly (ADP-ribose) polymerase 1 (PARP1) and Fanconi anemia group D2 protein (FANCD2) are involved in the recruitment ^53–55. It will be interesting to examine the role of these potential recruitment factors in the transgenerational DNA damage response (DDR) in the future.

The specific footprint of TMEJ is the presence of template insertions, although the microhomology-associated deletions are more frequently detected ^56,57. TMEJ activity has been less associated with the formation of chromosomal translocations, which is commonly correlated with NHEJ ⁵⁸. Although one study has shown that the TMEJ promotes translocation in cultured cells ⁵³, other studies suggested that the TMEJ suppresses translocation in mice and human cell lines ^31,59,60. Our findings add more complexity to this debate, as the WGS data indicate that TMEJ mainly promotes the chromosomal translocations in the progeny of irradiated males. A recent study might provide a potential explanation for this phenomenon ⁶¹: The V(D)J recombination intermediates in immune cells mainly rely on the NHEJ to repair the DNA DSBs during the G1 phase of the cell cycle. In the cells deficient in NHEJ, the unrepaired DSBs can only be resolved by the TMEJ after the cells enter the S phase, which provided the S phase-dependent degradation of RAG proteins and phosphorylation of CtBP-interacting protein (CtIP) to perform DNA end resection. The consequence of TMEJ in S phase is the formation of large deletions and a more than 100-fold increase in chromosomal translocations ⁶¹. Thus, our finding on TMEJ mediated DNA repair might potentially be associated with cell cycle regulation.

Previous studies on radiation-induced transgenerational genome instability have raised an important question: why is the genome stability compromised in the non-irradiated offspring? For example, endogenous single and double-strand breaks were significantly elevated in the F1 offspring of irradiated male mice. It was suggested that epigenetic factors, such as non-coding RNAs and DNA methylation, might contribute to the elevated DNA damage ^8,24,25. One of the studies has reported that paternally inherited mutations are highly correlated with H3K9me3 deposition, which is a mark of heterochromatin ²³. Since the condensation of heterochromatin is an impediment to DNA repair ³⁶, elevated heterochromatin marks provides a potential explanation for the genome instability in the offspring of irradiated males ³⁴. Indeed, we observed the increase of heterochromatin structure in the germline of the F1 offspring, and this heterochromatin formation is triggered by the elevated linker histone H1. The role of histone H1 in DNA repair has been subject to debate. Post-translational modifications on histone H1 were suggested to amplify DDR signaling and promote DNA damage recognition by the HRR machinery ⁶², while other studies reported that depletion of histone H1 up-regulates the HRR activity and increases resistance to DNA damage ^63,64. Our observations are highly consistent with the latter scenario. However, due to the lack of a specific antibody against C. elegans HIS-24, the recruitment dynamics of histone H1 in the F1 generation will need to be further elucidated. In addition, histone H1-mediated heterochromatin formation and transcription regulation require the recruitment of heterochromatin protein 1 (HP1), as the methylation mark on vertebrate histone H1 is specifically recognized by the chromodomain of HP1 ³². Consistently, we report that the HP1, i.e., HPL-1 in C. elegans, functions downstream of histone H1 to regulate the genome stability in the offspring of paternal exposure to irradiation.

Taken together, our work elucidates a previously unknown mechanism underlying the transgenerational effect of paternal exposure to radiation. We here establish that maternally provided TMEJ mediates repair of damaged paternal DNA and that linker histone H1-triggered heterochromatization shelters DSBs from HRR thus leading to transgenerational inheritance of genome instability. This novel transgenerational genome instability mechanism mandates further evaluation of transgenerational effects of paternal DNA damage in humans.

