DAF-16/FOXO maintains genome integrity following genotoxic stress

DAF-16/FOXO maintains genome integrity following genotoxic stress | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results DAF-16/FOXO maintains genome integrity following genotoxic stress View ORCID Profile Umanshi Rautela , View ORCID Profile Oviya Devendran , View ORCID Profile Gautam Chandra Sarkar , View ORCID Profile KR Ranjisha , View ORCID Profile Rashi Mittal , View ORCID Profile Anita Goyala , View ORCID Profile Arnab Mukhopadhyay doi: https://doi.org/10.1101/2025.10.27.684284 Umanshi Rautela 1 Molecular Ageing Laboratory, National Institute of Immunology , Aruna Asaf Ali Marg, New Delhi 110067, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Umanshi Rautela Oviya Devendran 1 Molecular Ageing Laboratory, National Institute of Immunology , Aruna Asaf Ali Marg, New Delhi 110067, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Oviya Devendran Gautam Chandra Sarkar 1 Molecular Ageing Laboratory, National Institute of Immunology , Aruna Asaf Ali Marg, New Delhi 110067, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gautam Chandra Sarkar KR Ranjisha 1 Molecular Ageing Laboratory, National Institute of Immunology , Aruna Asaf Ali Marg, New Delhi 110067, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for KR Ranjisha Rashi Mittal 1 Molecular Ageing Laboratory, National Institute of Immunology , Aruna Asaf Ali Marg, New Delhi 110067, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rashi Mittal Anita Goyala 1 Molecular Ageing Laboratory, National Institute of Immunology , Aruna Asaf Ali Marg, New Delhi 110067, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anita Goyala Arnab Mukhopadhyay 1 Molecular Ageing Laboratory, National Institute of Immunology , Aruna Asaf Ali Marg, New Delhi 110067, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Arnab Mukhopadhyay For correspondence: arnab{at}nii.ac.in Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Preserving genomic integrity is crucial for the accurate transmission of genetic information across generations, as well as for preventing precocious ageing. The DNA Damage Response (DDR) safeguards the genome from genotoxic stress through a coordinated system of sensors, relay proteins, and repair mechanisms. Since DNA repair is an energy-intensive activity, the process is tightly regulated and coordinated with various metabolic pathways. The nutrient-sensing insulin/IGF signalling (IIS) pathway has been extensively studied for its role in ageing and lifespan regulation in C. elegans through its downstream FOXO transcription factor DAF-16. However, there is limited understanding of its involvement in maintaining genomic integrity through the regulation of the DDR. In this study, we demonstrate the role of DAF-16/FOXO in preserving genome integrity by activating the expression of DDR repair genes in C. elegans . Activated DAF-16/FOXO directly binds to the promoter of DDR genes under conditions of low IIS, ensuring that their expression is maintained at a higher level, which is crucial for prompt DNA damage repair. Interestingly, we find that DAF-16 functions both cell autonomously as well as non-autonomously to support DNA integrity. We also determine that the DAF-16(d/f) isoform, but not the DAF-16(a) isoform, is essential for maintaining germline genome integrity. Furthermore, DAF-16 activation enhances the DDR primarily through the canonical DDR components and, to a lesser extent, via apoptosis-mediated clearance of damaged cells. Overall, our study highlights a new role for DAF-16/FOXO in the DDR and the preservation of genome integrity. Introduction Maintaining genome stability in the face of environmental and endogenous genotoxic stressors is essential for organismal survival, fertility, and longevity. The organism’s DNA damage response (DDR) comprises pathways that detect DNA lesions, halt cell cycle progression, and facilitate repair to prevent genome instability [ 1 ]. As DDR is an energetically demanding process, it is tightly linked to various metabolic pathways such as the pentose-phosphate pathway to provide nucleotide precursors [ 2 ] or the fatty acid oxidation-dependent DNA break detection via poly (ADP-ribose) polymerase 1 (PARP1) [ 3 ]. Additionally, NAD⁺ metabolism impacts the activity of PARPs and sirtuins, which are essential for chromatin relaxation and DNA strand break repair [ 4 ] [ 5 ]. Among key metabolic regulators, the nutrient-sensing insulin/IGF-1 signaling (IIS) pathway serves as a conserved modulator of stress resistance and lifespan [ 6 ]. Central to the IIS pathway is the FOXO transcription factor (TF), which remains cytoplasmic and inactive under normal/high insulin signaling, but translocates to the nucleus under low IIS to initiate the transcription of a wide array of protective genes [ 7 – 9 ]. While FOXO transcription factors are well known for their roles in metabolism, stress resilience, and lifespan extension [ 10 ], emerging studies over the past decade have identified them as important modulators of genome stability. In mammalian systems, FOXO3a regulates cell-cycle checkpoint activation (p21/Kip-1 and p27/Cip-1) [ 11 , 12 ], oxidative DNA damage repair [ 13 ] and also DNA damage-induced apoptosis [ 14 – 16 ]. FOXO3 is linked to enhanced DNA damage repair and has been shown to physically interact with the ataxia-telangiectasia mutated (ATM) protein kinase, a master regulator of DDR [ 17 ]. Additionally, FOXO is implicated in transcriptional regulation of Gadd45a (a growth arrest and DNA damage response gene)[ 18 ] and is known to influence varying modes of DDR pathways (BER: base excision repair; HR: Homologous recombination)[ 13 , 19 ]. However, how the FOXO activation mechanistically contributes to the DNA repair efficiency has not been comprehensively studied. The nematode Caenorhabditis elegans is a powerful model to study DDR due to its genetic tractability, conserved DDR pathways, and accessible germline, enabling real-time visualization and quantification of DNA damage responses. Importantly, the distinct utilization of homologous recombination (HR)-mediated DDR in the germline and non-homologous end joining (NHEJ) in somatic tissues provides a unique opportunity to investigate tissue-specific DDR mechanisms [ 20 , 21 ]. To probe the role of insulin/IGF-1-FOXO signalling in DDR, we employed the temperature-sensitive kinase-domain mutant daf-2(e1370), which mimics low IIS and activates the FOXO ortholog DAF-16 [ 22 – 24 ]. In the current study, we show that DAF-16 directly regulates DDR gene expression and improves damage resolution. Using tissue-specific knockdown and isoform-rescue experiments, we found that DAF-16 acts both cell-autonomously and cell non-autonomously to maintain germline genomic integrity, and the DAF-16(d/f) isoform is specifically required for enhancing germline DDR. Finally, we demonstrate that the improved DDR in daf-2 mutants is mediated by homologous recombination (HR)-mediated DNA repair rather than increased apoptotic clearance. Together, our findings reinforce the role of FOXO/DAF-16 as a guardian of genome integrity, mechanistically linking the metabolic status of the cell to DNA damage repair. Results DAF-16 directly regulates DNA damage response genes in daf-2 mutants The IIS pathway modulates longevity and stress resistance in C. elegans , largely through the FOXO transcription factor DAF-16. Under reduced IIS (as in daf-2 mutants), DAF-16 translocates to the nucleus and activates target genes involved in stress resistance, metabolism, and lifespan extension [ 23 – 26 ]. While the role of DAF-16 in stress resistance and longevity [ 27 ] is well-established, its regulation of the DNA damage response (DDR) remains less explored. To investigate whether DAF-16 influences DDR pathways, we analyzed transcriptomic data from late-L4 staged daf-2 and daf-16;daf-2 mutants (previously published transcriptomics data) [ 28 ]. Strikingly, DDR genes were significantly upregulated in daf-2 mutants compared to daf-16;daf-2 double mutants ( Fig. 1A ), suggesting that their induction depends on DAF-16. We validated these findings by qRT-PCR, confirming elevated expression of key DDR genes such as rad-50, rad-51, brc-1, hus-1 in the daf-2 mutants ( Fig. 1B ). Moreover, in comparison to the wild-type (WT) worms, we observed an increased expression in the basal levels of DDR gene expression in daf-2 worms ( Fig. S1A ). Download figure Open in new tab Fig 1: DAF-16/FOXO regulates DNA damage repair pathway gene expression (A) Heat map representation of the levels of DNA damage repair (DDR) genes from transcriptomics data comparing daf-2(1370) and daf-16(mgdf50);daf-2(e1370) late-L4 staged worms grown on control RNAi. (B) Quantitative RT-PCR analysis showing mRNA fold change in DDR gene expression in daf-2(1370) and daf-16(mgdf50);daf-2(e1370) late-L4 staged worms grown on control RNAi. Expression levels were normalized to actin . The average of three biological replicates is shown. Unpaired t -test with Welch’s correction. (C) UCSC browser view of DAF-16/FOXO peaks on the DDR gene ( rad-51, rad-50 , wee-1.3 , mrt-2, brc-1 or pch-2) promoters as determined by ChIP-seq using an anti-DAF-16 antibody [ 29 ]. Red boxes indicate the binding on promoter regions of DDR genes. The upper panel shows daf-2(e1370) , where DAF-16 peaks are observed, while lower panel shows daf-16(mgDf50);daf-2(e1370) (that lacks specific DAF-16 peaks). Experiments were performed at 20°C. Source data are provided in Table 1. Since DAF-16 is predominantly inactive in WT worms but nuclear-localized in daf-2 [ 23 ], we hypothesized that DAF-16 may directly regulate the DDR genes. To test this, we re-analyzed published DAF-16 ChIP-seq data [ 29 ] and identified strong DAF-16 binding peaks at the promoters of multiple DDR genes in daf-2 ( Fig. 1C ). These peaks were absent in daf-16;daf-2 mutants, indicating that DAF-16’s binding is specific and likely drives the transcriptional activation. Together, these results demonstrate that DAF-16, already known to govern stress and longevity pathways, also directly regulates DDR genes under reduced IIS, potentially linking DNA repair mechanisms to the extended lifespan of daf-2 . DAF-16 Activation Enhances DNA Damage Repair in Germline and Somatic Tissues In C. elegans hermaphrodites, oocytes pause at diakinesis, the final stage of meiotic prophase, prior to fertilization. At this stage, chromosomes are highly condensed, with homologous pairs no longer exhibiting