Full Text
<article class="scholarly-article">
<h2>Introduction</h2>
<p>Transgenerational epigenetic inheritance (TEI) describes the transmission of information that is not encoded in the DNA sequence across multiple generations [1,4,22]. In <em>Caenorhabditis elegans</em>, TEI has been extensively documented for small RNA species, including siRNAs and piRNAs, which can silence homologous sequences for several generations [2,6,19]. Histone post‑translational modifications (PTMs) such as methylation of lysine residues also participate in TEI, often through crosstalk with RNA interference pathways [1,12,14]. However, the extent to which environmentally induced changes in histone PTMs are stably inherited and the functional consequences of such inheritance remain active areas of investigation [9,18,21].</p><p>Stressors ranging from starvation and temperature shifts to exposure to toxicants can elicit epigenetic alterations that persist in subsequent generations [3,7,8,10,13,15,17,20,30]. For instance, starvation in early larval stages triggers transgenerational changes in small RNA populations that influence growth and stress resistance [19,20]. Similarly, pathogen infection induces heritable modifications to gene expression and fitness [3]. These observations raise the question of whether histone PTMs—especially repressive marks such as H3K9me3—serve as carriers of stress‑induced epigenetic information across generations.</p><p>In this study, we tested the hypothesis that a brief heat stress leads to transgenerational inheritance of histone methylation in <em>C. elegans</em>. We focused on two well‑characterized marks: H3K9me3, which is associated with heterochromatin formation and transcriptional silencing, and H3K4me3, a mark linked to active transcription [18]. Using ChIP‑qPCR, we quantified these modifications at selected loci in the parental generation and in three consecutive progeny generations. We also measured lifespan and brood size to assess whether any inherited chromatin changes were linked to organismal phenotypes. Our results demonstrate that heat‑induced H3K9me3 enrichment can endure for at least two generations, consistent with a model in which transgenerational histone marks are gradually diluted or erased through germline reprogramming.</p>
<h2>Literature Review</h2>
<p>The molecular machinery underlying TEI in <em>C. elegans</em> includes the Argonaute proteins, RNA‑dependent RNA polymerases, and chromatin regulators such as the H3K9 methyltransferases SET‑25 and SET‑32 [1,12,21]. A seminal study by Gu et al. [12] showed that synthetic siRNAs can trigger a transgenerational H3K9me3 footprint that spreads in <em>cis</em> and <em>trans</em>. More recently, Schwartz‑Orbach et al. [21] identified the nuclear RNAi factor SET‑32 as a key enzyme that deposits H3K23me3, a mark that can be inherited across generations. Meanwhile, Wan et al. [18] demonstrated that H3K4me3 modifications induced by dietary changes can serve as a transgenerational signal for lipid metabolism. These findings highlight the diversity of histone PTMs capable of TEI.</p><p>The stability of transmitted marks varies widely. Some modifications are stably inherited for more than five generations, while others are reset after one or two generations [4,14]. The mechanisms of resetting are thought to involve germline reprogramming during gametogenesis and early embryogenesis [14]. Small RNAs appear to be critical for the initial induction and subsequent maintenance of histone marks, as mutations in RNAi components abolish transgenerational silencing [2,6]. Environmental stressors can hijack these pathways. For example, crude oil exposure causes transgenerational reproductive defects associated with altered histone methylation [10], and nanoplastic particles upregulate germline methyltransferases that modify H3K9me3 [11]. Oxidative stress has also been implicated in transgenerational neurotoxicity [8].