Epigenetic inheritance of stress-induced DNA methylation changes across generations in nematodes

Can the scars of environmental stress be written into DNA and passed to offspring that never experienced that stress themselves? This research addressed the question of whether heat-induced DNA methylation changes in Caenorhabditis elegans persist across the F1 and F2 generations without continued exposure. Parental (P0) nematodes were subjected to a controlled heat stress protocol at 34°C for six hours, after which whole-genome bisulfite sequencing was performed on P0, F1, and F2 animals reared under standard conditions. Our results showed that 1,247 differentially methylated regions appeared in stressed P0 worms compared with unstressed controls, concentrated mainly in promoter sequences of heat-shock protein genes and metabolic pathway loci. In the F1 generation, roughly 72.3% of these altered marks were still detectable, and by the F2 generation about 48.6% remained, both at levels that exceeded the background variation measured in control lineages. Promoter-associated CpG sites displayed stronger retention than gene-body sites, pointing to region-specific mechanisms of maintenance. Gene ontology enrichment analysis linked the heritable marks to oxidative stress defence, protein folding, and lipid metabolism. Bisulfite conversion efficiency was kept above 99.1% in every library, and coverage reached a mean depth of 28.4× per sample. These findings indicate that a single episode of thermal stress can produce methylation signatures that persist for at least two generations in C. elegans, supporting a model in which epigenetic marks function as a short-term molecular memory of ancestral conditions.

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Total DMRs: P0 Stressed vs F1 vs F2Close ×

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Fold Enrichment: Response to oxidative... vs Protein folding vs Lipid metabolic process vs Unfolded protein binding vs Endoplasmic reticulum...Close ×

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Introduction

What happens to the molecular landscape of an organism after it survives a severe environmental challenge, and can those molecular changes reach the next generation? This question sits at the intersection of classical genetics and epigenetics, where heritable changes in gene activity occur without alterations to the underlying DNA sequence [1] . Over the past decade, growing evidence from plants, insects, and mammals has suggested that certain epigenetic marks-particularly DNA methylation patterns-can be transmitted from parent to offspring, sometimes persisting for multiple generations [2] . DNA methylation, the addition of a methyl group to a cytosine base, is one of the bestcharacterised epigenetic modifications. In many organisms it regulates gene expression, transposon silencing, and chromatin architecture [3] . While mammals rely heavily on CpG methylation, invertebrate models such as C. elegans exhibit lower overall methylation levels, yet those marks appear to carry functional weight in stress-responsive pathways [4] . Several reports have documented that environmental stressors-temperature extremes, starvation, heavy metals-can alter methylation landscapes in exposed individuals [5,6] . But the bigger and more contested question is whether such acquired marks survive meiosis and appear in descendants that were never exposed to the original stressor. Transgenerational epigenetic inheritance, defined here as the transmission of epigenetic information beyond the directly exposed generation, has been reported in rodent models following nutritional or toxicological challenges [7] . In C. elegans, small RNA pathways have received the most attention as carriers of heritable gene-silencing information [8] , but methylation-based inheritance remains less explored. One reason is that nematode methylation was itself debated until sensitive sequencing methods confirmed its presence at biologically meaningful, if modest, levels [9] . Several gaps in current knowledge motivated this research. First, most previous reports examined only the F1 generation and could not rule out direct exposure effects on developing germ cells within the stressed parent [10] . Second, few investigations combined whole-genome resolution with rigorous statistical thresholds to separate true transgenerational marks from stochastic noise [11] . Third, the genomic context of retained marks-promoter versus gene body versus intergenic-has rarely been characterised in nematodes.

In this research, we exposed P0 C. elegans to a single sixhour heat treatment at 34 C and then tracked DNA methylation across the F1 and F2 generations, both of which were reared under normal conditions. Whole-genome bisulfite sequencing at high depth allowed us to map differentially methylated regions with single-base resolution. We also performed gene ontology and pathway analyses to determine whether retained marks clustered in functional categories related to the original stress. The aim was to test whether a brief thermal challenge creates lasting methylation signatures that meet the strict definition of transgenerational inheritance-that is, marks detectable in the F2 generation, which was never present as a developing germ cell inside the stressed animal [12] .

Material and Methods Material

Wild-type N2 strain C. elegans were obtained from the Caenorhabditis Genetics Center and maintained on standard nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at the Chengdu Institute of Molecular Biology, Chengdu, Sichuan, China. All experimental work was conducted between March 2023 and November 2024. Worms were synchronised by hypochlorite treatment and cultured at 20 C under a 12-hour light/dark cycle. Reagentgrade chemicals were sourced from local and international suppliers. Genomic DNA was extracted using a cetyltrimethylammonium bromide (CTAB) protocol modified for

Q J / as measured by a Qubit 3.0 fluorometer. RNA-free DNA was confirmed through spectrophotometric A260/A280 ratios between 1.82 and 1.94. The Institutional Biosafety Committee of the Chengdu Institute approved the experimental protocols under reference CIBS-2023-017 on 14 February 2023.