the side-by-side pairing observed earlier in prophase. Instead, they remain physically linked through chiasmata formed by meiotic crossovers, enabling proper orientation on the meiotic spindle. In WT oocytes, six distinct DAPI-stained bivalents are typically visible, representing the six homologous chromosome pairs maintained by these chiasmata [ 30 ] To investigate the role of DAF-16 in DDR, we exploited this well-defined chromosomal organization and performed a chromosome fragmentation assay. For this, worms were exposed to ionizing radiation (IR; dose of 0 Gy and 100 Gy) at the late-L4 stage and DAPI-stained 48 hours post-irradiation (HPI) to observe the extent of chromosome fragmentation in the oocytes [ 31 ]. Irradiation (100 Gy) of WT worms induced severe chromosomal fragmentation and fusion in diakinesis-stage oocytes, disrupting the normal bivalent structure. Strikingly, daf-2 mutants, where DAF-16 is constitutively active, maintained intact chromosomal morphology, with clearly discernible bivalents ( Fig. S2A, B ). This protection was entirely DAF-16-dependent, as irradiated daf-16;daf-2 double mutants exhibited extensive chromosomal defects ( Fig. 2A, B ). While at 100 Gy IR dose, both WT and daf-16 worms displayed severe chromosomal abnormalities, at a reduced IR dose (70 Gy), daf-16 mutants showed significantly more chromosomal aberrations (fragmentation and fusion) than WT ( Fig. S2C, D ). This demonstrates a role of DAF-16 in preserving meiotic chromosome integrity. Download figure Open in new tab Fig. 2: Activated DAF-16 confers increased germline and somatic DDR efficiency following genotoxic stress (A) Representative fluorescence images of DAPI-stained gonads (48 HPI) of daf-2(e1370) and daf-16(mgdf50);daf-2(e1370) worms grown on control RNAi, irradiated (0 and 100 Gy) at late-L4 stage. Scale bar: 5 µm. (B) Quantification for the degree of chromosome fragmentation seen above. The oocyte chromosomes were categorized as normal, fragmented, or fused based on the number of oocyte chromosomes (normal = 6 chromosomes; fragmented = more than 6 chromosomes; fused = less than 6 chromosomes and fusion between two or more chromosomes). Combined data of three biological replicates ( n ≥45) are shown. Statistical comparisons between groups were performed using Chi-square analysis. (C) The percentage of hatched eggs of daf-2(e1370) and daf-16(mgdf50);daf-2(e1370) worms grown on control RNAi and irradiated (0, 30, and 60 Gy) at the late-L4 stage. Egg hatching was monitored for 12-24 HPI. The average of three biological replicates ( n ≥ 100 per replicate) is shown. Unpaired t -test with Welch’s correction. (D) The percentage of fertile worms in daf-2(e1370) and daf-16(mgdf50);daf-2(e1370) irradiated (0, 140, 200, and 300 Gy) at the L1 stage and grown on control RNAi. The percentage of fertile worms (with eggs in the uterus) was subsequently quantified post-IR (when unirradiated worms reached the late day-1 stage). The average of four biological replicates ( n ≥100 for each experiment) is shown. Unpaired t -test with Welch’s correction. (E) The percentage of larval development of daf-2(e1370) and daf-16(mgdf50);daf-2(e1370) worms grown on control RNAi and irradiated (0, 140, 200, and 300 Gy) at the L1 stage. The percentage of worms progressing to the L4 stage and beyond (when unirradiated worms reach the early day-1 stage) was subsequently quantified. The average of four biological replicates ( n ≥ 80 per replicate) is shown. Unpaired t -test with Welch’s correction. (F) Representative fluorescence images of DAPI-stained intestinal nuclei (48 HPI) of daf-2(e1370) and daf-16(mgdf50);daf-2(e1370) worms grown on control RNAi and irradiated (0 Gy and 100 Gy) at the late-L4 stage. The arrow points towards fused intestinal cell nuclei (intestinal karyokinesis defect). Scale bars: 10 µm. (G) Quantification of the degree of intestinal karyokinesis defect as seen above. The quality was categorized as normal or with defects based on its morphology, where normal indicates distinct intestinal nuclei without any chromatin bridges or intestinal nuclei fusion, and defect indicates intestinal chromatin bridge or fused intestinal nuclei. The combined data of four biological replicates ( n ≥120 intestinal cells) is shown. Statistical comparisons between groups were performed using Chi-square analysis. Experiments were performed at 20°C. Source data are provided in Table 1. Since proper chromosome segregation at diakinesis is essential for embryonic viability [ 32 ], we next assessed egg hatching efficiency after exposing the worms to IR. The IR-induced damage to oocyte chromosomes correlated with a dose-dependent (0, 30, and 60 Gy) reduction in hatching across all strains. However, daf-16;daf-2 and daf-16 mutants exhibited significantly lower hatching rates than daf-2 and WT, respectively ( Fig. 2C , S2E ), and daf-16;daf-2 mothers produced smaller broods post-IR ( Fig. S2F ). These results underscore the critical role of DAF-16 in maintaining germline genomic stability. Next, we assessed whether activated DAF-16 in daf-2 can protect the developing germline from IR-induced damage. At the L1 stage, the worm possesses 2 primordial germline cells and 2 somatic gonad precursor cells, which, during larval development, divide and differentiate to form the complete adult germline [ 33 ]. Irradiation (0, 140, 200, and 300 Gy) of worms at the L1 stage resulted in a dose-dependent reduction in fertility. After L1 irradiation, daf-2 and WT adults retained higher fertility than daf-16;daf-2 and daf-16 mutants, respectively ( Fig. 2D , S2G ), suggesting that DAF-16 safeguards germline development under genotoxic stress. We further explored whether DAF-16 also contributes to somatic DDR. Following L1 irradiation, daf-16;daf-2 worms exhibited severe developmental delays compared to daf-2 ( Fig. 2E ), and daf-16 mutants showed impaired progression to L4 or adulthood relative to WT ( Fig. S2H ). These results indicate that DAF-16 promotes somatic survival and development under DNA damage, consistent with its known role in stress resistance and longevity [ 34 ]. To further evaluate somatic DDR, we examined intestinal karyokinesis, a process critical for intestinal cell maturation [ 35 ]. Previous studies have linked DNA damage (e.g., IR or oxidative stress) and DDR gene mutations (such as atm-1 , dog-1 ) to intestinal karyokinesis defects [ 36 ] [ 28 ] Mirroring these findings, irradiated daf-16;daf-2 and daf-16 worms displayed increased intestinal chromatin bridges and binucleation, compared to daf-2 and WT, respectively ( Fig. 2F, G, S2I, J ), suggesting that amidst genotoxic insults, DAF-16 ensures proper chromosomal segregation during somatic cell division. Together, these results demonstrate that DAF-16 not only enhances germline DDR but also plays a critical role in somatic genome maintenance, expanding its known functions in stress resistance and aging to include direct modulation of DNA repair mechanisms. DAF-16 exerts cell-autonomous and cell non-autonomous control over germline DDR, predominantly through its d/f isoform DAF-16 exhibits tissue-specific functions to coordinate distinct physiological outcomes. For instance, DAF-16 in germ cells inhibits proliferation, while DAF-16 activation in the hypodermis promotes it [ 37 , 38 ]. The DAF-16 often performs functions in a cell non-autonomous fashion, as DAF-16 in the proximal gonad has been shown to influence the decline in germline stem cell pool with age [ 39 ]. Neuronal and intestinal DAF-16 have been found to be involved in a FOXO-to-FOXO feedback signalling that is required for longevity upon daf-2 inactivation in either tissue [ 40 ]. Interestingly, in a previous study, we elucidated the role of somatic DAF-16 in sensing DNA damage perturbations and influencing the reproductive decision [ 28 ]. Therefore, we sought to determine if DAF-16 regulates germline DDR cell-autonomously in the germ cells or cell non-autonomously from the somatic tissues. To achieve germline-specific daf-16 knockdown, we utilized a specialized RNAi system in which the rde-1 mutant background (deficient in the Argonaute protein RDE-1) is complemented by reintroducing rde-1 under a germline-specific sun-1 promoter [ 41 , 42 ]. In contrast, somatic knockdown was achieved using a ppw-1 mutant, which is defective in germline RNAi due to the loss of a PAZ/PIWI-domain protein essential for RNAi efficiency in germ cells [ 43 ]. We knocked down daf-16 in the whole body of daf-2 , exclusively in somatic tissues (using daf-2;ppw-1 ), or specifically in germ cells of the daf-2 mutant (using daf-2;rde-1;sun-1p::rde-1 ) by RNAi. We performed the chromosome fragmentation and the egg hatching assays to determine the germline DDR efficiency after control or daf-16 RNAi in each of these strains. Interestingly, in comparison to control RNAi-fed irradiated worms, we found that daf-16 knockdown in both germ cells as well as the somatic tissues of the daf-2 mutants increased chromosomal abnormalities ( Fig. 3A, B ) and dead eggs ( Fig. S3A ) upon irradiation. Notably, the effect on egg hatching was much more pronounced when daf-16 was knocked-down in the somatic tissues. This suggests that while DAF-16 is essential in both tissue types for implementing the proper DDR mechanism in the germ cells, somatic DAF-16 may play a more crucial role. The dual requirement for DAF-16 in both germline and somatic tissues emphasizes the integrated communication between these compartments, which is essential for maintaining genome stability in the face of genotoxic challenges. Download figure Open in new tab Fig 3: DAF-16 D/F, not A isoform, confers increased germline DDR efficiency following genotoxic stress (A) Representative fluorescence images of DAPI-stained gonads (48 HPI) of daf-2(e1370) , daf-2(e1370) ; rde-1(mkc36);sun-1p::rde-1 (germline-specific RNAi), and daf-2(e1370) ; ppw-1(pk1425) (soma-specific RNAi) worms grown on control or daf-16 RNAi, irradiated (0 and 100 Gy) at late-L4 stage. (B) Quantification for the degree of chromosome fragmentation as seen above. The oocyte chromosomes were categorized as normal, fragmented, or fused based on their morphology (normal = 6 chromosomes; fragmented = more than 6 chromosomes; fused = less than 6 chromosomes and fusion between two or more chromosomes). Combined data from three biological replicates ( n ≥ 42) are presented. Statistical comparisons between groups were performed using Chi-square analysis. (C) Representative fluorescence images