</p><p>The relevance of histone‑based TEI to evolution and adaptation has been debated [27,28]. Some argue that epigenetic changes provide a rapid, reversible mechanism for organisms to respond to fluctuating environments, while others view them as noise that is eventually filtered out by natural selection [4,27]. Work on corals suggests that epigenetic memory can enhance resilience to thermal stress [24], and similar principles may apply to <em>C. elegans</em> [30]. Understanding the constraints on TEI—such as the duration of memory and the interaction with genetic variation—is therefore important for predicting how populations cope with environmental change [16].</p>
<h2>Methodology</h2>
<h4>Strains and culture conditions</h4><p>The wild‑type N2 strain of <em>Caenorhabditis elegans</em> was maintained at 20 °C on nematode growth medium (NGM) plates seeded with <em>Escherichia coli</em> OP50. Age‑synchronized populations were obtained by hypochlorite treatment of gravid hermaphrodites and subsequent incubation of eggs in M9 buffer overnight.</p><h4>Heat stress exposure</h4><p>L4‑stage larvae were subjected to a 2‑h heat shock at 30 °C in a humidified incubator, followed by a 1‑h recovery at 20 °C. Control animals were kept at 20 °C throughout. After recovery, individual animals (F₀) were placed on fresh plates to lay eggs. Progeny were collected at each generation (F₁, F₂, F₃) by transferring single L4 hermaphrodites to new plates. Synchronous populations were generated for each generation.</p><h4>Chromatin immunoprecipitation (ChIP)</h4><p>For each generation, approximately 50,000 mixed‑stage embryos were harvested from the progeny of four replicate populations. Embryos were cross‑linked with 2 % formaldehyde, quenched, and chromatin was sheared to ~300‑bp fragments using a Diagenode Bioruptor. Immunoprecipitation was carried out with antibodies against H3K9me3 (Abcam ab8898) and H3K4me3 (Abcam ab8580). Normal rabbit IgG served as a negative control. Enrichment was measured by quantitative PCR at a repetitive element (CELE_T13F2), a stress‑response gene (<em>hsp‑70</em>), and a constitutively active control (<em>act‑1</em>). Data were normalized to input and expressed as fold‑change relative to the control (non‑stress) group for each generation.</p><h4>Lifespan and brood size assays</h4><p>Lifespan was assessed at 20 °C using 60 animals per condition, transferred every other day to fresh plates. Brood size was measured by placing individual L4 hermaphrodites (n = 10) on separate plates and counting total progeny after three days. All assays were performed in triplicate.</p><h4>Statistical analysis</h4><p>Differences in histone enrichment and phenotypic traits were analysed using one‑way ANOVA with Tukey post‑hoc tests. A mixed‑effects regression model examined the generational trend of H3K9me3 enrichment, with “generation” as a fixed effect and “biological replicate” as a random intercept. Analyses were conducted in R (version 4.3.2). Threshold for significance was set at p < 0.05.</p>
<h2>Results</h2>
<h4>Heat stress induces transient transgenerational H3K9me3 enrichment</h4><p>ChIP‑qPCR analysis revealed that H3K9me3 at the repetitive locus CELE_T13F2 was significantly enriched in the F₁ progeny of heat‑exposed parents compared to controls. As shown in Table 1, the fold‑change remained elevated in F₂ but returned to baseline by F₃. No significant enrichment was detected at <em>hsp‑70</em> or <em>act‑1</em> for H3K9me3, indicating locus specificity. H3K4me3 showed a transient increase only at <em>hsp‑70</em> in F₁ (Table 1), consistent with a rapid stress‑response activation that was not heritable beyond the first generation.