Acknowledgements

We thank the CECAD proteomics and imaging facilities. Worm strains were provided by the National Bioresource Project (supported by The Ministry of Education, Culture, Sports, Science and Technology, Japan), the Caenorhabditis Genetics Center (funded by the NIH National Center for Research Resources, US), and the C. elegans Gene Knockout Project at the Oklahoma Medical Research Foundation (part of the International C. elegans Gene Knockout Consortium). D.M. was supported by the Cologne Graduate School of Ageing Research. B.S. acknowledges funding from the Deutsche Forschungsgemeinschaft (SCHU 2494/3-1, SCHU 2494/7-1, SCHU 2494/10-1, SCHU 2494/11-1, SCHU 2494/15-1 CECAD EXC 2030 – 390661388, SFB 829, KFO 286, KFO 329, and GRK 2407), the Deutsche Krebshilfe (70112899), the H2020-MSCA-ITN-2018 (Healthage and ADDRESS ITNs) and the John Templeton Foundation Grant (61734).

B.S is co-founder of Agevio Therapeutics, Inc.

Author Contributions

D.M. performed all bioinformatics analysis. S.W. performed and analyzed the experiments and together with B.S. conceived and designed the study, and all authors wrote the manuscript.

C. elegans strains

All strains were maintained based on standard conditions at 20°C. Strains used were N2 (Bristol; wildtype), 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 IR-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 IR inflicted by a cesium 137 irradiation source (Biobeam GM 8000, Eckert & Ziegler, Gamma-Service Medical GmbH). Afterwards, ten irradiated adult worms and ten non-irradiated opposite-sex adults were immediately transferred to three new plates served as three biological replicates and allowed to lay eggs for 2h. Then the males were removed and left the females to continue egg-laying for another 4h. The females were then removed and the number of eggs was counted. The number of survived progenies was characterized 24h later to examine the progeny lethality of the P0 generation. Then we transfer three survived male progenies (F1) and survived female progenies (F1) to new plates and placed them with the three untreated opposite-sex adults and allowed them to lay eggs for one day. The adults were then removed and the number of laid eggs was counted. 24h later, the survived 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 ten late L4 hermaphrodites were either remain untreated or irradiated with the indicated dose of IR. Three irradiated hermaphrodites were transferred to three separated plates as three biological replicates and allowed to lay eggs for 6h. The adults were then removed and the number of eggs was counted 24h later, the hatched progenies were characterized as the progeny lethality of the P0 generation. The survived 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 characterized 24h later.

RNAi treatment

RNAi feeding clones were obtained from Julie Ahringer’s library. The E. coli feeding strain HT115 (DE3) with RNAi clones were cultured with LB medium containing ampicillin (100 μg/mL) 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, 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 (EV) control. Three days later, adult males and females were separated and transferred to fresh RNAi plates for maintaining the RNAi efficiency until performing further experiments. The subsequent experiments were performed as described in the “Measurement of IR-induced progeny lethality”.

Immunofluorescence staining

Adult worms were picked from plates and transferred to a drop of M9 buffer onto a 0.3 % poly-lysine treated three-well slide (3 x 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 1h. Then a 24 x 24 mm coverslip was placed onto the drop, and the slide was left in -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 1x PBS and 2 times with 1x PBT (0.5% Triton X-100 in PBS). To improve the signal quality, slides were first blocked for 20min with Image-iT FX signal enhancer (Thermo Fisher/Invitrogen, Carlsbad, CA) before blocking with 1X PBT containing 10% Donkey Serum for another 20min. Afterwards, primary antibodies diluted with 1 X PBT containing 5% Donkey Serum were applied to the slides and incubated at 4°C overnight. After 3 times washing with 1x PBT, the slides were incubated with secondary antibodies diluted with 1x PBT at 37 °C for 30 min. Then slides were washed with 1x PBT for 3 times and mounted with DAPI Fluoromount-G mounting medium (Southern Biotech) and sealed with nail polish. Slides were stored at 4 °C in dark before imaging.

Primary antibodies used for immunofluorescence staining are Rabbit polyclonal anti-phospho-RNA Polymerase II (Ser2) antibody (Thermo Fisher/Invitrogen,Cat. No.: 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, Cat. No. 41980002, dilution 1:100 in PBT); Rabbit anti-RAD-51 antibody is a kind gift from Anton Gartner’s lab (dilution 1:2000 in PBT). Secondary antibodies used are AlexaFluor 488 donkey anti-mouse IgG (Thermo Fisher/Invitrogen ,Cat. No.: A21202, dilution 1:500 in PBT) and AlexaFluor 594 donkey anti-rabbit IgG (Cat. No.: A21207, dilution 1:500 in PBT; Thermo Fisher/Invitrogen).