of DAPI-stained gonads (48 HPI) of daf-2(e1370), daf-16(mgdf50);daf-2(e1370) and different DAF-16 isoforms rescued (daf-16a::RFP, daf-16b::CFP, and daf-16d/f::GFP) in daf-16(mgdf50);daf-2(e1370) worms grown on control RNAi, irradiated (0 and 80 Gy) at late-L4 stage. (D) Quantification for the degree of chromosome fragmentation as seen above. The oocyte chromosomes were categorized as normal, fragmented, or fused based on their morphology (normal = 6 chromosomes; fragmented = more than 6 chromosomes; fused = less than 6 chromosomes and fusion between two or more chromosomes). Combined data from three biological replicates ( n ≥ 22) are presented. Statistical comparisons between groups were performed using Chi-square analysis. (E) The percentage of hatched eggs of daf-2(e1370), daf-16(mgdf50);daf-2(e1370) and different DAF-16 isoforms rescued (daf-16a::RFP, daf-16b::CFP , and daf-16d/f::GFP ) in daf-16(mgdf50);daf-2(e1370) worms grown on control RNAi, irradiated (0, 30, and 60 Gy) at the late-L4 stage. Egg hatching was monitored for 12-24 HPI. The average of seven biological replicates ( n ≥ 80 per replicate) is shown. Two-way ANOVA with Tukey’s multiple comparison test. Experiments were performed at 20°C. Scale bars 5 µm. Source data are provided in Table 1. The daf-16 gene codes for multiple isoforms that differ in their promoter regions and transcription start sites. The DAF-16(b) isoform is a smaller protein compared to the DAF-16(a) and DAF-16(d/f) isoforms. The expression pattern is varied amongst these isoforms, with both DAF-16(a) and DAF-16(d/f) expressed more widely than the DAF-16(b) isoform, which is more restricted in expression to the pharynx, a few neurons, and the somatic gonad [ 8 , 44 , 45 ]. The DAF-16(a) isoform predominantly functions to support dauer arrest and longevity of daf-2 . In the absence of the DAF-16(a) isoform, DAF-16(d/f) plays a role in dauer arrest and longevity [ 46 ]. We aimed to investigate which DAF-16 isoform may play a crucial role in DDR. For this, we utilized the different transgenic lines where daf-16 isoforms (a, b or d/f) are rescued in the daf-16;daf-2 mutant background. To evaluate the effect on germline DDR, we employed chromosome fragmentation and the egg hatching assays following IR exposure at the late-L4 stage. Interestingly, rescuing the daf-16(d/f) isoform in the daf-16;daf-2 led to a significant alleviation in the IR-induced chromosome fragmentation ( Fig. 3C, D ) and egg hatching ( Fig. 3E ), as compared to the irradiated daf-16;daf-2 worms. The other daf-16 isoforms (a or b) did not rescue these phenotypes ( Fig. 3C -E ). The level of chromosome fragmentation and egg hatching percentage was comparable in irradiated daf-16(d/f) isoform rescued lines and daf-2 ( Fig. 3C-E ), indicating a predominant role of DAF-16(d/f) isoform in the germline DDR in the daf-2 worms. Further, we wanted to assess the contribution of each of the daf-16 isoforms in somatic DDR, for which we employed the L1 larval IR sensitivity assay (development and fertility). However, none of the isoforms could rescue the developmental defects ( Fig. S3B ) or the sterility ( Fig. S3C ) of the worms that were irradiated at L1. These findings underscore the specific importance of the daf-16(d/f) isoform in maintaining germline DDR efficiency. It further suggests that somatic DDR may either require a distinct daf-16 isoform or involve a combinatorial action of multiple isoforms to fully preserve DNA repair capacity following genotoxic stress. DAF-16 mediates higher DDR efficiency in daf-2 following genotoxic stress We observed significantly higher frequencies of oocyte chromosomal defects (fragmentation and fusion) in daf-16;daf-2 compared to daf-2 mutants at 48 hours post-IR (100 Gy at late-L4 stage; Fig. 2A,B ). This DAF-16-dependent phenotype suggested either differential initial damage or repair efficiency between the strains. To distinguish these possibilities, we performed a comprehensive time-course analysis of chromosomal integrity at 12, 18, 24, 30, 36, 42, and 48 HPI. Intriguingly, while daf-2 oocytes consistently maintained low levels of chromosomal abnormalities throughout all time points ( Fig. 4A, S4A, B ), daf-16;daf-2 oocytes exhibited progressive accumulation of damage ( Fig. 4A, S4A, B ). Notably, the difference in chromosomal abnormalities between daf-2 and daf-16;daf-2 was minimal (19.2%) at 12 HPI, but became increasingly significant at later time points (72.65 at 18-48 hours) ( Fig. 4A S4A, B) . This temporal pattern suggests that, while both strains incur almost similar initial damage, activated DAF-16 may drive the subsequent repair processes efficiently in daf-2 . The progressive divergence in chromosomal integrity phenotypes supports a model where DAF-16 activity sustains DNA repair capacity over time, rather than simply preventing initial damage, consistent with a conserved role of FOXO3a in double-strand break repair [ 47 ] Download figure Open in new tab Fig 4: Kinetics of DNA damage repair following genotoxic stress is accelerated upon DAF-16 activation (A) Quantification of the chromosomal fragmentation kinetics of DAPI-stained gonads (12, 18, 24, 30, 36, 42, and 48 HPI) in daf-2(e1370) and daf-16(mgdf50);daf-2(e1370) worms grown on control RNAi, irradiated (100 Gy) at the late-L4 stage (as seen in Figure S4A,B). The oocyte chromosomes were categorized as normal, fragmented, or fused based on their morphology (normal = 6 chromosomes; fragmented = more than 6 chromosomes; fused = less than 6 chromosomes and fusion between two or more chromosomes). Combined data from three biological replicates ( n ≥ 22) are presented. Statistical comparisons between groups were performed using Chi-square analysis. (B) Representative fluorescence images showing HUS-1::GFP (DNA damage checkpoint protein) expression in daf-2(e1370) and daf-2(e1370);daf-16(mgdf50) (6-8, 24-28 and 44-48 HPI). Worms grown on control RNAi were irradiated (0 and 100 Gy) at the late-L4 stage. Yellow arrows pointed towards HUS-1::GFP foci formation. (C) Quantification for the HUS-1::GFP foci per 10 pachytene germ cells per gonad as seen above. Combined data from three biological replicates ( n ≥ 12) are presented. Unpaired t -test with Welch’s correction. (D) Quantification for the HUS-1::GFP foci per 10 pachytene germ cells per gonad as seen in Fig. 4B . Combined data from three biological replicates ( n ≥ 12) are presented. Two-way ANOVA with Tukey’s multiple comparison test. (E) Representative fluorescent images of DAPI-stained gonads (48 HPI) of daf-2(e1370) and daf-2(e1370);hus-1(op241) worms grown on control RNAi, irradiated (0 and 100 Gy) at the late-L4 stage. (F) Quantification for the degree of chromosome fragmentation as seen above. The oocyte chromosomes were categorized as normal, fragmented, or fused based on its morphology (normal = 6 chromosomes; fragmented = more than 6 chromosomes; fused = less than 6 chromosomes and fusion between two or more chromosomes). Combined data from three biological replicates ( n ≥ 48) are presented. Statistical comparisons between groups were performed using Chi-square analysis Experiments were performed at 20°C. Scale bars 5 µm. Source data are provided in Table 1. To further assess the role of FOXO in the damage repair efficiency, we examined the localization of the checkpoint protein HUS-1::GFP in germ cells following IR. We utilized the transgenic strain hus-1(op241);hus-1p::hus-1::gfp to visualize HUS-1 dynamics following IR. In untreated worm gonads, HUS-1 remains diffused in the cytoplasm, with only a few HUS-1 foci detected in the germ cell nuclei [ 48 ] However, upon IR treatment, HUS-1 accumulates at the sites of DNA damage, leading to the formation of HUS-1 foci, which are known to facilitate the recruitment of other DNA damage repair proteins [ 48 , 49 ]. Upon IR exposure, we observed an increase in HUS-1::GFP foci formation (8 hrs and 27 hrs post-IR) in both daf-2 and daf-16;daf-16 pachytene germ cells. However, the extent of HUS-1::GFP foci formation was markedly elevated in the daf-16;daf-2 gonads in comparison to the daf-2 worms ( Fig. 4B, C, D ), suggesting that the absence of DAF-16 exacerbates DNA damage accumulation. Moreover, at 48 hrs post-IR, while the number of HUS-1::GFP foci declined in the irradiated daf-2 germ cells, HUS-1::GFP foci were still increased in the daf-16;daf-2 germ cells ( Fig. 4B, C, D ). This, along with DAF-16-dependent lower chromosomal abnormalities in daf-2 gonads following irradiation, highlights the role of DAF-16 in conferring increased DDR efficiency under low insulin signalling. To further investigate the role of HUS-1 in daf-2 DDR in the germline, we analysed daf-2;hus-1 double mutants following IR. Reduction of HUS-1 function in daf-2 led to a massive increase in chromosome fragmentation following IR ( Fig. 4E, F ). This highlights the crucial role of the conserved DNA damage checkpoint protein, HUS-1, in maintaining higher DDR efficiency under conditions of low insulin signalling. DAF-16 employs canonical DDR, but not apoptosis, to enhance repair efficiency Upon exposure to a DNA-damaging agent, the DDR machinery in the germline is activated to repair the damaged DNA. If the damage remains unrepaired, germ cell apoptosis is triggered to eliminate the damaged/defective germ cells [ 1 ]. We speculated that the germ cell apoptosis in daf-2 gonads might also be elevated to support enhanced clearance of damaged nuclei. To monitor the kinetics for germ cell apoptosis in daf-2 and daf-16;daf-2 , we utilized the transgenic strain ced-1p::gfp , where CED-1 is a transmembrane protein on the surface of sheath cells that engulf the dying germ cells [ 50 ]. At 6 and 12 HPI, an increased number of apoptotic corpses was observed in both daf-2 and daf-16;daf-2 gonads. However, at 24 and 36 HPI, apoptosis was attenuated in irradiated daf-2 germlines, whereas a significant increase in germ cell apoptosis persisted in the irradiated daf-16;daf-2 gonads ( Fig. 5A, B, S5A, B ). This contrasted with our expectation of increased apoptotic clearance of germ cells in irradiated daf-2 germline. However, a faster kinetics of apoptosis in daf-2 may hint towards a lower burden of damaged germ cells due to an enhanced damage repair efficiency. To further explore the role of apoptosis, we examined daf-2;ced-4 [CED-4 is the C. elegans ortholog for mammalian APAF1 that activates the procaspase-9 [ 51 ]]. Despite the absence of apoptotic clearance, daf-2;ced-4 worms did not exhibit increased