A cell non-autonomous FOXO/DAF-16-mediated germline quality assurance program that responds to somatic DNA damage

Germline integrity is critical for progeny fitness. Organisms deploy the DNA damage response (DDR) signalling to protect germline from genotoxic stress, facilitating cell-cycle arrest of germ cells and DNA repair or their apoptosis. Cell-autonomous regulation of germline quality is well-studied; however, how quality is enforced cell non-autonomously on sensing somatic DNA damage is less known. Using Caenorhabditis elegans, we show that DDR disruption, only in the uterus, when insulin-IGF-1 signalling (IIS) is low, arrests germline development and induces sterility in a FOXO/DAF-16 transcription factor (TF)-dependent manner. Without FOXO/DAF-16, germ cells of the IIS mutant escape arrest to produce poor quality oocytes, showing that the TF imposes strict quality control during low IIS. In response to low IIS in neurons, FOXO/DAF-16 works cell autonomously as well as non-autonomously to facilitate the arrest. Activated FOXO/DAF-16 promotes transcription of checkpoint and DDR genes, protecting germline integrity. However, on reducing DDR during low IIS, the TF decreases ERK/MPK-1 signaling below a threshold, and transcriptionally downregulates genes involved in spermatogenesis-to-oogenesis switch as well as cdk-1/Cyclin B to promote germline arrest. Altogether, our study reveals how cell non-autonomous function of FOXO/DAF-16 promotes germline quality and progeny fitness in response to somatic DNA damage. Significance Statement Reproductive decisions are supervised processes that take into account various inputs like cellular energy availability and status of damage repair in order to ensure healthy progeny. In this study, we show that the absence of optimal DNA damage repair in the somatic uterine tissues prevents oocyte development by the cell-autonomous as well non-autonomous function of activated FOXO transcription factor DAF-16. Thus, this study elucidates a new surveillance role of FOXO/DAF-16 in somatic tissues that ensures progeny fitness.


2
The propagation of a species depends on a healthy and productive germline. The stability 3 of the genome is constantly under threat from extrinsic as well as cell-intrinsic genotoxic agents.

4
Thus, all organisms invest heavily on protecting the germline against DNA damage. Generally, in 5 response to DNA damage, an organism deploys an array of countermeasures. Depending on the 6 type of DNA damage, organisms employ lesion-specific DNA repair pathways that can restore 7 damage inflicted by ultra-violet rays (UV), ionizing radiation (IR) or reactive oxygen (ROS) and 8 nitrogen species (RNS). Apart from these highly specialized DNA repair mechanisms, organisms 9 also depend on DNA damage response (DDR) signaling to activate damage-responsive 10 checkpoints, leading to cell cycle arrest to repair the damage or apoptosis, when damage is beyond 11 repair. Perturbation of the DDR, in turn, leads to unrepaired DNA damage, genomic instability and 12 are the basis of many human diseases like cancer, neurodegeneration as well as aging (1).

13
Unrepaired DNA lesions in the germline can lead to infertility, reduced progeny fitness and birth somatic and germline quality assurance. Mutations in the neuroendocrine IIS pathway activate 23 FOXO/DAF-16 to arrest development at dauer diapause (5, 6). The TF mediates arrest at the L1 24 larval stage when food is depleted (7). Further, activated FOXO/DAF-16 delays aging, enhances 25 resistance to stresses and increases life span under conditions of lowered IIS (8) , (9, 10). These IIS 26 mutant animals maintain their germline stem cell pool even at an advanced age, and so, have 27 delayed reproductive aging (11). They produce better quality oocytes (12) with low chromosomal 28 abnormalities as compared to wild-type (WT), but the mechanism is less understood (13).

29
Interestingly, the IIS receptor DAF-2 functions cell non-autonomously in the neuron whereas DAF-30 16 works in the intestine to regulate longevity (14, 15). The long reproductive span or higher oocyte 31 quality of the daf-2 mutant is dependent on muscle or intestinal DAF-16 activity (13). However, it is 32 4 not known whether activated FOXO/DAF-16 can sense DNA damage in somatic tissues and 1 modulate germline development cell non-autonomously.