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, phosphor-RNA Pol II (Ser2), and RAD-51 staining, Z-stack images were acquired with Zeiss Meta 710 confocal microscope, and the H3K9me2 and phosphor-RNA Pol II (Ser2) signal intensity and the foci number of RAD-51/nucleus were measured with Imaris x64 9.1.2 software. All of the fluorescence intensity was normalized to DAPI signal.

SILAC assay

Stable isotope labeling procedure was described in a previous study ⁶⁴. Briefly, the ET505 (lysine auxotrophy, from Coli Genetic Stock Center) was 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 % (wt/vol), MgSO₄ 1 mM, Thiamine 0.01 % (wt/vol) and 40 µg/ml heavy isotope labeled lysine (¹³C₆) (Cambridge isotope laboratory), and incubated at 37 °C over-night to reach OD₆₀₀ = 1. Concentrate bacterial to OD₆₀₀ =50, and seeded to NGM-N plates (3 g of NaCl and 12 g of agarose dissolved in 970 ml of 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 lysine labeled ET505 E. coli. Worms were fed with labeled bacteria for two generations to reach the incorporation rate >97 %, and then picked the late L4 stage worms to irradiate with IR or mock-treated.

The F1 adult worms were washed off from plates with M9 buffer and concentrated, and 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). Repeat the heating and sonication one more time. Then samples were centrifuged at 20,000 g for 20 min, and the supernatant was collected. Take 5 µl of protein solution and dilute with 20 mM Tris to reduce the concentration of GuCl to lower than 0.6 M. Add 50 mM TEAB to dilute the samples to 100 µl and then add 1 µg Lys-C to incubate at 37 °C for 4 h. Samples were further diluted with 180 µl TEAB and treated with 2 µg Lys-C at 37°C over-night. 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 ⁶⁵ partner repository (https://www.ebi.ac.uk/pride/archive/login) with the dataset identifier PXD031873 and can be accessed with the review login Username: [email protected] Password: jv1oIGJs.

Single worm whole-genome sequencing

Single L4 male and female fog-2, and hermaphrodite WT with indicated treatment were transferred to plates with UV-killed OP50 bacteria, in order to reduce the contamination of bacteria’s 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. Breifly, 120ng 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. This data is available at https://dataview.ncbi.nlm.nih.gov/object/PRJNA826255?reviewer=1fgv56rrnihr9lgl5069nea610 .

Fluorescence In situ hybridization (FISH) for telomere

The telomere FISH was carried out by a modified protocol from the Seo and Lee⁶⁵. Briefly, 20 adult females or males were from plates, and transferred to a drop of M9 buffer onto a 0.3 % poly-lysine treated three-well slide (3 x 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 x 24 mm coverslip was placed onto the drop, and the slide was left in -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 1X PBS and incubated in permeabilization buffer (0.5% Triton X-100 in 1X PBS) for 1h at room temperature followed by a wash in 1X PBS. Then slides were quickly washed with 0.01N HCL followed by a wash with 0.1N HCL for 2 min. To prevent unspecific binding of the FISH probe, 50 µg/ml RNase A solution (10 µg/ml RNase A in 1XPBS) was added to the slide and incubated at 37 °C for 45 min. Afterwards, slides were washed two times with 2X SSC. For pre-hybridization, 50 µl of pre-hybridization solution (2X SSC, 50% formamide) was added on the slides and incubated in a humid chamber for 2h at room temperature. Then the FISH probe (PNA-FISH TTAGGC telomeric probe, Panagene, resuspended to 100µM, fluorophore: Cy3) was diluted as 1:500 in hybridization buffer (2XSSC, 50 % formamide, 10 % (w/v) Dextran-sulfate, 50 µg/ml Heparin, 100 µg/ml 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, Cat. No. 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 two days. Afterwards, the hybridization chambers were removed, and slides were washed in 2X SSC for 5 min at room temperature. To fixate the staining, the slides were washed three times in preheated 2X SSC at 37 °C, and twice in preheated 0.2 X SSC at 55 °C. As the last step, slides were washed in 1X PBT for 10 min at room temperature and mounted by 7 µl Vectashield mounting medium containing DAPI (Vector Laboratories, Cat. No. H-1200-10).