chromosomal abnormalities compared to daf-2 alone ( Fig. 5C, D ). Moreover, knock-down of the worm caspase, CED-3 [ 52 ] by RNAi also did not exacerbate the chromosomal defects in daf-2 mutant worms upon irradiation (100Gy) ( Fig. S5C, D ). These results suggests that the enhanced DDR in daf-2 is not mediated by apoptosis but rather by improved DNA repair. Download figure Open in new tab Fig 5: DAF-16 requires canonical DDR components, and not apoptosis, to enhance DDR efficiency. (A) Kinetics of apoptosis (6, 12, 24, and 36 HPI) showing mean apoptotic corpse numbers per gonadal arm of daf-2(e1370);ced-1::GFP worms grown on control RNAi, irradiated (0, 30, and 60 Gy) at the late-L4 stage (as seen in Figure S5A). The average of three biological replicates is shown (n≥17 for each replicate). Two-way ANOVA with Tukey’s multiple comparison test. (B) Kinetics of apoptosis (6, 12, 24, and 36 HPI) showing mean apoptotic corpse numbers per gonadal arm of daf-16(mgdf50);daf-2(e1370);ced-1::GFP worms grown on control RNAi, irradiated (0, 30, and 60 Gy) at late-L4 stage (as seen in Figure S5B). An average of three biological replicates (n≥22 for each replicate). Two-way ANOVA with Tukey’s multiple comparison test. (C) Representative fluorescence images of DAPI-stained gonads (48 HPI) of daf-2(e1370) and daf-2(e1370);ced-4(n1162) worms grown on control RNAi, irradiated (0 and 100 Gy) at the late-L4 stage. Scale bars: 5 μm. (D) The quantification of the degree of chromosome fragmentation as seen above. The oocyte chromosomes were categorized as normal, fragmented, or fused based on their morphology (normal = 6 chromosomes; fragmented = more than 6 chromosomes; fused = less than 6 chromosomes and fusion between two or more chromosomes). Combined data from three biological replicates ( n ≥ 36) are presented. Statistical comparisons between groups were performed using Chi-square analysis. (E) Quantification of the extent of chromosomal fragmentation of DAPI-stained gonads (48 HPI) in daf-2(e1370) worms grown for two generations on control, hus-1 , brc-1 , rad-50 , cku-80 , and lig-4 RNAi, irradiated (0 and 100 Gy) at the late-L4 stage (as seen in Figure S5E). Based on morphology, the oocyte chromosomes were categorized as normal, fragmented, or fused (normal = 6 chromosomes; fragmented = more than 6 chromosomes; fused = less than 6 chromosomes and fusion between two or more chromosomes). Combined data from four biological replicates ( n ≥ 58) are presented. Statistical comparisons between groups were performed using Chi-square analysis. (F) The percentage of hatched eggs of daf-2(e1370) worms grown for two generations on control, brc-1 , and rad-50 RNAi, irradiated (0, 30, and 60 Gy) at the late-L4 stage. Egg hatching was monitored for 12-24 HPI. Average of five biological replicates ( n ≥ 120 per replicate). Two-way ANOVA with Tukey’s multiple comparison test. Experiments were performed at 20°C. Source data are provided in Table 1. As HUS-1 is required to maintain germline DDR in daf-2 ( Fig. 4D, E ), we next wanted to ascertain the requirement of other key proteins involved in the canonical DDR. For this, we exposed daf-2 worms to RNAi-expressing bacteria, followed by IR exposure at the late-L4 stage, and examined chromosome fragmentation in the oocytes. We were able to validate the functionality of our KD experiments since hus-1 KD by RNAi led to a significant increase in chromosome fragmentation ( Fig. 5E , S5E ) post-IR, similar to that observed in the irradiated daf-2;hus-1 mutant ( Fig. 4E, F ). Also, rad-51 KD led to chromosomal abnormalities even in the unirradiated daf-2 worms ( Fig. S5E ). Compared to worms grown on control RNAi, daf-2 mutants subjected to knockdown of DDR genes ( hus-1, brc-1 or rad-50 ) exhibited significantly higher levels of chromosomal abnormalities ( Fig. 5E , S5E ) and reduced egg hatchability ( Fig. 5F ) following irradiation. Notably, homologous recombination (HR)-mediated DNA repair, which depends on components such as HUS-1, BRC-1, and RAD-50, appears to be essential for maintaining genome integrity under these conditions. In contrast, non-homologous end joining (NHEJ) was found to be largely dispensable, as RNAi-mediated depletion of cku-80 and lig-4 [cku-80 is part of the double-strand breaks (DSBs) recognizing CKU70/CKU80 heterodimer and lig-4 is the NHEJ ligase [ 31 ]] did not lead to a detectable increase in chromosomal defects ( Fig. 5E , S5E ). This is consistent with previous findings showing that HR is the predominant repair pathway for DSBs in the germline, whereas post-mitotic somatic cells primarily rely on NHEJ for DSB repair [ 31 ] Importantly, these findings indicate that the enhanced DNA damage response observed due to activation of DAF-16 (in daf-2 mutants) is not due to increased apoptotic clearance of damaged germ cells post-IR, but rather reflects an improved capacity for repair, mediated by the robust function of the core DDR machinery. Discussion In the current study, we uncover a previously underexplored role of the longevity-linked IIS axis in the DDR. Using Caenorhabditis elegans long-lived Insulin-IGF-1 receptor mutant ( daf-2 ), we show up-regulation in the basal levels of DDR genes due to binding of activated worm FOXO transcription factor, DAF-16, to DDR gene promoters. We used IR to elicit DNA damage and find a DAF-16-dependent increase in the DNA-repair capacities in both mitotically/meiotically active (germline cells) and post-mitotic cells (somatic cells). Our findings reinforce an evolutionarily well-conserved role of FOXO as custodian of the genome that helps maintain DNA integrity ( Fig. 6 ). Download figure Open in new tab Fig 6. Model depicting the role of DAF-16 in somatic and germline DDR Upon genotoxic stress (such as IR: Ionizing radiation), DNA damage such as double-strand break is elicited. In daf-2 mutant worms (characterised by low insulin signalling), the presence of the activated DAF-16/FOXO transcription factor results in an increased level of DNA damage response (DDR) genes. This leads to a heightened DDR efficiency and genomic stability in both somatic and germ cells. However, the absence of DAF-16 in daf-2 mutant animals compromises DDR efficiency, leading to unresolved DNA damage that manifests as karyokinesis defects in intestinal cells and as fragmented chromosomes in oocytes. Thus, DAF-16 safeguards genome integrity in both somatic and germ cells. The Schematic was created using BioRender.com . Previously, in mammalian cell line models, FOXO has been shown to protect against DNA damage by inducing cell-cycle arrest [ 11 ], thus allowing time for DNA damage to be repaired [ 53 ] or by triggering cell cycle exit or apoptosis in case of unresolved DNA damage [ 14 , 16 , 54 , 55 ]. Overexpression of FOXO3a in primary human fibroblasts has been shown to decrease DSB-induced γH2AX foci and suppress genomic rearrangements [ 47 ]. FOXO3 has also been shown to protect the hematopoietic stem and progenitor cells from oxidative stress-induced DNA damage [ 13 ]. We wanted to test whether and how the worm FOXO transcription factor, DAF-16, plays a functional role in DNA damage repair. For examining germ-line DDR, we observed oocyte chromosome fragmentation and egg hatching following IR. In germline cells, daf-2 mutants maintained intact bivalent chromosome structures post-IR exposure, in contrast to wild-type (WT) or daf-16;daf-2 double mutants, which displayed significant chromosomal fragmentation. This DAF-16-dependent protection also translated into improved embryonic viability following IR-induced stress. These results underscore a functional role for DAF-16 in promoting germline genome stability. While the germline DDR is relatively well characterized, the understanding of somatic DDR in C. elegans is limited and partially repressed by the DREAM complex [ 56 , 57 ]. We evaluated somatic repair by quantifying IR-induced developmental arrest and intestinal karyokinesis defects [ 36 ]. We found increased larval development post-IR in daf-2 compared to the daf-16;daf-2 mutant. In many DDR mutants ( atm-1, dog-1 ) or on treating animals with chemicals that generate reactive oxygen species (ROS), the formation of intestinal chromatin bridges or fusion of intestinal nuclei have been noticed [ 36 ]. Utilizing the intestinal karyokinesis defects post-IR as a readout to assess somatic DDR efficiency, we found fewer karyokinesis defects in irradiated daf-2 mutants than in daf-16;daf-2 worms. Interestingly, our findings contrast with prior studies reporting that DAF-16 promotes larval development despite unresolved UV-induced DNA lesions [ 58 ], suggesting that under IR stress, DAF-16 enhances DNA repair rather than bypassing damage. Even in the WT background, where DAF-16 is predominantly cytoplasmic, removal of the transcription factor compromised somatic and germline DDR capabilities. Since germline maintenance is an energetically demanding process, the somatic tissues often take inputs from the environment to influence germline development [ 59 ], proliferation [ 60 ], apoptosis [ 61 ], oocyte growth and maturation [ 62 ]. Increasing evidence supports the regulatory role of soma in germline DDR. For instance, the somatic niche cells have been shown to influence CEP-1/p53-mediated DDR in primordial germ cells (PGC) and mammalian stem cells [ 63 ]. Similarly, the stress-responsive PMK-1/p38 functions in the intestine to promote stress-induced (heat, IR) apoptosis in germline [ 64 ]. Neuronal HIF-1, IRE-1, and intestinal KRI-1 function upstream or in parallel to CEP-1/p53 to regulate IR-induced germ cell apoptosis [ 65 – 67 ]. Quite strikingly, in our study, we found knocking down daf-16 , either in the soma or the germline, led to increased chromosomal defects and embryonic lethality post-IR exposure. Strikingly, these effects were more contrasting upon somatic DAF-16 depletion. These findings establish that while DAF-16 functions both cell-autonomously in germ cells and cell non-autonomously in the somatic cells to influence germline DDR, somatic DAF-16 appears to play a predominant role in germline DDR repair. Additionally, using isoform-specific rescue lines, we show DAF-16 (d/f) isoform to be crucial for germline DDR, whereas somatic DDR appears to require the combined activity of multiple DAF-16 isoforms, suggesting a tissue-specific complexity in isoform function. In the context of lifespan regulation, the neuronal and the intestinal DAF-16 have been shown to form a feedback loop, and both are required