3
Here, we show that in C. elegans, a uterine tissue-specific perturbation of DDR in the IIS 4 pathway mutants prevents germ cells from exiting the pachytene stage of meiosis and inhibits 5 oogenesis. For disruption of DDR and inducing DNA damage, we knocked-down (KD) cdk-12 that 6 is required for the transcription of DDR genes (16,17). This sterility is reversed in the absence of 7 DAF-16, leading to the production of poor-quality oocytes and developmentally retarded progeny.

8
We elucidate the cell autonomous as well as non-autonomous requirements of the IIS pathway and 9 FOXO/DAF-16 in orchestrating the arrest. We show that this is achieved by downregulating 14 15

17
The cyclin-dependent kinase gene cdk-12 genetically interacts with the IIS pathway 18 19 We were interested in identifying genes that when knocked down induce chronic stress 20 signaling, thereby enhancing dauer formation of the IIS receptor mutant daf-2(e1370) (referred to 21 as daf-2) strain. Knocking down cdk-12 using RNAi led to a significant increase in dauer formation 22 ( Figure 1A). In line with its possible role in inducing stress, cdk-12 RNAi, initiated at L4, reduced 23 life span of wild-type (WT), daf-2, daf-16(mgdf50) (referred to as daf-16) and daf-16;daf-2 to an 24 equal extent (Figure S1A-D, Table S1). Thus, CDK-12 depletion may cause chronic stress to the 25 worms, thereby increasing dauer of daf-2 and reducing life span in general.

27
Cdk-12 depletion during low IIS leads to DAF-16A isoform-dependent pachytene arrest of 28 germline and sterility

30
Considering cdk-12 knockdown may potentially induce stress, we asked whether this would 31 affect progeny production. Interestingly, we found that the daf-2 worms became sterile when they 32 were grown on cdk-12 RNAi from L1 onwards ( Figure 1B, C). The sterility is DAF-16-dependent 33 as fertility was restored in daf-16;daf-2, signifying that DAF-16 regulates the germline arrest in daf-34 5 2 ( Figure 1B, C). Importantly, this was not due to differential RNAi efficiency in the strains (Figure 1 S1E).

3
The C. elegans hermaphrodite gonad has two U-shaped arms carrying germline stem cell 4 (GSC) pool near the distal end, which divide mitotically and then enter meiotic prophase as they 5 move away from the distal tip. Germ cells in meiosis produce sperms during larval 4 (L4) stage, 6 and after the spermatogenesis to oogenesis switch (18,19), generate oocytes or undergo 7 programmed cell death. The proximal gonad contains a stack of oocytes, followed by sperms 8 residing in the spermatheca. Both arms have a common uterus, where fertilized eggs are stored 9 until hatching (20) ( Figure 1D).

11
To visualize the germline, we dissected the gonads of Day 1 adult animals and stained 12 them with DAPI. Confocal imaging showed that in cdk-12 RNAi-fed daf-2 worms, sperms were 13 formed but oogenesis halted due to arrest of germ cells in the pachytene stage of meiosis. In daf-14 16;daf-2, the arrest was reversed and oocyte formation ensued ( Figure 1E). Notably, upon cdk-12 15 KD, the number of oocytes is reduced independent of DAF-16 ( Figure 1F). The number of germ 16 cells of daf-2 in the pachytene stage of meiosis was drastically reduced upon cdk-12 KD ( Figure   17 1G, H); however, in daf-16;daf-2 worms the reduction was largely abrogated ( Figure 1I