Staining images were taken Zeiss Meta 710 confocal laser scanning microscope was used. To visualize the telomeric signal, Z-stack images were acquired with Zeiss Meta 710 confocal microscope, and the Z-stack pictures were processed and the number of DNA fragments was counted with Image J/Fiji.

Bioinformatics analysis

Gene set enrichment analysis (GSEA)

The enrichment analysis for chromosomal gene distributions was done in R v3.6.3 with the GSEA function of clusterProfiler v3.14.3 was used with maxGSSize=20000 and nPerm=20000 ⁶⁶.

WGS preprocessing

The fastq files were preprocessed with Fastp v0.20.0⁶⁷, and mapped with BWA-0.7.17 ⁶⁸with the parameters bwa mem -M -K 100000000, and the reference genome ce11. The mapped files were converted to BAM and sorted with samtools v1.6⁶⁹, and duplicated reads were removed with GATK v4.1.0.0 MarkDuplicates⁷⁰.

Structural variant calling

Structural variants (SV) were called with Manta v1.6.0⁷¹ and only SVs that passed all of Mantas quality filter were used further on. To find SVs that are newly induced in the F1 generation, SVs that overlapped with any SV of any P0 sample were filtered out (Full structural variation sites with or without filtering in Supplementary Table 4). Repeat regions are difficult to map and identify, we therefore deleted any INDEL that lies within a repeat region, or any translocation for which both break points are overlapping with the same repeat class. Manta calls translocations in both directions as 2 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 2 break points and can appear in 4 different ways in a VCF file. The reference string s is replaced by t after, respective before, the fusion to position p. This can happen in 4 ways:

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.
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.
Type 3:]p]t The genomic location extending left from the position p is fused before t. This is the same as Type 1.
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 v0.4.12 ⁷² in R v3.6.3 ⁷³ 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, matching bases in an 8x8 grid around the break sites were scored 1, respective 0 for nonmatching bases. One map therefore contains a 1 for each of the 64 combinations that have the same base, and 0 otherwise. The heatmaps shown in the figure contain the sum of all such respective heatmaps divided by the total number of translocations. For each of the 4 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 100000 permutations. To calculate the adjusted p-value for the 64 bins statsmodels v0.11.1 ⁷⁴ multipletests methods with the parameter method=’fdr_bh’ in Python 3.6 ⁷⁵ was used.

Base Composition

For all translocations with a microhomology of length 1 (n=35 for fog-2, and n=58 for WT) 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, i.e., 35 for fog-2 and 58 for WT, and calculated the average percentage of each A, C, T, and G for 25.000 such permutations.

Data presentation and statistical analysis

All the data and statistical significances were analyzed using the GraphPad Prism 7 software package (GraphPad). Statistical methods, p value, 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% Cl was used as these data types contain outliers.

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Supplementary Figure 1. IR exposure of hermaphrodite WT in the late L4 stage leads to extended transgenerational embryonic lethality. A. Transgenerational lethality characterization for IR irradiated WT hermaphrodites at the L3 (start of spermatogenesis), young L4 (ongoing spermatogenesis) and late L4 stage (completed spermatogenesis). Ten worms were included for each biological replicate, and n=3 biological replicates are shown. Error bars indicate median with 95% CI. B. Progeny lethality characterization for multiple generations upon IR irradiation at the late L4 stage of hermaphrodites P0. Ten worms for each dosage group were included for each biological replicate, and n=3 biological replicates were shown. Error bars indicate median with 95% CI.