for longevity induced by neuronal or intestinal IIS reduction [ 40 ]. It is therefore plausible that similar inter-tissue communication underlies the DAF-16-mediated coordination of DDR. Whether this cross-talk is mediated by insulin-like peptides, neuropeptides, hormonal signals, or other small molecules remains to be explored. To monitor the DDR kinetics, we utilized HUS-1::GFP (a checkpoint protein fused to gfp [ 48 ]) foci detection, chromosomal abnormalities, and apoptosis kinetics following IR exposure. Through the chromosome fragmentation kinetics, we observed that, while both strains incur almost similar initial damage, activated DAF-16 may drive the subsequent repair processes efficiently in daf-2 . This indicates an enhanced repair capacity owing to heightened DAF-16 activity in daf-2, rather than simply preventing initial damage. Lower levels of HUS-1::GFP foci formation and accelerated resolution of IR-induced apoptosis in daf-2 support these findings. Interestingly, the canonical components of the HR arm of DSB repair, such as hus-1 , brc-1 , and rad-50 , were found to be critical for robust germline DDR, while the NHEJ ( cku-80 and lig-4 ) arm of DSB repair [ 31 ] or apoptotic machinery were found to be dispensable. These results suggest that DAF-16 may selectively promote HR-mediated repair over alternative pathways during genotoxic stress in germ cells, where the preservation of genomic integrity is critical for reproductive success and organismal fitness. Previously, FOXO3 has been shown to physically interact with the Ataxia-Telangiectasia Mutated (ATM: a protein kinase and a master regulator of DDR [ 17 ] and promote intra-S-phase or G2/M cell-cycle checkpoint, and repair of damaged DNA [ 68 ]. In line with this, it would be worthwhile to test if, apart from transcriptional regulation of DDR genes, DAF-16 also physically interacts with the DDR components to enhance damage recognition and resolution, thereby orchestrating a rapid and effective DNA damage response. With its role in cell cycle arrest [ 69 , 70 ] and apoptosis [ 54 , 71 , 72 ], FOXO is generally regarded as a tumor suppressor [ 73 – 75 ] However, emerging studies complicate this narrative [ 76 – 78 ]. In cancers such as neuroblastoma, colon and thyroid carcinoma, FOXO activity has been linked to poor prognosis [ 77 , 79 , 80 ]. As an extension of our current findings, we speculate that FOXO activation may enhance the DDR efficiency in cancer cells and may cause resistance against chemotherapeutic drugs which normally induce cancer cell death by causing DNA damage. Therefore, while FOXO activation may be beneficial in normal cells for genome maintenance, its role in cancer must be carefully contextualized to avoid unintended therapeutic resistance. Materials and Methods C. elegans strain maintenance Unless otherwise mentioned, all the C. elegans strains were maintained and propagated at 20°C on E. coli OP50 using standard procedures [ 81 ]. The strains used in this study are provided in Table 2. Preparation of RNAi plates RNAi plates were prepared using an autoclaved nematode growth medium supplemented with 100 μg/ml ampicillin and 2 mM IPTG. The plates were allowed to dry at room temperature for 24 hours. Bacterial cultures containing the specific RNAi construct were grown overnight in Luria-Bertani (LB) medium supplemented with 100 μg/ml ampicillin and 12.5 μg/ml tetracycline at 37°C in a shaker incubator. The following day, the saturated primary culture was diluted 1:50 in fresh LB medium containing 100 μg/ml ampicillin and incubated at 37°C in a shaker until the optical density at 600 nm (OD₆₀₀) reached 0.5–0.6. Bacterial cells were then pelleted by centrifugation at 3214 × g for 5 minutes at 4°C and resuspended in 1/10th of the original volume in M9 buffer containing 100 μg/ml ampicillin and 1 mM IPTG. Approximately 350 μl of this bacterial suspension was seeded onto RNAi plates and allowed to dry at room temperature for 48 hours before being stored at 4°C until further use. Hypochlorite treatment for synchronizing worm population Gravid adult worms, initially grown on E . coli OP50 bacteria, were collected using M9 buffer in a 15 ml falcon tube. Worms were washed twice by first centrifuging at 652 g for 60 seconds followed by resuspension of the worm pellet in 1X M9 buffer. After the second wash, the worm pellet was resuspended in 3.5 ml of 1X M9 buffer and 0.5 ml 5N NaOH and 1 ml of 4% Sodium hypochlorite solution was added. The mixture was vortexed for 4–5 minutes until the entire worm body dissolved, leaving behind the eggs. The eggs were washed 5–6 times, by first centrifuging at 1258 g, the 1X M9 was aspirated out, followed by resuspension in a fresh 1X M9 buffer to remove traces of bleach and alkali. After the final wash, eggs were kept in 15 ml falcons with ∼ 10 ml of 1X M9 buffer and kept on rotation ∼15 r.p.m for 17 hours to obtain L1 synchronized worms for all strains. The L1 worms were obtained by centrifugation at 805 g followed by resuspension in approximately 200–300 μl of M9 and added to respective RNAi plates. RNA isolation L4-staged worms were collected using 1X M9 buffer and washed thrice to remove bacteria. Trizol reagent (200 μl; Takara Bio, Kusatsu, Shiga, Japan) was added to the 50 μl worm pellet and subjected to three freeze-thaw cycles in liquid nitrogen with intermittent vortexing for 1 minute to break open the worm bodies. The samples were then frozen in liquid nitrogen and stored at -80°C till further use. Later, 200 μl of Trizol was again added to the worm pellet and the sample was vigorously vortexed for 1 minute. To this, 200 μl of chloroform was added and the tube was gently inverted several times followed by 5 minutes of incubation at room temperature. The sample was then centrifuged at 12000 g for 15 minutes at 4°C. The RNA containing the upper aqueous phase was gently removed into a fresh tube without disturbing the bottom layer and interphase. To this aqueous solution, an equal volume of isopropanol was added and the reaction was allowed to sit for 10 minutes at room temperature followed by centrifugation at 12000 g for 15 minutes at 4°C. The supernatant was carefully discarded without disturbing the RNA-containing pellet. The pellet was washed using 1 ml 70% ethanol solution followed by centrifugation at 8000g for 10 minutes at 4°C. The RNA pellet was further dried at room temperature and later dissolved in autoclaved MilliQ water followed by incubation at 65°C for 10 minutes with intermittent tapping. The concentration of RNA was determined by measuring absorbance at 260 nm using a NanoDrop UV spectrophotometer (Thermo Scientific, Waltham, USA) and the quality was checked using denaturing formaldehyde-agarose gel. Gene expression analysis using quantitative real-time PCR (QRT-PCR) First-strand cDNA synthesis was carried out using the Iscript cDNA synthesis kit (Biorad, Hercules, USA) following the manufacturer’s guidelines. The prepared cDNA was stored at -20°C. Gene expression levels were determined using the Brilliant III Ultra-Fast SYBR Green QPCR master mix (Agilent, Santa Clara, USA) and Agilent AriaMx Real-Time PCR system (Agilent, Santa Clara, USA), according to manufacturer’s guidelines. The relative expression of each gene was determined by normalizing the data to actin expression levels. The list of primers is provided in Table 3. Measurement of cell corpses using CED-1::GFP Engulfed cell corpses were analyzed using transgenic C. elegans expressing CED-1::GFP, a transmembrane protein localized to surrounding sheath cells responsible for engulfing apoptotic cell corpses. daf-2(e1370);ced-1p::ced-1::GFP ( smIs34 ) and daf-16(mgdf50);daf-2(e1370);ced-1p::ced-1::GFP ( smIs34 ) worms were bleached, and their eggs were incubated in a 1X M9 buffer for 17 hours to obtain synchronized L1 larvae. Approximately 100 L1 worms were then transferred onto control RNAi plates in triplicate. The worms were irradiated (0, 30 and 60 gy) at the late-L4 stage and imaged (6, 12, 24 and 36 HPI) in Z-stacks using a 488 nm laser to excite GFP on an LSM980 confocal microscope (Carl Zeiss, Oberkochen, Germany). The images were processed into maximum intensity projections (MIPs), and the number of cell corpses per gonadal arm was manually quantified. Chromosomal fragmentation assay Approximately 100 L1 worms were placed onto control or respective RNAi plates in triplicate. The worms were irradiated with ionizing radiation (IR) of different doses at the L4 stage. At the desired time point post-irradiation, the worms were stained with DAPI, and the oocyte chromosomes were imaged in z -stack using a using a 405 nm laser to LSM980 confocal microscope (Zeiss). For scoring chromosome fragmentation, images were converted into maximum intensity projections (MIPs) and scored. The oocyte chromosomes were categorized as normal, fragmented, or fused based on its morphology. Normal oocytes contain six bivalent chromosomes. Fragmented oocytes exhibit more than six and clustered chromosomes, while fused oocytes appear as aggregates with fewer than six bivalent chromosomes. DAPI staining Worms were cultured on specific RNAi plates from the L1 stage onward. Adult worms were collected in a 1.5 ml Eppendorf tube containing 1X M9 buffer and allowed to settle. The buffer was carefully removed using a glass Pasteur pipette, leaving approximately 100 μl of worm suspension. Ice-cold 100% methanol (1 ml) was then added to the worm pellet, followed by incubation at -20°C for 30 minutes. The methanol-fixed worm pellet was placed on a glass slide, and after the methanol had evaporated, Fluoroshield with DAPI (Invitrogen, Carlsbad, USA) was applied for staining. A coverslip was carefully placed on top, ensuring no air bubbles, and sealed with transparent nail polish. Finally, images were acquired using a 405 nm laser excitation for DAPI on an LSM980 confocal microscope (Carl Zeiss, Oberkochen, Germany). L1 larval development and fertility assay Almost 100 L1 worms were irradiated with different dose of gamma (0, 140, 200 and 300 Gy) and scored for worms progressing to the L4-stage or beyond (when the unirradiated worms reached early Day-1 adult). The larval development was assessed by determining the percentage of developed larvae that reached L4 stage or above. In the same experiment, fertile worms were counted (with eggs in the uterus) when the unirradiated controls reached late-day1 of adulthood. The percentage fertile worms for each condition were then calculated. Egg hatching To