</p><figure class="table-figure"><table><thead><tr><th>Histone mark</th><th>Target locus</th><th>F₀</th><th>F₁</th><th>F₂</th><th>F₃</th></tr></thead><tbody><tr><td>H3K9me3</td><td>CELE_T13F2</td><td>1.02 (0.08)</td><td>1.64 (0.12) **</td><td>1.31 (0.09) *</td><td>1.03 (0.06)</td></tr><tr><td>H3K9me3</td><td><em>hsp‑70</em></td><td>0.97 (0.05)</td><td>1.12 (0.07)</td><td>1.01 (0.04)</td><td>0.99 (0.05)</td></tr><tr><td>H3K4me3</td><td><em>hsp‑70</em></td><td>1.05 (0.09)</td><td>1.58 (0.11) *</td><td>1.10 (0.08)</td><td>0.98 (0.06)</td></tr><tr><td>H3K4me3</td><td><em>act‑1</em></td><td>0.98 (0.06)</td><td>1.03 (0.04)</td><td>0.99 (0.05)</td><td>1.02 (0.07)</td></tr></tbody></table><figcaption>Table 1. Histone modification enrichment (fold‑change relative to generation‑matched controls) measured by ChIP‑qPCR. Values are mean (SEM) from three biological replicates. *p < 0.05; **p < 0.01 (ANOVA with Tukey post‑hoc).</figcaption></figure><p><figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/transgenerational-inheritance-of-stress-induced-histone-modifications-in-caenorhabditis-elegans-tkexx/figure-1-1779095887252.octet-stream" alt="Dot plot showing H3K9me3 enrichment at CELE_T13F2 across generations F₀–F₃, with individual replicate points and group means highlighted." loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 1. Dot plot showing H3K9me3 enrichment at CELE_T13F2 across generations F₀–F₃, with individual replicate points and group means highlighted.</figcaption></figure></p><p>To quantify the generational decline in H3K9me3 enrichment, we fitted a mixed‑effects regression model with generation as a continuous predictor. Table 2 summarises the regression coefficients. The significant negative slope for generation (β = −0.12, p = 0.02) confirms that the signal decays with each generation, consistent with a process of gradual resetting.</p><figure class="table-figure"><table><thead><tr><th>Predictor</th><th>Estimate</th><th>SE</th><th>t‑value</th><th>p‑value</th></tr></thead><tbody><tr><td>(Intercept)</td><td>0.81</td><td>0.09</td><td>9.00</td><td><0.001</td></tr><tr><td>Generation (numeric)</td><td>−0.12</td><td>0.05</td><td>−2.40</td><td>0.02</td></tr><tr><td>Stress (binary)</td><td>0.34</td><td>0.10</td><td>3.40</td><td>0.001</td></tr><tr><td>Stress × Generation</td><td>−0.09</td><td>0.06</td><td>−1.50</td><td>0.14</td></tr></tbody></table><figcaption>Table 2. Mixed‑effects regression coefficients for H3K9me3 enrichment (log‑transformed fold‑change) at CELE_T13F2. Random intercept for biological replicate; n = 24 observations (4 generations × 3 replicates × 2 conditions).</figcaption></figure><h4>Lifespan and brood size</h4><p><figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/transgenerational-inheritance-of-stress-induced-histone-modifications-in-caenorhabditis-elegans-tkexx/figure-2-1779095892787.octet-stream" alt="Kaplan–Meier survival curves for heat‑stressed and control lineages across generations." loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 2. Kaplan–Meier survival curves for heat‑stressed and control lineages across generations.</figcaption></figure></p><p>We next asked whether the observed chromatin changes were associated with differences in lifespan or fecundity. The mean lifespan of heat‑exposed lineages was slightly increased in F₁ and F₂ compared to controls, but the differences did not reach statistical significance (Table 3). Brood size was comparable across all conditions. A correlation analysis between H3K9me3 enrichment at CELE_T13F2 and lifespan in individual replicates revealed a positive but non‑significant trend (Pearson’s r = 0.34, p = 0.12).</p><figure class="table-figure"><table><thead><tr><th>Generation</th><th>Treatment</th><th>Mean lifespan (days)</th><th>SEM</th><th>p‑value (vs. control</th><th>Mean brood size</th><th>SEM</th></tr></thead><tbody><tr><td>F₀</td><td>Control</td><td>18.2</td><td>0.9</td><td>–</td><td>285</td><td>12</td></tr><tr><td>F₀</td><td>Heat</td><td>18.5</td><td>1.0</td><td>0.82</td><td>278</td><td>14</td></tr><tr><td>F₁</td><td>Control</td><td>17.9</td><td>0.8</td><td>–</td><td>290</td><td>11</td></tr><tr><td>F₁</td><td>Heat</td><td>19.6</td><td>1.1</td><td>0.07</td><td>282</td><td>13</td></tr><tr><td>F₂</td><td>Control</td><td>18.0</td><td>0.8</td><td>–</td><td>288</td><td>10</td></tr><tr><td>F₂</td><td>Heat</td><td>19.1</td><td>1.0</td><td>0.15</td><td>275</td><td>15</td></tr><tr><td>F₃</td><td>Control</td><td>18.1</td><td>0.9</td><td>–</td><td>287</td><td>9</td></tr><tr><td>F₃</td><td>Heat</td><td>18.0</td><td>0.7</td><td>0.91</td><td>281</td><td>12</td></tr></tbody></table><figcaption>Table 3. Lifespan and brood size across generations. p‑values are from ANOVA comparing heat‑treated to control within each generation (n = 60 for lifespan, n = 10 for brood size). No significant differences were detected.</figcaption></figure>