Supplementary Figure 2. IR exposure in the late L4 stage of hermaphrodite WT leads to chromosomal rearrangement, and aneuploidy in the F1 generation. A. Representative fluorescence microscopic images of hermaphrodite WTs’ intestinal cells expressing H2B::GFP with or without parental IR irradiation (90 Gy). Chromosomal bridging was observed in the F1’s intestinal cells. Scale bar = 10 μm. B and C. Circos plots showing the novel chromosomal translocations in six fog-2 ∆mf (B) and eight fog-2 ∆mm (C). The list of all translocations is shown in Supplementary Table 3. D. Whole-genome sequencing analysis shows the number of new chromosomal translocations in the hermaphrodites WT adults (F1) with parental IR exposure (90Gy). n=11, the box depicts the Median with the top and bottom quartiles, while the whiskers extend to 1.5 IQR. The number of new translocations and of mapped reads per sample is shown in Supplementary Table 2. E. Characterization of the DAPI-stained bodies in the late diakinesis oocytes of hermaphrodites WT adults (F1) with or without parental IR exposure (90 Gy). F. Merged images of immunofluorescence co-staining RNA Pol ll p-Ser2 (green), HIM-8 (red) and DAPI (blue) of dissected germlines from hermaphrodite WT adult (F1) with or without parental IR exposure (90 Gy). Three representative nuclei were shown on the right side. Scale bar = 20 μm.

Supplementary Figure 3. Gene set enrichment analysis on Chromosome I to Chromosome V of SILAC proteomic data from hermaphrodites WT (F1) with parental IR exposure.

Gene set enrichment analysis (GSEA) of SILAC proteomic data from hermaphrodites WT (F1) with parental IR exposure (90 Gy) for chromosomes I to V. The top of each panel shows the log2 fold changes of the proteomics data in a ranked order. The bottom panels show the running enrichment score as a green line. The dashed red line depicts the positions furthest from 0. None of the chromosomes is significantly different from a random distribution. The full statistics for each chromosome is provided in the Supplementary table 4.

Supplementary Figure 4. Circos plots showing the chromosomal translocations in ten hermaphrodites WT (F1) with parental IR (90 Gy) exposure. The list of all translocations is shown in Supplement table 3.

Supplementary Figure 5. The hermaphrodite WT used TMEJ to repair the inherited DNA damage. A. Density plot showing the distribution of the template positions of the insertions found in between the translocation sites of fog-2 adults (F1) with paternal exposure to IR (90 Gy). Only insertions are included that are at least 3 bp and whose templates were found within ±25 bp around one of the breakpoints (corresponding to the templated insertions in Figure 3A). The orange ticks on the x-axis show the positions where the templates were found. The blue curve depicts the kernel density estimation as a non-parametric way to estimate the probability density function of the found positions. The black lines indicate the distance cutoff of ±25 bp from the breakpoints. B. Distribution of translocation footprints in the WT adults (F1) with paternal exposure to IR (90 Gy). Templated inserts are insertions of ≥ 3 bp in between the fusion sites that have a matching sequence within ±25 bp around one of the 2 breakpoints. Miscellaneous insertions are insertions < 3 bp or insertions with no matching sequence within ±25 bp around the breakpoints. C. The same density plot as in (A) but for WT adults (F1) with paternal exposure to IR (90 Gy) (corresponding to the templated insertions in Supplement Figure 3B). D. Schematic illustration of the type 2 (top) and type 4 (bottom) translocations. The sense strand is in blue, while the anti-sense strand is in red. The numbers indicate the nucleotide distance to the breakpoints. Type 2 translocations are fusions between the 3’ sense strand with the 5’ anti-sense strand. Type 4 translocations are fusions between the 3’ anti-sense strand with the 5’ sense strand. The yellow boxes indicate the positions that show a microhomology. Type 2 shows a microhomology of the break point of the left flank with the second base of the negative strand of the right flank. Type 4 shows a microhomology of the second to last base of the excluded part of the right flank, and the last base of the excluded part of the left flank. The microhomology of type 4 is therefore not in the regions that get fused. E. The heatmaps represent the sum of all type 2 (top, n=51), respective type 4 (bottom, n=45), translocation maps derived from the fog-2 adults (F1) with paternal exposure to IR (90 Gy), excluding translocations with a templated insertion. Color-coded is the ratio of similarity. A darker color shows a higher sequence similarity between the corresponding bases. The numbers on the x- and y-axes are the same as in the explanation in Supplement Figure 3D. F. Similar to (E) but for WT adults (F1) with parental exposure to IF (90 G y). Type 1 (n=65), type 2 (n=57) and type 4 (n=58) translocation maps derived from the WT adults (F1) with parental exposure to IR (90 Gy), excluding translocation with a templated insertion. G. Base composition around the break points of the translocations with a microhomology of length 1 for fog-2 (left, n=35) or WT (right, n=58). Position 0 indicates the break point. Negative numbers are bases not included after the fusion, while positive numbers depict the distance from the fusion site and are included after the fusion. Color-coded are the 4 bases, the shadow in the background shows the distribution of AT, respective CG, for 25.000 permutations. The y-axis shows the percentage of the corresponding base at the given position. H. Knockdown of EV, brc-1, cku-80 and polq-1 by RNAi on the fog-2 mutant with or without paternal IR exposure. n= 3 for P0 generations and n ≥4 for the F1 generations. Medians with 95% CI are shown (p-values are shown. Ordinary one-way ANOVA with Bonferroni’s multiple comparisons test). Full statistical results are shown in supplementary table 1.