assess the extent of DNA damage repair in the germline the percentage of hatched eggs was determined. Worms were grown on respective RNAi L1 onwards, and gamma irradiated (0, 30 and 60 gy) at the late-L4 stage. Almost 15 adult worms per condition were allowed to lay eggs for 12-24 hr window post IR. The mother worms were then sacrificed and the number of eggs on respective plates were counted. After 48 hours the number of hatched progenies were counted, and the percentage of hatched eggs were calculated for each condition. Brood size Worms were grown on control RNAi from L1 onwards and gamma irradiated (0, 30 and 60 gy) at the late-L4 stage. Upon reaching the young adult stage, five worms were picked onto fresh RNAi plates, in triplicates, and allowed to lay eggs for 24 hours. The worms were then transferred to fresh plates every day until worms ceased to lay eggs, and these plates were counted after 48 hours to document the number of hatched worms. The total number of hatched progenies per worm is defined as brood size. Intestinal karyokinesis Approximately 100 L1 worms were placed onto control RNAi plates in triplicate. The worms were irradiated (0 and 100 Gy) at the L4 stage. At 48 HPI, the worms were stained with DAPI, and the intestinal nuclei were imaged in z -stack using a using a 405 nm laser to LSM980 confocal microscope (Zeiss). For scoring intestinal karyokinesis defects, images were converted into maximum intensity projections (MIPs) and scored. The quality was categorized as normal, or with defects based on its morphology. A normal score was given to distinct intestinal nuclei without any chromatin bridges or fusion; a defect indicated intestinal chromatin bridges or intestinal nuclei fusion. HUS-1::GFP foci formation HUS-1::GFP transgenic worms [ daf-2(e1370);hus-1(op241);hus-1p::hus-1::gfp and daf-16(mgdf50);daf-2(e1370);hus-1(op241);hus-1p::hus-1::gfp ] were grown on control RNAi L1 onwards. At the late-L4 stage, the worms were subjected to IR (0 and 100 gy) and incubated for a duration of 6-8 or 24-27 hours at 20°C. The adults were subsequently mounted on microscope slides coated with a 2% agarose pad in 20 mM sodium azide, and z-stacked images were acquired using a 488 nm laser channel on a confocal microscope (LSM980 Zeiss). Under baseline conditions, HUS-1::GFP exhibits a diffuse presence in the cytoplasm and shows only weak localization in the chromatin of germ cells. However, in response to DNA damage (e.g., gamma radiation), HUS-1::GFP accumulates in nuclear foci across all germ cells, appearing as prominent bright foci. The bright HUS-1::GFP foci were quantified in 10 pachytene germ cells per gonad for each condition. Statistical tests All statistical tests were performed utilizing the built-in functions of GraphPad Prism 10.1.0. In instances where we compared two conditions (continuous data), we applied a Student’s t -test with Welch’s correction, making no assumptions about consistent standard deviations. When comparing categorical data, we used the chi-square test. When comparing multiple conditions (continuous), we employed a Two-way ANOVA with Tukey’s multiple comparison test. Funding This project was partly funded by the National Bioscience Award for Career Development (BT/HRD/NBA/38/04/2016) and extramural grant (BT/PR27603/GET/119/267/2018) from the Department of Biotechnology, Government of India ( https://dbtindia.gov.in/ ), Science and Engineering Research Board-Science and Technology Award for Research (SERB-STAR) award (STR/2019/000064), Jagadish Chandra Bose National Fellowship (JCB/2022/000021) and extramural grant (CRG/2022/000525) from the Ministry of Science and Technology, Government of India ( https://serb.gov.in/page ), as well as core funding from the National Institute of Immunology (to AM). GCS is supported by an ICMR SRF fellowship (RMBH/FW/2020/19), and UR by DBT-JRF fellowship DBT/2018/NII/1035. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions UR, GCS, AM conceptualized the project. UR, GCS, OD, RKR, RM and AG performed the experiments and analyzed data. UR, GCS, OD, AM wrote the manuscript. AM supervised the project and acquired funding. Competing Interest Statement The authors disclose that there is no competing interest. Acknowledgements We thank the members of the Molecular Ageing Laboratory, the Central Confocal Facility, and the Central Instrumentation Facility of the National Institute of Immunology. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Funder Information Declared Department of Biotechnology , BT/HRD/NBA/38/04/2016 , BT/PR27603/GET/119/267/2018 , DBT/2018/NII/1035 Science and Engineering Research Board , STR/2019/000064 , JCB/2022/000021 , CRG/2022/000525 Indian Council of Medical Research , RMBH/FW/2020/19 References 1. ↵ Jackson SP , Bartek J . The DNA-damage response in human biology and disease . Nature . 2009 ; 461 ( 7267 ): 1071 – 8 . Epub 2009/10/23. doi: 10.1038/nature08467 . PubMed PMID: 19847258 ; PubMed Central PMCID: PMCPMC2906700 . OpenUrl CrossRef PubMed Web of Science 2. ↵ Cosentino C , Grieco D , Costanzo V . ATM activates the pentose phosphate pathway promoting anti-oxidant defence and DNA repair . EMBO J . 2011 ; 30 ( 3 ): 546 – 55 . Epub 2010/12/16. doi: 10.1038/emboj.2010.330 . PubMed PMID: 21157431 ; PubMed Central PMCID: PMCPMC3034007 . OpenUrl Abstract / FREE Full Text 3. ↵ Yang S , Hwang S , Kim B , Shin S , Kim M , Jeong SM . Fatty acid oxidation facilitates DNA double-strand break repair by promoting PARP1 acetylation . Cell Death Dis . 2023 ; 14 ( 7 ): 435 . Epub 2023/07/16. doi: 10.1038/s41419-023-05968-w . PubMed PMID: 37454129 ; PubMed Central PMCID: PMCPMC10349888 . OpenUrl CrossRef PubMed 4. ↵ Murata MM , Kong X , Moncada E , Chen Y , Imamura H , Wang P , et al. NAD+ consumption by PARP1 in response to DNA damage triggers metabolic shift critical for damaged cell survival . Mol Biol Cell . 2019 ; 30 ( 20 ): 2584 – 97 . Epub 2019/08/08. doi: 10.1091/mbc.E18-10-0650 . PubMed PMID: 31390283 ; PubMed Central PMCID: PMCPMC6740200 . OpenUrl CrossRef PubMed 5. ↵ Vazquez BN , Thackray JK , Serrano L . Sirtuins and DNA damage repair: SIRT7 comes to play . Nucleus . 2017 ; 8 ( 2 ): 107 – 15 . Epub 2017/04/14. doi: 10.1080/19491034.2016.1264552 . PubMed PMID: 28406750 ; PubMed Central PMCID: PMCPMC5403131 . OpenUrl CrossRef PubMed 6. ↵ van Heemst D. Insulin, IGF-1 and longevity . Aging Dis . 2010 ; 1 ( 2 ): 147 – 57 . Epub 2010/10/01. PubMed PMID: 22396862 ; PubMed Central PMCID: PMCPMC3295030 . OpenUrl PubMed 7. ↵ Gottlieb S , Ruvkun G . daf-2, daf-16 and daf-23: genetically interacting genes controlling Dauer formation in Caenorhabditis elegans . Genetics . 1994 ; 137 ( 1 ): 107 – 20 . Epub 1994/05/01. doi: 10.1093/genetics/137.1.107 . PubMed PMID: 8056303 ; PubMed Central PMCID: PMCPMC1205929 . OpenUrl Abstract / FREE Full Text 8. ↵ Ogg S , Paradis S , Gottlieb S , Patterson GI , Lee L , Tissenbaum HA , et al. The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans . Nature . 1997 ; 389 ( 6654 ): 994 – 9 . doi: 10.1038/40194 . PubMed PMID: 9353126 . OpenUrl CrossRef PubMed Web of Science 9. ↵ Mukhopadhyay A , Oh SW , Tissenbaum HA . Worming pathways to and from DAF-16/FOXO . Experimental gerontology . 2006 ; 41 ( 10 ): 928 – 34 . Epub 2006/07/15. doi: 10.1016/j.exger.2006.05.020 . PubMed PMID: 16839734 . OpenUrl CrossRef PubMed Web of Science 10. ↵ Martins R , Lithgow GJ , Link W . Long live FOXO: unraveling the role of FOXO proteins in aging and longevity . Aging Cell . 2016 ; 15 ( 2 ): 196 – 207 . Epub 2015/12/09. doi: 10.1111/acel.12427 . PubMed PMID: 26643314 ; PubMed Central PMCID: PMCPMC4783344 . OpenUrl CrossRef PubMed 11. ↵ Lei H , Quelle FW . FOXO transcription factors enforce cell cycle checkpoints and promote survival of hematopoietic cells after DNA damage . Mol Cancer Res . 2009 ; 7 ( 8 ): 1294 – 303 . Epub 2009/08/13. doi: 10.1158/1541-7786.MCR-08-0531 . PubMed PMID: 19671690 . OpenUrl Abstract / FREE Full Text 12. ↵ Seoane J , Le HV , Shen L , Anderson SA , Massague J . Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation . Cell . 2004 ; 117 ( 2 ): 211 – 23 . Epub 2004/04/16. doi: 10.1016/s0092-8674(04)00298-3 . PubMed PMID: 15084259 . OpenUrl CrossRef PubMed Web of Science 13. ↵ Bigarella CL , Li J , Rimmele P , Liang R , Sobol RW , Ghaffari S . FOXO3 Transcription Factor Is Essential for Protecting Hematopoietic Stem and Progenitor Cells from Oxidative DNA Damage . The Journal of biological chemistry . 2017 ; 292 ( 7 ): 3005 – 15 . Epub 2016/12/21. doi: 10.1074/jbc.M116.769455 . PubMed PMID: 27994057 ; PubMed Central PMCID: PMCPMC5314194 . OpenUrl Abstract / FREE Full Text 14. ↵ Sunters A , Fernandez de Mattos S , Stahl M , Brosens JJ , Zoumpoulidou G , Saunders CA , et al. FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines . The Journal of biological chemistry . 2003 ; 278 ( 50 ): 49795 – 805 . Epub 2003/10/07. doi: 10.1074/jbc.M309523200 . PubMed PMID: 14527951 . OpenUrl Abstract / FREE Full Text 15. Huang H , Regan KM , Lou Z , Chen J , Tindall DJ . CDK2-dependent phosphorylation of FOXO1 as an apoptotic response to DNA damage . Science . 2006 ; 314 (5797): 294 -7. Epub 2006/10/14. doi: 10.1126/science.1130512 . PubMed PMID: 17038621 . OpenUrl Abstract / FREE Full Text 16. ↵ Chung YM , Park SH , Tsai WB , Wang SY , Ikeda MA , Berek JS , et al. FOXO3 signalling links ATM to the p53 apoptotic pathway following DNA damage . Nature communications . 2012 ; 3 : 1000 . Epub 2012/08/16. doi: 10.1038/ncomms2008 . PubMed PMID: 22893124 ; PubMed Central PMCID: PMCPMC3589124 . OpenUrl CrossRef PubMed 17. ↵ Paull TT . Mechanisms of ATM Activation . Annu Rev Biochem . 2015 ; 84 : 711 – 38 . Epub 2015/01/13. doi: 10.1146/annurev-biochem-060614-034335 . PubMed PMID: 25580527 . OpenUrl CrossRef PubMed 18. ↵ Tran H , Brunet A , Grenier JM , Datta SR , Fornace AJ , Jr. , DiStefano PS , et al. DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein . Science . 2002 ; 296 ( 5567 ): 530 – 4 . doi: 10.1126/science.1068712 . PubMed PMID: 11964479 . OpenUrl Abstract / FREE Full Text 19. ↵ Inci G , Warkad MS , Kang BG , Lee NK , Suh HW , Lim SS , et al. FOXO3a Mediates Homologous Recombination Repair (HRR) via Transcriptional Activation of MRE11, BRCA1, BRIP1, and RAD50 . Molecules . 2022 ; 27 ( 23 ). Epub 2022/12/12. doi: 10.3390/molecules27238623 . PubMed PMID: 36500714 ; PubMed Central PMCID: PMCPMC9741359 . OpenUrl CrossRef PubMed 20. ↵ Craig AL , Moser SC , Bailly AP , Gartner A . Methods for studying the DNA damage response in the Caenorhabdatis elegans germ line . Methods Cell Biol . 