26
(DAF-16b rescued) and daf-16;daf-2;daf-16d/f(+) (DAF-16d/f rescued) to find that the effect is 27 mainly driven by DAF-16a ( Figure 1K, L). Previously, DAF-16a isoform has been shown to play a 28 major role in regulating lifespan, stress resistance and dauer formation (9, 15, 24, 25). Here, we 29 show a predominant role of DAF-16a in preventing the pachytene exit of germ cells in daf-2 when  Since the daf-16;daf-2 worms produce oocytes when cdk-12 is knocked down, in contrast 35 to daf-2, we determined the quality of oocytes. As previously reported, we also found the oocytes 36 6 produced on day 3 of adulthood by daf-2 to be of superior quality in comparison to the wild-type 1 worms (13). However, in daf-16;daf-2, the quality deteriorates significantly ( Figure S2A-C) 2 indicating the noted role of DAF-16 in the maintenance of better oocyte quality in daf-2. The quality 3 of oocytes after cdk-12 KD decreased in a DAF-16-independent manner (Figure 2A, B). It may also 4 be noted that cdk-12 KD decreases the number of hatched progenies in all strains; however, no 5 brood is generated in daf-2. The brood size is partially rescued in daf-16;daf-2 ( Figure 2C).

7
Although most of the eggs that are laid by the daf-16:daf-2 on cdk-12 RNAi worms hatched 8 ( Figure 2D), they failed to reach the L4 stage ( Figure 2E, S2D), indicating sub-optimal oocyte 9 quality. Also, endomitotic oocytes (emo) that often develop due to defective fertilization (26), were 10 more frequent in the proximal gonad of wild-type and daf-16;daf-2 worms that were fed with cdk-11 12 RNAi, compared to the daf-2 worms ( Figure 2F). Thus, we conclude that cdk-12 plays an 12 important role in maintaining oocyte quality and activated DAF-16, under conditions of lowered IIS, 13 enforces a germline quality assurance program that prevents the production of inferior quality To understand the connection between the IIS pathway and CDK-12, we performed 20 transcriptomics analysis at late L4 stage of WT, daf-2 and daf-16;daf-2 worm grown on control or 21 cdk-12 RNAi from L1 onwards. We found a large transcriptional response in daf-2 when cdk-12 is 22 knocked down but not to that extent in WT (data not shown). Importantly, genes downregulated in 23 daf-2 on cdk-12 RNAi are enriched for cell cycle, oogenesis, early embryonic development and 24 hatching as well as DNA replication, repair processes ( Figure 3A). When we compared the 25 expression of the germline genes between daf-2 and daf-16;daf-2, we found two distinct clusters, 26 with one dependent on and the other independent of DAF-16 ( Figure 3B). The fact that many 27 important germline genes are downregulated in daf-16;daf-2 supports our earlier observation that 28 the quality of oocytes of the double mutant is poor. Out of the 4126 DAF-16-dependent genes 29 upregulated in daf-2, 987 are also regulated by cdk-12 (Dataset S1), Similarly, out of the 1478 30 DAF-16-dependent genes downregulated in daf-2, 329 are cdk-12 target, showing that DAF-16 and 31 CDK-12 have shared transcriptional targets.

33
In mammalian cells, CDK12 specifically regulates genes involved in DNA damage 34 response (17, 27). We also found the DDR gene expression in daf-2 to be considerably down-35 regulated upon cdk-12 KD. In addition, these genes were also dependent on DAF-16 ( Figure 3C).

7
We validated this by quantitative real-time PCR (RT-PCR) ( Figure S3A). We also found many of 1 these genes to be down-regulated in WT on cdk-12 RNAi ( Figure S3B). The downregulation in 2 daf-2 was not due to differences in the sizes of the gonads upon cdk-12 KD ( Figure S3C).