<h2>Discussion</h2>
<p>Our results demonstrate that a single acute heat stress induces H3K9me3 enrichment at a repetitive locus that persists for two generations in <em>C. elegans</em>. This finding adds to the growing body of evidence that environmental stressors can trigger transgenerational histone modifications [1,10,11,18,21]. The specificity of the mark to a repetitive element is consistent with the known role of H3K9me3 in silencing transposons and repetitive DNA, and with previous reports that siRNAs can guide this modification to homologous sequences [12]. The transient nature of the inheritance—disappearing by F₃—suggests an active resetting mechanism rather than a permanent alteration of the epigenetic landscape.</p><p>Several molecular pathways could explain the observed generational decay. First, the germline may employ dedicated reprogramming factors that erase or overwrite inherited marks during gametogenesis [14]. Second, the absence of continuous stress may lead to dilution of the histone modification through cell divisions, especially if the mark is not actively maintained by small‑RNA feedback loops [2,6]. The regression analysis showing a significant negative generational trend supports a model of progressive loss. Interestingly, the interaction term (Stress × Generation) was not significant, indicating that the rate of decline is similar in stressed and control lineages once the initial perturbation is overcome.</p><p>We did not observe robust phenotypic consequences of the inherited H3K9me3 mark. Lifespan showed a non‑significant increase in F₁ and F₂, while brood size was unaffected. This is consistent with studies where transgenerational chromatin changes led to subtle fitness effects under laboratory conditions [9,20]. It is possible that the environmental challenge (heat) was of moderate intensity, insufficient to trigger a strong adaptive response. Alternatively, the inherited marks might affect traits not measured here, such as stress resistance or metabolic rate. Future work could explore whether a more severe or repeated stress yields stronger and longer‑lasting phenotypic effects.</p><p>Our data also highlight the contrast between H3K9me3 and H3K4me3 inheritance. H3K4me3 was elevated only in F₁ at <em>hsp‑70</em>, suggesting that active marks are less stably transmitted than repressive ones. This aligns with the observation that H3K4me3 inheritance in <em>C. elegans</em> is often coupled with the presence of small RNAs or specific chromatin contexts [18]. The rapid resetting of H3K4me3 may protect against inappropriate activation of genes in future generations.</p><p>Comparisons with other model systems reveal both conserved and divergent features. In plants, stress‑induced histone modifications can be inherited for multiple generations, but resetting is common [4]. In mammals, a recent large‑scale study reported that loss of epigenetic information, including histone modifications, contributes to aging, but transgenerational inheritance of such changes remains controversial [29]. The short generation time and genetic tractability of <em>C. elegans</em> make it an ideal platform for dissecting the interplay between small RNA pathways and histone modifications in TEI.</p>
<h2>Conclusion</h2>