2012 ; 107 : 321 – 52 . Epub 2012/01/10. doi: 10.1016/B978-0-12-394620-1.00011-4 . PubMed PMID: 22226529 . OpenUrl CrossRef PubMed 21. ↵ Lans H , Vermeulen W . Tissue specific response to DNA damage: C. elegans as role model . DNA Repair (Amst) . 2015 ; 32 : 141 – 8 . Epub 2015/05/11. doi: 10.1016/j.dnarep.2015.04.025 . PubMed PMID: 25957488 . OpenUrl CrossRef PubMed 22. ↵ Kimura KD , Tissenbaum HA , Liu Y , Ruvkun G. daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans . Science . 1997 ; 277 ( 5328 ): 942 – 6 . Epub 1997/08/15. doi: 10.1126/science.277.5328.942 . PubMed PMID: 9252323 . OpenUrl Abstract / FREE Full Text 23. ↵ Lin K , Hsin H , Libina N , Kenyon C . Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling . Nat Genet . 2001 ; 28 ( 2 ): 139 – 45 . Epub 2001/05/31. doi: 10.1038/88850 . PubMed PMID: 11381260 . OpenUrl CrossRef PubMed Web of Science 24. ↵ Murphy CT , McCarroll SA , Bargmann CI , Fraser A , Kamath RS , Ahringer J , et al. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans . Nature . 2003 ; 424 ( 6946 ): 277 – 83 . doi: 10.1038/nature01789 . OpenUrl CrossRef PubMed Web of Science 25. Kenyon C , Chang J , Gensch E , Rudner A , Tabtiang R . A C. elegans mutant that lives twice as long as wild type . Nature . 1993 ; 366 ( 6454 ): 461 – 4 . Epub 1993/12/02. doi: 10.1038/366461a0 . PubMed PMID: 8247153 . OpenUrl CrossRef PubMed Web of Science 26. ↵ Lee SS , Kennedy S , Tolonen AC , Ruvkun G . DAF-16 target genes that control C. elegans life-span and metabolism . Science . 2003 ; 300 ( 5619 ): 644 – 7 . Epub 2003/04/12. doi: 10.1126/science.1083614 . PubMed PMID: 12690206 . OpenUrl Abstract / FREE Full Text 27. ↵ Lin XX , Sen I , Janssens GE , Zhou X , Fonslow BR , Edgar D , et al. DAF-16/FOXO and HLH-30/TFEB function as combinatorial transcription factors to promote stress resistance and longevity . Nature communications . 2018 ; 9 ( 1 ): 4400 . Epub 2018/10/26. doi: 10.1038/s41467-018-06624-0 . PubMed PMID: 30353013 ; PubMed Central PMCID: PMCPMC6199276 . OpenUrl CrossRef PubMed 28. ↵ Sarkar GC , Rautela U , Goyala A , Datta S , Anand N , Singh A , et al. DNA damage signals from somatic uterine tissue arrest oogenesis through activated DAF-16 . Development . 2023 ; 150 ( 17 ). Epub 20230912. doi: 10.1242/dev.201472 . PubMed PMID: 37577954 . OpenUrl CrossRef PubMed 29. ↵ Kumar N , Jain V , Singh A , Jagtap U , Verma S , Mukhopadhyay A . Genome-wide endogenous DAF-16/FOXO recruitment dynamics during lowered insulin signalling in C. elegans . Oncotarget . 2015 ; 6 ( 39 ): 41418 – 33 . Epub 2015/11/06. doi: 10.18632/oncotarget.6282 . PubMed PMID: 26539642 ; PubMed Central PMCID: PMCPMC4747164 . OpenUrl CrossRef PubMed 30. ↵ Pazdernik N , Schedl T . Introduction to germ cell development in Caenorhabditis elegans . Advances in experimental medicine and biology . 2013 ; 757 : 1 – 16 . Epub 2012/08/09. doi: 10.1007/978-1-4614-4015-4_1 . PubMed PMID: 22872472 ; PubMed Central PMCID: PMCPMC3781019 . OpenUrl CrossRef PubMed 31. ↵ Clejan I , Boerckel J , Ahmed S . Developmental modulation of nonhomologous end joining in Caenorhabditis elegans . Genetics . 2006 ; 173 ( 3 ): 1301 – 17 . Epub 2006/05/17. doi: 10.1534/genetics.106.058628 . PubMed PMID: 16702421 ; PubMed Central PMCID: PMCPMC1526663 . OpenUrl Abstract / FREE Full Text 32. ↵ Dernburg AF , McDonald K , Moulder G , Barstead R , Dresser M , Villeneuve AM . Meiotic recombination in C. elegans initiates by a conserved mechanism and is dispensable for homologous chromosome synapsis . Cell . 1998 ; 94 ( 3 ): 387 – 98 . doi: 10.1016/s0092-8674(00)81481-6 . PubMed PMID: 9708740 . OpenUrl CrossRef PubMed Web of Science 33. ↵ Mata-Cabana A , Perez-Nieto C , Olmedo M . Nutritional control of postembryonic development progression and arrest in Caenorhabditis elegans . Adv Genet . 2021 ; 107 : 33 – 87 . Epub 2021/03/02. doi: 10.1016/bs.adgen.2020.11.002 . PubMed PMID: 33641748 . OpenUrl CrossRef PubMed 34. ↵ Melcher M , Schmid M , Aagaard L , Selenko P , Laible G , Jenuwein T . Structure-function analysis of SUV39H1 reveals a dominant role in heterochromatin organization, chromosome segregation, and mitotic progression . Mol Cell Biol . 2000 ; 20 ( 10 ): 3728 – 41 . Epub 2000/04/25. doi: 10.1128/MCB.20.10.3728-3741.2000 . PubMed PMID: 10779362 ; PubMed Central PMCID: PMCPMC85674 . OpenUrl Abstract / FREE Full Text 35. ↵ Hedgecock EM , White JG . Polyploid tissues in the nematode Caenorhabditis elegans . Dev Biol . 1985 ; 107 ( 1 ): 128 – 33 . Epub 1985/01/01. doi: 10.1016/0012-1606(85)90381-1 . PubMed PMID: 2578115 . OpenUrl CrossRef PubMed Web of Science 36. ↵ Kniazeva M , Ruvkun G . Rhizobium induces DNA damage in Caenorhabditis elegans intestinal cells . Proc Natl Acad Sci U S A . 2019 ; 116 ( 9 ): 3784 – 92 . Epub 2019/02/28. doi: 10.1073/pnas.1815656116 . PubMed PMID: 30808764 ; PubMed Central PMCID: PMCPMC6397575 . OpenUrl Abstract / FREE Full Text 37. ↵ Michaelson D , Korta DZ , Capua Y , Hubbard EJ . Insulin signaling promotes germline proliferation in C. elegans . Development . 2010 ; 137 ( 4 ): 671 – 80 . doi: 10.1242/dev.042523 . PubMed PMID: 20110332 ; PubMed Central PMCID: PMCPMC2827619 . OpenUrl Abstract / FREE Full Text 38. ↵ Qi W , Huang X , Neumann-Haefelin E , Schulze E , Baumeister R . Cell-nonautonomous signaling of FOXO/DAF-16 to the stem cells of Caenorhabditis elegans . PLoS Genet . 2012 ; 8 ( 8 ): e1002836 . Epub 2012/08/24. doi: 10.1371/journal.pgen.1002836 . PubMed PMID: 22916022 ; PubMed Central PMCID: PMCPMC3420913 . OpenUrl CrossRef PubMed 39. ↵ Qin Z , Hubbard EJ . Non-autonomous DAF-16/FOXO activity antagonizes age-related loss of C. elegans germline stem/progenitor cells . Nature communications . 2015 ; 6 : 7107 . Epub 20150511. doi: 10.1038/ncomms8107 . PubMed PMID: 25960195 ; PubMed Central PMCID: PMCPMC4432587 . OpenUrl CrossRef PubMed 40. ↵ Uno M , Tani Y , Nono M , Okabe E , Kishimoto S , Takahashi C , et al. Neuronal DAF-16-to-intestinal DAF-16 communication underlies organismal lifespan extension in C. elegans . iScience . 2021 ; 24 ( 7 ): 102706 . Epub 20210610. doi: 10.1016/j.isci.2021.102706 . PubMed PMID: 34235410 ; PubMed Central PMCID: PMCPMC8246587 . OpenUrl CrossRef PubMed 41. ↵ Qadota H , Inoue M , Hikita T , Koppen M , Hardin JD , Amano M , et al. Establishment of a tissue-specific RNAi system in C. elegans . Gene . 2007 ; 400 ( 1-2 ): 166 – 73 . Epub 20070803. doi: 10.1016/j.gene.2007.06.020 . PubMed PMID: 17681718 ; PubMed Central PMCID: PMCPMC3086655 . OpenUrl CrossRef PubMed 42. ↵ Zou L , Wu D , Zang X , Wang Z , Wu Z , Chen D . Construction of a germline-specific RNAi tool in C. elegans . Sci Rep . 2019 ; 9 ( 1 ): 2354 . Epub 20190220. doi: 10.1038/s41598-019-38950-8 . PubMed PMID: 30787374 ; PubMed Central PMCID: PMCPMC6382888 . OpenUrl CrossRef PubMed 43. ↵ Tijsterman M , Okihara KL , Thijssen K , Plasterk RH . PPW-1, a PAZ/PIWI protein required for efficient germline RNAi, is defective in a natural isolate of C. elegans . Curr Biol . 2002 ; 12 ( 17 ): 1535 – 40 . doi: 10.1016/s0960-9822(02)01110-7 . PubMed PMID: 12225671 . OpenUrl CrossRef PubMed Web of Science 44. ↵ Lin K , Dorman JB , Rodan A , Kenyon C. daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans . Science (New York, NY) . 1997 ; 278 (5341): 1319 – 22 . doi: 10.1126/science.278.5341.1319 . OpenUrl Abstract / FREE Full Text 45. ↵ Kwon ES , Narasimhan SD , Yen K , Tissenbaum HA . A new DAF-16 isoform regulates longevity . Nature . 2010 ; 466 ( 7305 ): 498 – 502 . Epub 2010/07/09. doi: 10.1038/nature09184 . PubMed PMID: 20613724 ; PubMed Central PMCID: PMCPMC3109862 . OpenUrl CrossRef PubMed Web of Science 46. ↵ Chen AT , Guo C , Itani OA , Budaitis BG , Williams TW , Hopkins CE , et al. Longevity Genes Revealed by Integrative Analysis of Isoform-Specific daf-16/FoxO Mutants of Caenorhabditis elegans . Genetics . 2015 ; 201 ( 2 ): 613 – 29 . Epub 2015/07/30. doi: 10.1534/genetics.115.177998 . PubMed PMID: 26219299 ; PubMed Central PMCID: PMCPMC4596673 . OpenUrl Abstract / FREE Full Text 47. ↵ White RR , Maslov AY , Lee M , Wilner SE , Levy M , Vijg J . FOXO3a acts to suppress DNA double-strand break-induced mutations . Aging Cell . 2020 ; 19 ( 9 ): e13184 . Epub 2020/07/29. doi: 10.1111/acel.13184 . PubMed PMID: 32720744 ; PubMed Central PMCID: PMCPMC7511859 . OpenUrl CrossRef PubMed 48. ↵ Hofmann ER , Milstein S , Boulton SJ , Ye M , Hofmann JJ , Stergiou L , et al. Caenorhabditis elegans HUS-1 is a DNA damage checkpoint protein required for genome stability and EGL-1-mediated apoptosis . Curr Biol . 2002 ; 12 ( 22 ): 1908 – 18 . Epub 2002/11/26. doi: 10.1016/s0960-9822(02)01262-9 . PubMed PMID: 12445383 . OpenUrl CrossRef PubMed Web of Science 49. ↵ Lim PX , Patel DR , Poisson KE , Basuita M , Tsai C , Lyndaker AM , et al. Genome Protection by the 9-1-1 Complex Subunit HUS1 Requires Clamp Formation, DNA Contacts, and ATR Signaling-independent Effector Functions . The Journal of biological chemistry . 2015 ; 290 ( 24 ): 14826 – 40 . Epub 2015/04/26. doi: 10.1074/jbc.M114.630640 . PubMed PMID: 25911100 ; PubMed Central PMCID: PMCPMC4463431 . OpenUrl Abstract / FREE Full Text 50. ↵ Zhou Z , Caron E , Hartwieg E , Hall A , Horvitz HR . The C. elegans PH domain protein CED-12 regulates cytoskeletal reorganization via a Rho/Rac GTPase signaling pathway . Dev Cell . 2001 ; 1 ( 4 ): 477 – 89 . Epub 2001/11/13. doi: 10.1016/s1534-5807(01)00058-2 . PubMed PMID: 11703939 . OpenUrl CrossRef PubMed Web of Science 51. ↵ Zou H , Henzel WJ , Liu X , Lutschg A , Wang X . Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3 . Cell . 1997 ; 90 ( 3 ): 405 – 13 . Epub 1997/08/08. doi: 10.1016/s0092-8674(00)80501-2 . PubMed PMID: 9267021 . OpenUrl CrossRef PubMed Web of Science 52. ↵ Yuan J , Shaham S , Ledoux S , Ellis HM , Horvitz HR . The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme . Cell . 1993 ; 75 ( 4 ): 641 – 52 . Epub 1993/11/19. doi: 10.1016/0092-8674(93)90485-9 . PubMed PMID: 8242740 . OpenUrl CrossRef PubMed Web of Science 53. ↵ Hornsveld M , Feringa FM , Krenning L , van den Berg J , Smits LMM , Nguyen NBT , et al. A FOXO-dependent replication checkpoint restricts proliferation of damaged cells . Cell reports . 2021 ; 34 ( 4 ): 108675 . Epub 2021/01/28. doi: 10.1016/j.celrep.2020.108675 . PubMed PMID: 33503422 . OpenUrl CrossRef PubMed 54. ↵ Brunet A , Bonni A , Zigmond MJ , Lin MZ , Juo P , Hu LS , et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor . Cell . 