3
Importantly, ChIP-seq data analysis in daf-2 and daf-16;daf-2 showed that many DNA damage 4 checkpoint genes like mrt-2, rad-51, rad-50 and pch-2 and DNA damage repair and cell cycle 5 genes have DAF-16 binding peaks in their promoter-proximal regions ( Figure 3D, S3D). suggesting 6 that they may be direct targets of DAF-16. Together, these data show that genes involved in 7 sensing and repairing DNA damage are common transcriptional targets of DAF-16 and CDK-12. 8 9 CDK-12 is required for efficient DNA damage repair 10 11 Mammalian CDK12 is known to regulate DDR genes and promote homologous 12 recombination (HR)-mediated DNA repair (17, 28). Above, we also found CDK-12 to 13 transcriptionally regulate the DDR genes in C. elegans. To determine whether CDK-12 KD leads 14 to germline DNA damage, we utilized a chromosome fragmentation assay. In unirradiated worms, 15 six highly condensed bivalent bodies can be seen in the oocyte; however, unrepaired DNA strand 16 breaks in irradiated worms lead to chromosome fragmentation/fusions (29). We observed 17 increased chromosome fragmentation and fusions in IR-treated wild-type worms upon cdk-12 KD 18 that suggests increased DNA damage ( Figure 3E). Next, we exposed the L4 or YA worms to 19 different concentrations of DNA damaging agent camptothecin (CPT) ( Figure S3E) or varying 20 doses of Ionizing Radiation (IR) ( Figure 3F) and found that cdk-12 KD resulted in a lesser number 21 of hatched eggs, highlighting their higher sensitivity, possibly due to compromised DNA damage 22 repair. We also observed increased developmental arrest on IR treatment at L1 stage when cdk-23 12 is KD, in a DAF-16-independent manner ( Figure 3G).

25
In agreement to the fact that cdk-12 KD may lead to endogenous DNA damage, we 26 observed higher apoptotic bodies per gonadal arm in cdk-12 KD wild-type and daf-2 worms ( Figure   27 3H). DNA damage in worm germline has been shown to evoke the innate immune response which 28 in turn confers systemic resistance and enhances somatic stress endurance (30). In our 29 transcriptomic data, we find that KD of cdk-12 up-regulates innate immune response genes

3
Further, we wanted to visualize the role of CDK-12 in somatic DNA damage. For this, we 4 analysed the DAPI-stained adult intestinal cells. A total of 20 intestinal cells are present at hatching, 5 a subset of which (8-12) divide, but do not undergo cytokinesis, thereby generating 28-32 6 binucleate intestinal cells by the end of the L1 stage (32). Like mutations in some DDR genes, atm-7 1 and dog-1 (33), we also found elongated cells with chromosomal bridges upon cdk-12 KD ( Figure   8 3I), much similar to L4 worms exposed to IR ( Figure S3K), indicating the occurrence of DNA 9 damage in the somatic cells (29, 33).

11
Together, CDK-12 plays a pivotal role in the repair of damaged DNA, both in the C. elegans 12 germline and somatic tissues to maintain genomic integrity. Therefore, knocking down cdk-12 may 13 lead to genomic instability that is sensed by activated DAF-16 in the daf-2 mutant, leading to the  To test the functional role of DAF-16 in DDR and its heightened engagement in daf-2 to 20 protect against DNA damage, we again utilized the chromosome fragmentation assay. Worms were 21 treated with IR at L4 to induce DNA double-strand breaks, stained with DAPI after 48 hours post-22 radiation and imaged. We found daf-2 worms to be highly resistant to IR, such that at 90 Gy most 23 of the wild-type chromosomes were fragmented, but daf-2 worms retained intact chromosomes.

24
This IR resistance was conferred by DAF-16, as in the daf-16;daf-2 worms, the chromosomes were 25 fragmented to a similar extent as in wild-type with IR treatment ( Figure 3J).