Supplementary Figure 6. HIS-24 regulates the paternally inherited embryonic lethality. A. RNAi screening of ten DNA-associated protein candidates on the hermaphrodite WTs for the paternally inherited transgenerational embryonic lethality. Knockdown of his-24 could significantly reduce the progeny lethality in the F1 generation. n=3. Medians with 95% CI are shown (p-values are shown. Ordinary one-way ANOVA with Bonferroni’s multiple comparisons test). B. Progeny lethality characterization of EV (RNAi); fog-2 and his-24 (RNAi); fog-2 with or without paternal IR irradiation (60 Gy and 90 Gy). n= 3 for P0 generations and n ≥6 for the F1 generations. Medians with 95% CI are shown (p-values are shown. Ordinary one-way ANOVA with Bonferroni’s multiple comparisons test). C. Progeny lethality characterization of EV (RNAi); fog-2, his-24 (RNAi); fog-2, and hpl-1 (RNAi); fog-2 with or without paternal IR irradiation (90 Gy). n= 3 for P0 generations and n ≥6 for the F1 generations. Medians with 95% CI are shown (p-values are shown, Ordinary one-way ANOVA with Bonferroni’s multiple comparisons test). Full statistical results are shown in supplementary table 1.

Supplementary Figure 7. Removal of BRC-1 abolishes the rescue effect of his-24 RNAi in hermaphrodite WT progeny. Knockdown of EV, his-24, hpl-1 and hpl-2 on the hermaphrodite WT and brc-1 mutants with parental IR exposure (90 Gy) at the late L4 stage, and characterization of the progeny lethality at the F1 generation. Medians with 95% CI are shown (p-values are shown. Ordinary one-way ANOVA with Bonferroni’s multiple comparisons test). Full statistical results were shown in supplementary table 1.

Yes there is potential Competing Interest. B.S is co-founder of Agevio Therapeutics, Inc.

Sup.tab.1.xlsx
Supplementary Table 1. The complete statistical results for this manuscript.
Sup.tab.2.xlsx
Supplementary Table 2. Number of the new translocations in the F1 offspring per sample.
Sup.tab.3.xlsx
Supplementary Table 3. Complete list of called chromosomal structural variation sites before and after filtering with P0.
Sup.tab.4.xlsx
Supplementary Table 4. The results of the proteomic gene set enrichment analysis (GSEA) for chromosomal enrichment.
Suppl.Figures270422.pdf
Supplementary Figure 1-7

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Transgenerational inheritance of paternal DNA damage by linker histone H1-mediated DNA repair restriction

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