1999 ; 96 ( 6 ): 857 – 68 . Epub 1999/04/02. doi: 10.1016/s0092-8674(00)80595-4 . PubMed PMID: 10102273 . OpenUrl CrossRef PubMed Web of Science 55. ↵ Sunters A , Madureira PA , Pomeranz KM , Aubert M , Brosens JJ , Cook SJ , et al. Paclitaxel-induced nuclear translocation of FOXO3a in breast cancer cells is mediated by c-Jun NH2-terminal kinase and Akt . Cancer research . 2006 ; 66 ( 1 ): 212 – 20 . Epub 2006/01/07. doi: 10.1158/0008-5472.CAN-05-1997 . PubMed PMID: 16397234 . OpenUrl Abstract / FREE Full Text 56. ↵ Vermezovic J , Stergiou L , Hengartner MO, d’Adda di Fagagna F. Differential regulation of DNA damage response activation between somatic and germline cells in Caenorhabditis elegans . Cell Death Differ . 2012 ; 19 ( 11 ): 1847 – 55 . Epub 2012/06/19. doi: 10.1038/cdd.2012.69 . PubMed PMID: 22705849 ; PubMed Central PMCID: PMCPMC3469062 . OpenUrl CrossRef PubMed 57. ↵ Bujarrabal-Dueso A , Sendtner G , Meyer DH , Chatzinikolaou G , Stratigi K , Garinis GA , et al. The DREAM complex functions as conserved master regulator of somatic DNA-repair capacities . Nat Struct Mol Biol . 2023 ; 30 ( 4 ): 475 – 88 . Epub 2023/03/25. doi: 10.1038/s41594-023-00942-8 . PubMed PMID: 36959262 ; PubMed Central PMCID: PMCPMC10113156 declare no competing interests. OpenUrl CrossRef PubMed 58. ↵ Mueller MM , Castells-Roca L , Babu V , Ermolaeva MA , Muller RU , Frommolt P , et al. DAF-16/FOXO and EGL-27/GATA promote developmental growth in response to persistent somatic DNA damage . Nature cell biology . 2014 ; 16 ( 12 ): 1168 – 79 . Epub 2014/11/25. doi: 10.1038/ncb3071 . PubMed PMID: 25419847 ; PubMed Central PMCID: PMCPMC4250074 . OpenUrl CrossRef PubMed 59. ↵ Killian DJ , Hubbard EJ . Caenorhabditis elegans germline patterning requires coordinated development of the somatic gonadal sheath and the germ line . Dev Biol . 2005 ; 279 ( 2 ): 322 – 35 . Epub 2005/03/01. doi: 10.1016/j.ydbio.2004.12.021 . PubMed PMID: 15733661 . OpenUrl CrossRef PubMed Web of Science 60. ↵ Gopal S , Amran A , Elton A , Ng L , Pocock R . A somatic proteoglycan controls Notch-directed germ cell fate . Nature communications . 2021 ; 12 ( 1 ): 6708 . Epub 2021/11/20. doi: 10.1038/s41467-021-27039-4 . PubMed PMID: 34795288 ; PubMed Central PMCID: PMCPMC8602670 . OpenUrl CrossRef PubMed 61. ↵ Li X , Johnson RW , Park D , Chin-Sang I , Chamberlin HM . Somatic gonad sheath cells and Eph receptor signaling promote germ-cell death in C. elegans . Cell Death Differ . 2012 ; 19 ( 6 ): 1080 – 9 . Epub 2012/01/14. doi: 10.1038/cdd.2011.192 . PubMed PMID: 22240896 ; PubMed Central PMCID: PMCPMC3354057 . OpenUrl CrossRef PubMed 62. ↵ Govindan JA , Nadarajan S , Kim S , Starich TA , Greenstein D . Somatic cAMP signaling regulates MSP-dependent oocyte growth and meiotic maturation in C. elegans . Development . 2009 ; 136 ( 13 ): 2211 – 21 . Epub 2009/06/09. doi: 10.1242/dev.034595 . PubMed PMID: 19502483 ; PubMed Central PMCID: PMCPMC2729340 . OpenUrl Abstract / FREE Full Text 63. ↵ Ou HL , Kim CS , Uszkoreit S , Wickstrom SA , Schumacher B . Somatic Niche Cells Regulate the CEP-1/p53-Mediated DNA Damage Response in Primordial Germ Cells . Dev Cell . 2019 ; 50 ( 2 ): 167 – 83 e8. Epub 2019/07/25. doi: 10.1016/j.devcel.2019.06.012 . PubMed PMID: 31336098 . OpenUrl CrossRef PubMed 64. ↵ Soltanmohammadi N , Wang S , Schumacher B . Somatic PMK-1/p38 signaling links environmental stress to germ cell apoptosis and heritable euploidy . Nature communications . 2022 ; 13 ( 1 ): 701 . Epub 2022/02/06. doi: 10.1038/s41467-022-28225-8 . PubMed PMID: 35121747 ; PubMed Central PMCID: PMCPMC8816960 competing interests. OpenUrl CrossRef PubMed 65. ↵ Levi-Ferber M , Salzberg Y , Safra M , Haviv-Chesner A , Bulow HE , Henis-Korenblit S . It’s all in your mind: determining germ cell fate by neuronal IRE-1 in C. elegans . PLoS Genet . 2014 ; 10 ( 10 ): e1004747 . Epub 2014/10/24. doi: 10.1371/journal.pgen.1004747 . PubMed PMID: 25340700 ; PubMed Central PMCID: PMCPMC4207656 . OpenUrl CrossRef PubMed 66. Sendoel A , Kohler I , Fellmann C , Lowe SW , Hengartner MO . HIF-1 antagonizes p53-mediated apoptosis through a secreted neuronal tyrosinase . Nature . 2010 ; 465 ( 7298 ): 577 – 83 . Epub 2010/06/04. doi: 10.1038/nature09141 . PubMed PMID: 20520707 ; PubMed Central PMCID: PMCPMC3328299 . OpenUrl CrossRef PubMed Web of Science 67. ↵ Chapman EM , Lant B , Ohashi Y , Yu B , Schertzberg M , Go C , et al. A conserved CCM complex promotes apoptosis non-autonomously by regulating zinc homeostasis . Nature communications . 2019 ; 10 ( 1 ): 1791 . Epub 2019/04/19. doi: 10.1038/s41467-019-09829-z . PubMed PMID: 30996251 ; PubMed Central PMCID: PMCPMC6470173 . OpenUrl CrossRef PubMed 68. ↵ Tsai WB , Chung YM , Takahashi Y , Xu Z , Hu MC . Functional interaction between FOXO3a and ATM regulates DNA damage response . Nature cell biology . 2008 ; 10 ( 4 ): 460 – 7 . Epub 2008/03/18. doi: 10.1038/ncb1709 . PubMed PMID: 18344987 ; PubMed Central PMCID: PMCPMC2674111 . OpenUrl CrossRef PubMed Web of Science 69. ↵ Medema RH , Kops GJ , Bos JL , Burgering BM . AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1 . Nature . 2000 ; 404 ( 6779 ): 782 – 7 . Epub 2000/04/28. doi: 10.1038/35008115 . PubMed PMID: 10783894 . OpenUrl CrossRef PubMed Web of Science 70. ↵ Kops GJ , Medema RH , Glassford J , Essers MA , Dijkers PF , Coffer PJ , et al. Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors . Mol Cell Biol . 2002 ; 22 ( 7 ): 2025 – 36 . Epub 2002/03/09. doi: 10.1128/MCB.22.7.2025-2036.2002 . PubMed PMID: 11884591 ; PubMed Central PMCID: PMCPMC133681 . OpenUrl Abstract / FREE Full Text 71. ↵ Brunet A , Sweeney LB , Sturgill JF , Chua KF , Greer PL , Lin Y , et al. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase . Science . 2004 ; 303 ( 5666 ): 2011 – 5 . Epub 2004/02/21. doi: 10.1126/science.1094637 . PubMed PMID: 14976264 . OpenUrl Abstract / FREE Full Text 72. ↵ Fu Z , Tindall DJ . FOXOs, cancer and regulation of apoptosis . Oncogene . 2008 ; 27 ( 16 ): 2312 – 9 . Epub 2008/04/09. doi: 10.1038/onc.2008.24 . PubMed PMID: 18391973 ; PubMed Central PMCID: PMCPMC2819403 . OpenUrl CrossRef PubMed Web of Science 73. ↵ Bullock MD , Bruce A , Sreekumar R , Curtis N , Cheung T , Reading I , et al. FOXO3 expression during colorectal cancer progression: biomarker potential reflects a tumour suppressor role . Br J Cancer . 2013 ; 109 ( 2 ): 387 – 94 . Epub 2013/07/06. doi: 10.1038/bjc.2013.355 . PubMed PMID: 23828518 ; PubMed Central PMCID: PMCPMC3721407 . OpenUrl CrossRef PubMed 74. Tsuji T , Maeda Y , Kita K , Murakami K , Saya H , Takemura H , et al. FOXO3 is a latent tumor suppressor for FOXO3-positive and cytoplasmic-type gastric cancer cells . Oncogene . 2021 ; 40 ( 17 ): 3072 – 86 . Epub 2021/04/03. doi: 10.1038/s41388-021-01757-x . PubMed PMID: 33795838 ; PubMed Central PMCID: PMCPMC8084732 . OpenUrl CrossRef PubMed 75. ↵ Smit L , Berns K , Spence K , Ryder WD , Zeps N , Madiredjo M , et al. An integrated genomic approach identifies that the PI3K/AKT/FOXO pathway is involved in breast cancer tumor initiation . Oncotarget . 2016 ; 7 ( 3 ): 2596 – 610 . Epub 2015/11/26. doi: 10.18632/oncotarget.6354 . PubMed PMID: 26595803 ; PubMed Central PMCID: PMCPMC4823058 . OpenUrl CrossRef PubMed 76. ↵ Storz P , Doppler H , Copland JA , Simpson KJ , Toker A . FOXO3a promotes tumor cell invasion through the induction of matrix metalloproteinases . Mol Cell Biol . 2009 ; 29 ( 18 ): 4906 – 17 . Epub 2009/07/01. doi: 10.1128/MCB.00077-09 . PubMed PMID: 19564415 ; PubMed Central PMCID: PMCPMC2738298 . OpenUrl Abstract / FREE Full Text 77. ↵ Tenbaum SP , Ordonez-Moran P , Puig I , Chicote I , Arques O , Landolfi S , et al. beta-catenin confers resistance to PI3K and AKT inhibitors and subverts FOXO3a to promote metastasis in colon cancer . Nat Med . 2012 ; 18 ( 6 ): 892 – 901 . Epub 2012/05/23. doi: 10.1038/nm.2772 . PubMed PMID: 22610277 . OpenUrl CrossRef PubMed 78. ↵ Hornsveld M , Dansen TB , Derksen PW , Burgering BMT . Re-evaluating the role of FOXOs in cancer . Seminars in cancer biology . 2018 ; 50 : 90 – 100 . Epub 2017/11/28. doi: 10.1016/j.semcancer.2017.11.017 . PubMed PMID: 29175105 . OpenUrl CrossRef PubMed 79. ↵ Hagenbuchner J , Rupp M , Salvador C , Meister B , Kiechl-Kohlendorfer U , Muller T , et al. Nuclear FOXO3 predicts adverse clinical outcome and promotes tumor angiogenesis in neuroblastoma . Oncotarget . 2016 ; 7 ( 47 ): 77591 – 606 . Epub 2016/10/22. doi: 10.18632/oncotarget.12728 . PubMed PMID: 27769056 ; PubMed Central PMCID: PMCPMC5363607 . OpenUrl CrossRef PubMed 80. ↵ Marlow LA , von Roemeling CA , Cooper SJ , Zhang Y , Rohl SD , Arora S , et al. Foxo3a drives proliferation in anaplastic thyroid carcinoma through transcriptional regulation of cyclin A1: a paradigm shift that impacts current therapeutic strategies . Journal of cell science . 2012 ; 125 ( Pt 18 ): 4253 – 63 . Epub 2012/06/22. doi: 10.1242/jcs.097428 . PubMed PMID: 22718346 ; PubMed Central PMCID: PMCPMC3516436 . OpenUrl Abstract / FREE Full Text 81. ↵ Stiernagle T . Maintenance of C. elegans . WormBook . 2006 : 1 – 11 . Epub 2007/12/01. doi: 10.1895/wormbook.1.101.1 . PubMed PMID: 18050451 ; PubMed Central PMCID: PMCPMC4781397 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted October 28, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following DAF-16/FOXO maintains genome integrity following genotoxic stress Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share DAF-16/FOXO maintains genome integrity following genotoxic stress Umanshi Rautela , Oviya Devendran , Gautam Chandra Sarkar , KR Ranjisha , Rashi Mittal , Anita Goyala , Arnab Mukhopadhyay bioRxiv 2025.10.27.684284; doi: https://doi.org/10.1101/2025.10.27.684284 Share This Article: Copy Citation Tools DAF-16/FOXO maintains genome integrity following genotoxic stress Umanshi Rautela , Oviya Devendran , Gautam Chandra Sarkar , KR Ranjisha , Rashi Mittal , Anita Goyala , Arnab Mukhopadhyay bioRxiv 2025.10.27.684284; doi: https://doi.org/10.1101/2025.10.27.684284 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (7618) Biochemistry (17633) Bioengineering (13856) Bioinformatics (41841) Biophysics (21399) Cancer Biology (18529) Cell Biology (25422) Clinical Trials (138) Developmental Biology (13352) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24282) Genetics (15582) Genomics (22462) Immunology (17700) Microbiology (40295) Molecular Biology (17140) Neuroscience (88419) Paleontology (666) Pathology (2823) Pharmacology and Toxicology (4813) Physiology (7632) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4284) Systems Biology (9808) Zoology (2267)