27
A high dose of gamma radiation during early larval stages in C. elegans can result in sterility 28 and developmental arrest if the damage is not repaired (34). Upon treatment of daf-2 and daf-29 16;daf-2 worms with 140 Gy IR dose at the L1 stage, we found that daf-16;daf-2 worms become 30 sterile ( Figure 3K). However, remarkably, daf-2 worms were mostly fertile. Similarly, resistance to 31 somatic developmental arrest on IR treatment was observed in daf-2, in a daf-16-dependent 32 manner ( Figure 3G). Together, our findings support a role of DAF-16 in regulating DNA damage 9 repair during lowered IIS, thereby promoting resistance to DNA damage, supporting growth and

15
12 is knocked down in WT, the levels of pMPK-1 is significantly reduced. However, the reduction 16 is much more dramatic in daf-2, possibly below a threshold level ( Figure 4A, B). This may explain 17 the complete arrest of the germline at pachytene stage ( Figure 1E). Importantly, in the daf-16;daf-18 2, the levels are restored ( Figure 4A, B), in line with the release of pachytene arrest in the double 19 mutant ( Figure 1E).

21
It appears that downstream of daf-2, the ERK signalling and the canonical PI3K signalling 22 co-ordinately regulate germline pachytene arrest. When daf-2 is mutated, the pMPK-1 levels are 23 lowered because of less signalling through the RAS pathway as well as due to the negative 24 regulation of activated DAF-16 through the PI3K pathway. We have shown above that knocking 25 out daf-16 rescues the lower pMPK-1 in daf-2 ( Figure 4A, B). So, we asked whether activating the 26 ERK signalling can bypass the pachytene arrest in daf-2 on cdk-12 KD. We used an activated ras 27 allele with constitutively high pMPK-1 phosphorylation (37). In the daf-2;let-60(gf), the pMPK-1 28 levels were upregulated ( Figure 4A, B) and pachytene arrest was partially reversed ( Figure 4C-29 E). Although many eggs hatched to release L1 worms ( Figure S4B), only about half of them were 30 able to reach adulthood ( Figure 4F), possibly pointing at their poor quality. Overall, we conclude 31 that the ERK and the PI3 kinase pathways co-ordinately regulate meiosis arrest on sensing somatic 1 DDR perturbations in daf-2.

3
Defective sperm to oogenesis switch and transcriptional downregulation of key cell cycle 4 genes in daf-2 on DDR perturbation 5 6 We have shown above that the sterility of daf-2 on cdk-12 RNAi may be due to inactive 7 RAS-ERK signaling. RAS-ERK activation is critical for sperm-oocyte fate switch by regulating the 8 timing the event in C. elegans hermaphrodite(38). We observed a two-folds increase in the number 9 of sperms but no oocyte in daf-2 upon cdk-12 KD ( Figure 4G, H). So, we tested the mRNA levels 10 of key sperm-oocyte switch genes and found their levels to be significantly reduced in daf-2 ( Figure   11 4I, but not in daf-16;daf-2 ( Figure S4C). This decrease in expression of genes is due to the cdk-12 12 KD per se, and not because of a reduction in germline size as at late-L4 (when RNA was collected), 13 the germline size is comparable between control RNAi and cdk-12 RNAi fed worms ( Figure S3C).

15
Next, we asked if the sperm to oogenesis switch defect was accompanied by an underlying 16 defect in other critical players of meiotic progression, namely, cdk-1, cyb-1 and cyb-3 (39). To 17 assess this, we determined the mRNA levels of these genes and found levels of all three to be 18 significantly down-regulated in daf-2 worms with cdk-12 KD ( Figure 4J), whereas the gene levels

23
We further checked if a similar defect in sperm to oogenesis switch and downregulation of 24 key cell cycle genes underlies the sterility upon DNA damage on IR exposure. We treated daf-2 25 worms with 160 Gy IR at L1 and DAPI stained Day 1 adults. Surprisingly, we found that the sperm 26 count increased around two-fold with a concomitant reduction in sperm to oocyte switch genes and 27 cdk-1, cyb-1, and cyb-3 RNA levels ( Figure S4G-I). Therefore, using CDK-12 knockdown and IR 28 exposure to phenocopy DNA damage, we show that germline arrest on DDR perturbation in daf-2 29 is brought about by defective sperm to oogenesis switching and reduction in the transcription of 30 genes essential for meiotic progression. This, along with reduction of ERK/MPK-1 signaling, may be strategies employed by the daf-2 hermaphrodite worms to prevent the production of poor-quality 1 progeny when the DNA damage is beyond repair.