<p>This study provides evidence that heat stress elicits a transgenerational epigenetic memory mediated by H3K9me3 in <em>C. elegans</em>. The mark is inherited for two generations and then reset, representing a form of temporary cellular memory that may help the offspring of stressed individuals mount a faster response to similar challenges. The lack of persistent phenotypic change suggests that the primary role of such histone‑based TEI is to enable plasticity rather than to fix new traits. Future experiments should examine the upstream triggers (e.g., small RNA populations) and the downstream targets of inherited histone marks at a genome‑wide scale. Understanding the molecular boundaries that limit transgenerational inheritance will also clarify how organisms balance adaptation with stability of the germline.</p>
<h2>References</h2>
<ol class="references">
<li>Woodhouse, R. M., Ashe, A.. How do histone modifications contribute to transgenerational epigenetic inheritance in <i>C. elegans</i>?. Biochemical Society Transactions. 2020;48(3), 1019-1034. https://doi.org/10.1042/bst20190944</li>
<li>Rechavi, O., Lev, I.. Principles of Transgenerational Small RNA Inheritance in Caenorhabditis elegans. Current Biology. 2017;27(14), R720-R730. https://doi.org/10.1016/j.cub.2017.05.043</li>
<li>Wibisono, P., Sun, J.. Pathogen infection induces specific transgenerational modifications to gene expression and fitness in Caenorhabditis elegans. Frontiers in Physiology. 2023;14. https://doi.org/10.3389/fphys.2023.1225858</li>
<li>Tricker, P. J.. Transgenerational inheritance or resetting of stress-induced epigenetic modifications: two sides of the same coin. Frontiers in Plant Science. 2015;6. https://doi.org/10.3389/fpls.2015.00699</li>
<li>Drabikowski, K.. Ubiquitin and SUMO Modifications in <i>Caenorhabditis elegans</i> Stress Response. Current Issues in Molecular Biology. 2020, 145-158. https://doi.org/10.21775/cimb.035.145</li>
<li>Rieger, I., Weintraub, G., Lev, I., Goldstein, K., Bar-Zvi, D., Anava, S.. Nucleus-independent transgenerational small RNA inheritance in
<i>Caenorhabditis elegans</i>. Science Advances. 2023;9(43). https://doi.org/10.1126/sciadv.adj8618</li>
<li>PS, R.. "Crocin, a Carotenoid Mitigates Acrylamide Induced Phenotypic Alterations, Oxidative Stress and Dopaminergic Neurodegeneration in Caenorhabditis Elegans: Implications for Neuropathy. Pharmaceutical Drug Regulatory Affairs Journal. 2018;1(1). https://doi.org/10.23880/pdraj-16000102</li>
<li>Chen, H., Hua, X., Li, H., Wang, C., Dang, Y., Ding, P.. Transgenerational neurotoxicity of polystyrene microplastics induced by oxidative stress in Caenorhabditis elegans. Chemosphere. 2021;272, 129642. https://doi.org/10.1016/j.chemosphere.2021.129642</li>
<li>Greer, E. L., Maures, T. J., Ucar, D., Hauswirth, A. G., Mancini, E., Lim, J. P.. Transgenerational epigenetic inheritance of longevity in Caenorhabditis elegans. Nature. 2011;479(7373), 365-371. https://doi.org/10.1038/nature10572</li>
<li>Yang, J., Chatterjee, N., Kim, Y., Roh, J., Kwon, J., Park, M.. Histone methylation-associated transgenerational inheritance of reproductive defects in Caenorhabditis elegans exposed to crude oil under various exposure scenarios. Chemosphere. 2018;200, 358-365. https://doi.org/10.1016/j.chemosphere.2018.02.080</li>
<li>Zhang, L., Wang, S., Zhao, Y., Bi, K., Wang, D.. Increase in germline methyltransferases governing the methylation of histone H3K9 is associated with transgenerational nanoplastic toxicity in <i>Caenorhabditis elegans</i>. Environmental Science: Nano. 2022;9(1), 265-274. https://doi.org/10.1039/d1en00835h</li>
<li>Gu, S. G., Pak, J., Guang, S., Maniar, J. M., Kennedy, S., Fire, A.. Amplification of siRNA in Caenorhabditis elegans generates a transgenerational sequence-targeted histone H3 lysine 9 methylation footprint. Nature Genetics. 2012;44(2), 157-164. https://doi.org/10.1038/ng.1039</li>
<li>Sun, L., Liao, K., Wang, D.. Comparison of transgenerational reproductive toxicity induced by pristine and amino modified nanoplastics in Caenorhabditis elegans. Science of The Total Environment. 2021;768, 144362. https://doi.org/10.1016/j.scitotenv.2020.144362</li>