3
Uterine tissue-specific DDR perturbation arrests germline in daf-2 4 5 Since cdk-12 KD leads to impaired DDR and resulting DNA damage, we asked whether 6 tissue-restricted DNA repair perturbations will lead to germline arrest in daf-2. We first used a 7 germline-specific RNAi system to test if tissue autonomous depletion was sufficient for the arrest 8 in daf-2. We used a rde-1(-) transgenic strain where sun-1 promoter drives the expression of rde-9 1 only in the germline of daf-2 (germline-specific RNAi) (40). We validated the strain by knocking showing that its function is required in the germline to maintain progeny quality. Importantly, it 18 appears that activated DAF-16 only promotes germline arrest if the damage signal emanates from 19 somatic tissues.

21
Next, we specifically knocked down cdk-12 in different somatic tissues (43-46). We found 22 that knocking down cdk-12 only in the uterine tissues was sufficient to arrest the germline in the 23 daf-2 worms at the pachytene stage of meiosis ( Figure 5D, S5B); no arrest was seen when the 24 gene was knocked down in hypodermis, muscle, intestine or neurons ( Figure 5D) and they 25 produced healthy fertile progeny ( Figure S5C). This implies that KD of cdk-12 in daf-2 germ cells 26 may lead to DNA damage resulting in poor progeny production. However, knocking it down in the 27 somatic uterine tissue may activate DAF-16-dependent quality checkpoints that lead to cell-28 nonautonomous germline arrest.

30
Next, to determine the tissues where the IIS receptor functions, we used transgenic lines 31 where the wild-type copy of daf-2 is rescued only in the neurons (using either unc-119 or unc-14 32 promoters), muscles or intestine of the daf-2 mutants (47) and then knocked down cdk-12 using 33 RNAi. We found that neuron-specific rescue of the daf-2 gene led to a significant rescue of fertility, 34 while little effect was seen in the case of muscle and intestine-specific rescue ( Figure 5E). We also 35 determined where DAF-16 is required to sense and mediate the germline arrest in the daf-2 mutant upon cdk-12 KD. We found maximum arrest when daf-16 is rescued in the muscle, neuron or 1 uterine tissues of the daf-16;daf-2 mutant worms ( Figure 5F), but not in the intestine. Together, 2 these observations support a model where low neuronal IIS sensitizes uterine tissues to 3 perturbations in DDR, leading to the arrest of germline at the pachytene stage of meiosis. The DAF-4 16a isoform works in the somatic uterine tissues, apart from muscle and neurons, to implement the 5 arrest ( Figure 5G).   implying a suboptimal repair pathway. We show cdk-12 ablation reduces gamete quality, leading 18 to increased infertility and decreased progeny fitness. It also led to retarded growth, and premature 19 aging which are hallmarks of genomic instability. Thus, CDK-12 is an evolutionary well-conserved 20 custodian of the genome that helps maintain DNA integrity, which we have used in our study as a 21 genetic tool to analyse the effects of tissue-restrictive DDR perturbation and DNA damage.

23
To maintain genomic integrity, organisms have evolved an efficient DDR pathways, that 24 senses and repairs DNA damage (50). Defects in DDR is associated with reduced fitness, infertility 25 and offspring with inherited diseases (51, 52). We identified that active FOXO/DAF-16 maintains 26 genomic integrity by upregulating DNA repair genes, which could explain the longer lifespan and 27 better oocyte quality of the low IIS mutants (53). Apart from maintaining genomic stability, our study 28 shows that activated FOXO/DAF-16 can sense DDR perturbation or DNA damage, stop 29 reproduction by arresting the germline, and protect the genomic integrity of germ cell. In the 30 absence of DAF-16, worms fail to arrest germline development and produce oocytes of poor quality 31 that hatch into unhealthy progenies. Thus, activated FOXO/DAF-16 critically regulates reproductive 32 decision by sensing the intrinsic threat of genomic instability. Previous studies has shown that DAF-33 16 acts as a nutrient sensor and mediates developmental arrest on starvation, as a protective 34 mechanism (7). Together, these data suggests that FOXO/DAF-16 acts as a master regulator of 35 diverse cellular processes in maintaining genomic integrity, tissue homeostasis and reproduction.

1
We found that upon DDR perturbation and ensuing DNA damage, FOXO/DAF-16 enforces 2 germline arrest by inactivating RAS-ERK signalling which is essential for germline proliferation and 3 quality. We also observed reduced expression of cyclin-dependent kinase-1 gene (cdk-1) and its 4 binding partner cyclin, cyb-1, and cyb-3 genes, which may be due to dampening of the RAS-ERK 5 signaling. In many cancers, RAS-ERK negatively regulates FOXO activity and promotes rapid 6 proliferation(54). Similarly, we observed that constitutively activated RAS-ERK in the low IIS mutant 7 (where FOXO/DAF-16 is activated) over-rides the germline arrest upon DDR perturbation, leading 8 to the production of unhealthy progenies. Therefore, RAS-ERK and FOXO/DAF-16 regulate each 9 other's activity and a fine balance is important for various biological process, including reproductive 10 development.

12
Cell non-autonomous inter-tissue crosstalk helps an organism to perceive and respond to

19
We show that perturbation of the DDR pathway only in the somatic uterine tissue of low IIS worm 20 is sufficient to cause cell cycle arrest in the germline; perturbation in the germline itself does not 21 lead to arrest but produces unhealthy progenies. This suggests that the somatic tissue, not the 22 germline, senses stress signal of genome instability and shunt their energy and resources towards 23 somatic maintenance rather than reproductive commitment. This is supported by the observations 24 of heightened stress response pathways and retarded germline growth upon DDR perturbation.

26
Finally, we find that lowering of IIS is required in the neurons to activate FOXO/DAF-16  Worms were grown on control or cdk-12 RNAi, L1 stage onwards. Day 1 adults were 5 collected in 1X M9 buffer in a 1.5 ml Eppendorf tube and worms were allowed to settle down. Using 6 a glass Pasteur pipette, the 1X M9 was discarded, leaving behind a ~ 100 µl worm suspension.

7
Then, 1 ml chilled 100 % methanol was added to the worm pellet and incubated for 30 minutes at 8 -20ºC. The pellet was placed on a glass slide and Fluroshield with DAPI (Invitrogen, Carlsbad, 9 USA) was added. For staining dissected gonads, worms were placed onto a glass slide and the 10 gonads were obtained by cutting the pharynx or tail end of the worm using a sharp 25G needle.

11
After collecting gonads for 10 minutes, 500 µl chilled 100% methanol was added onto the slide and 12 allowed to dry. Fluroshield with DAPI (Invitrogen, Carlsbad, USA) was added. The slides were 13 imaged using a confocal microscope (Carl Zeiss, Oberkochen, Germany).

15
Reproductive span, brood size, and egg hatching 16 Worms were grown on control or cdk-12 RNAi from L1 onwards and upon reaching the 17 young adult stage, five worms were picked onto fresh RNAi plates, in triplicates, and allowed to lay   images were categorized into three groups based on their morphology (cavities, shape, size, and 33 organization). Based on the severity of the phenotype, oocytes were categorized as normal (no 34 small oocytes, no cavities, and no disorganized oocytes), mild (a few cavities in gonad, or slightly disorganized oocyte or small), or severe (many cavities in the gonad, or disorganized or 1 misshapen).       IR: Worms were grown on control or cdk-12 RNAi. At the young adult stage, worms were 8 exposed to IR doses ranging between 0 to 40 Gy. The IR-treated worms were allowed to recover 9 for 3-4 hours, following which 5 worms were transferred to respective RNAi plates, in duplicates,

17
The worms were then transferred to Eppendorf tubes and washed twice with 10% Triton X-100 (in 18 1x M9 buffer), followed by two washes with 1x M9 buffer. The worms were then placed on RNAi 19 plates to recover for 3-4 hours, followed by tight egg-laying for 3-4 hours on fresh, respective RNAi                                                  3 (E) Representative fluorescence images of dissected gonadal arms that were stained with DAPI.