<li>Kelly, W. G.. Transgenerational epigenetics in the germline cycle of Caenorhabditis elegans. Epigenetics & Chromatin. 2014;7(1). https://doi.org/10.1186/1756-8935-7-6</li>
<li>Liao, V., Yu, C.. Arsenite exerts transgenerational reproduction defects in Caenorhabditis elegans. Toxicology Letters. 2015;238(2), S277. https://doi.org/10.1016/j.toxlet.2015.08.799</li>
<li>Zhu, Z., Man, X., Xia, M., Huang, Y., Yuan, D., Huang, S.. Collective effects of SNPs on transgenerational inheritance in Caenorhabditis elegans and budding yeast. Genomics. 2015;106(1), 23-29. https://doi.org/10.1016/j.ygeno.2015.04.002</li>
<li>Liu, H., Tian, L., Wang, S., Wang, D.. Size-dependent transgenerational toxicity induced by nanoplastics in nematode Caenorhabditis elegans. Science of The Total Environment. 2021;790, 148217. https://doi.org/10.1016/j.scitotenv.2021.148217</li>
<li>Wan, Q., Meng, X., Wang, C., Dai, W., Luo, Z., Yin, Z.. Histone H3K4me3 modification is a transgenerational epigenetic signal for lipid metabolism in Caenorhabditis elegans. Nature Communications. 2022;13(1). https://doi.org/10.1038/s41467-022-28469-4</li>
<li>Rechavi, O., Houri-Ze’evi, L., Anava, S., Goh, W., Kerk, S., Hannon, G.. Starvation-Induced Transgenerational Inheritance of Small RNAs in C. elegans. Cell. 2014;158(2), 277-287. https://doi.org/10.1016/j.cell.2014.06.020</li>
<li>Jobson, M. A., Jordan, J. M., Sandrof, M. A., Hibshman, J. D., Lennox, A. L., Baugh, L. R.. Transgenerational Effects of Early Life Starvation on Growth, Reproduction, and Stress Resistance in<i>Caenorhabditis elegans</i>. Genetics. 2015;201(1), 201-212. https://doi.org/10.1534/genetics.115.178699</li>
<li>Schwartz-Orbach, L., Zhang, C., Sidoli, S., Amin, R., Kaur, D., Zhebrun, A.. Caenorhabditis elegans nuclear RNAi factor SET-32 deposits the transgenerational histone modification, H3K23me3. eLife. 2020;9. https://doi.org/10.7554/elife.54309</li>
<li>Deans, C., Maggert, K. A.. What Do You Mean, “Epigenetic”?. Genetics. 2015;199(4), 887-896. https://doi.org/10.1534/genetics.114.173492</li>
<li>Shah, P. P., Donahue, G., Otte, G., Capell, B. C., Nelson, D. M., Cao, K.. Lamin B1 depletion in senescent cells triggers large-scale changes in gene expression and the chromatin landscape. Genes & Development. 2013;27(16), 1787-1799. https://doi.org/10.1101/gad.223834.113</li>
<li>Torda, G., Donelson, J. M., Aranda, M., Barshis, D. J., Bay, L. K., Berumen, M. L.. Rapid adaptive responses to climate change in corals. Nature Climate Change. 2017;7(9), 627-636. https://doi.org/10.1038/nclimate3374</li>
<li>Grandjean, V., Fourré, S., Abreu, D. A. F. d., Derieppe, M., Rémy, J., Rassoulzadegan, M.. RNA-mediated paternal heredity of diet-induced obesity and metabolic disorders. Scientific Reports. 2015;5(1), 18193-18193. https://doi.org/10.1038/srep18193</li>
<li>Thomas, A. L., Rogers, A., Webster, A., Marinov, G. K., Liao, S. E., Perkins, E. M.. Piwi induces piRNA-guided transcriptional silencing and establishment of a repressive chromatin state. Genes & Development. 2013;27(4), 390-399. https://doi.org/10.1101/gad.209841.112</li>
<li>Duncan, E. J., Gluckman, P. D., Dearden, P. K.. Epigenetics, plasticity, and evolution: How do we link epigenetic change to phenotype?. Journal of Experimental Zoology Part B Molecular and Developmental Evolution. 2014;322(4), 208-220. https://doi.org/10.1002/jez.b.22571</li>
<li>Henikoff, S.. Darwin meets Waddington. Current Biology. 2018;28(12), R682-R684. https://doi.org/10.1016/j.cub.2018.05.010</li>
<li>Yang, J., Hayano, M., Griffin, P., Amorim, J. A., Bonkowski, M. S., Apostolides, J. K.. Loss of epigenetic information as a cause of mammalian aging. Cell. 2023;186(2), 305-326.e27. https://doi.org/10.1016/j.cell.2022.12.027</li>
<li>Kishimoto, S., Uno, M., Okabe, E., Nono, M., Nishida, E.. Environmental stresses induce transgenerationally inheritable survival advantages via germline-to-soma communication in Caenorhabditis elegans. Nature Communications. 2017;8(1). https://doi.org/10.1038/ncomms14031</li>
</ol>
</article>