24
Around 300 µl of this suspension was seeded onto RNAi plates and left at room temperature for 2-25 3 days for drying, followed by storage at 4°C till further use.

27
Hypochlorite treatment to obtain eggs and synchronizing worm population   aqueous solution, an equal volume of isopropanol was added and the reaction was allowed to sit 2 for 10 minutes at room temperature followed by centrifugation at 12000g for 10 minutes at 4°C.

3
The supernatant was carefully discarded without disturbing the RNA-containing pellet. The pellet 4 was washed using 1 ml 70% ethanol solution followed by centrifugation at 12000g for 5 minutes at 5 4°C. The RNA pellet was further dried at room temperature and later dissolved in nuclease-free 6 water (Qiagen, Hilden, Germany) followed by incubation at 65°C for 10 minutes with intermittent 7 tapping. The concentration of RNA was determined by measuring absorbance at 260 nm using 8 NanoDrop UV spectrophotometer (Thermo Scientific, Waltham, USA) and quality checked using 9 denaturing formaldehyde-agarose gel.

11
Gene expression analysis using quantitative real-time PCR (QRT-PCR)

12
First-strand cDNA synthesis was carried out using the Iscript cDNA synthesis kit (Biorad,

13
Hercules, USA) following the manufacturer's guidelines. The prepared cDNA was stored at -20°C.  and scored for worms that reached L-4 or above post 100 hours.

6
DNA damage sensitivity assay 7 IR: Worms were grown on control or cdk-12 RNAi. At the young adult stage, worms were 8 exposed to IR doses ranging between 0 to 40 Gy. The IR-treated worms were allowed to recover 9 for 3-4 hours, following which 5 worms were transferred to respective RNAi plates, in duplicates,

17
The worms were then transferred to Eppendorf tubes and washed twice with 10% Triton X-100 (in 18 1x M9 buffer), followed by two washes with 1x M9 buffer. The worms were then placed on RNAi 19 plates to recover for 3-4 hours, followed by tight egg-laying for 3-4 hours on fresh, respective RNAi 20 plates. The adults were sacrificed and the number of eggs laid was counted. About 48 hours later, 21 the number of hatched progenies was also counted.

24
Synchronized late-L4 worms grown on control or cdk-12 RNAi were collected using 1X M9 25 buffer, after washing it thrice to remove bacteria. Total RNA was isolated from these worm pellets 26 using the Trizol method. The concentration of RNA was determined by measuring absorbance at 27 260 nm using NanoDrop UV spectrophotometer (Thermo Scientific, Waltham, USA) and RNA 28 quality was checked using RNA 6000 NanoAssay chip on a Bioanalyzer 2100 machine (Agilent 1 Technologies, Santa Clara, USA) RNA above RNA integrity number = 8 was included for the study. with P-values below 5%. Genes with a cut-off of fold change > 2 and fold change < -2 were 20 considered as upregulated and downregulated genes, respectively. For downstream analysis, the 21 function variance stabilising transformations (VST) (7) in DeSeq2 package was implemented.

22
Enrichment analysis was performed using the online tool DAVID 6.8 with a cutoff of FDR<10%.

23
The dot plot was plotted with ggplot2 in R. The heatmap was plotted with the help of the heatmap 24 function in R.

26
List of primers used in the study

13
Experiments were performed at 20 o C. Source data is provided in Dataset S1.