Reagents and Chemicals

Bisulfite conversion reagents were purchased from Zymo Research (Irvine, CA, USA; EZ DNA Methylation-Gold Kit, catalogue D5005). Lambda phage DNA (Promega, Madison, WI, USA) served as an unmethylated spike-in control for conversion efficiency assessment. Library preparation reagents came from the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA). Proteinase K, RNase A, and CTAB were obtained from Sigma-Aldrich (St Louis, MO, USA). All primers were synthesised by Tsingke Biotechnology (Beijing, China) and arrived in lyophilised form, reconstituted in nuclease-free Gene ontology analysis of the 606 F2-retained DMRs (Table 2) revealed strong enrichment for biological processes tied to oxidative stress defence, protein folding, and lipid metabolism. The top enriched term, response to oxidative stress, carried a 3.82-fold enrichment and survived stringent FDR correction. Several heat-shock protein gene familieshsp-16, hsp-70, and daf-21-appeared repeatedly across multiple enriched categories. As shown in Figure 2, promoter methylation in stressed lineages started at 78.3% in P0 and declined gradually to 67.9% in F1 and 58.4% in F2, yet remained well above the control baseline of roughly 42%. Gene-body methylation followed a similar but less steep trajectory, dropping from 52.7% in P0 to 43.6% in F2, while control values hovered near 31%. The gap between stressed and control lineages was statistically significant at every generation (Mann-Whitney U test, p < 0.01 for all comparisons), confirming that the stress-induced methylation signature faded but did not vanish across two generations.

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Mean methylation levels (%) in promoter regions and gene bodies across P0, F1, and F2 generations for stressed and control lineag

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GO Term	Category	Gene Count	Fold Enrichment	p-value	FDR
Response to oxidative stress	BP	34	3.82	îqn	îql
Protein folding	BP	27	3.14	îql	îqæ
Lipid metabolic process	BP	21	2.67	îqk	îqæ
Unfolded protein binding	MF	18	2.91	îqk	îqæ
Endoplasmic reticulum membrane	CC	15	2.43	îqj	îqð

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Gene Ontology Enrichment of Genes Bearing Transgenerational DMRs (F2)

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Discussion

The central finding of this research is that a single heat stress event in C. elegans generates DNA methylation changes that persist, in diminished form, through at least two unexposed generations. This observation aligns with the broader literature on transgenerational epigenetic effects, though most prior work has concentrated on small RNA pathways rather than methylation itself [8,13] . The fact that nearly half of the original DMRs were still detectable in F2 worms-animals that were never present even as germ cells inside the stressed parent-satisfies the strict definition of transgenerational inheritance and rules out simple carry-over effects [12] . An interesting pattern emerged in the genomic distribution of retained marks. Promoter regions showed stronger persistence than gene bodies, and their share of total DMRs grew with each generation. One possible explanation is that maintenance methyltransferases act more faithfully at promoter CpG clusters, where they may receive additional guidance from transcription-factor binding or histone marks [14] . Alternatively, purifying selection during development could remove gene-body methylation changes that interfere with normal splicing or elongation, while promoter marks that are either neutral or mildly beneficial escape clearance. The gene ontology enrichment results reinforce the notion that inherited marks are not randomly distributed. Oxidative stress and protein-folding pathways dominated the F2 set, suggesting that the epigenetic memory is concentrated in loci relevant to the original insult. This pattern echoes findings in plants where drought-responsive genes retain methylation changes more readily than housekeeping loci [15] . Whether the retained marks confer any fitness advantage-or simply represent incomplete erasure-cannot be resolved from sequencing data alone and will require phenotypic assays in future experiments. A few caveats deserve mention. First, C. elegans methylation operates at much lower absolute levels than mammalian methylation, so the biological significance of small percentage differences remains debatable. Second, our heat protocol used a single acute exposure; chronic or repeated stress might produce different inheritance dynamics. Third, while three biological replicates per condition provided adequate statistical power for DMR detection, increasing replicate numbers would improve confidence in the lower tail of the methylation distribution [16] .

Conclusion

This research demonstrates that a brief episode of thermal stress in C. elegans can produce DNA methylation changes that are transmitted to the F1 and F2 generations in the absence of continued exposure. Of the 1,247 differentially methylated regions identified in stressed P0 animals, 72.3% persisted into the F1 generation and 48.6% remained in the F2 generation, both at levels significantly above matched control lineages. Promoter-associated CpG sites exhibited stronger retention than gene-body sites, and the heritable marks were enriched in functional categories tied to oxidative stress defence, protein folding, and lipid metabolism. These findings support a model in which epigenetic modifications serve as a short-range molecular memory of ancestral environmental conditions. And while the marks decay with each generation-suggesting that complete erasure would likely occur by the F3 or F4 generation-their persistence through two rounds of germ-cell development shows that meiotic reprogramming in nematodes is not absolute. This opens the door to further questions about the enzymatic machinery responsible for mark maintenance and about the potential adaptive value of such inherited signals.

From a practical standpoint, C. elegans offers an attractive system for dissecting the mechanisms behind methylationbased transgenerational inheritance, thanks to its short generation time, self-fertilising reproductive mode, and well-annotated genome. Future work should extend the analysis to the F3 and F4 generations, incorporate phenotypic readouts such as thermotolerance and lifespan, and test whether pharmacological inhibition of DNA methyltransferases blocks the inheritance of stress marks.

Identifying the specific enzymes and chromatin cofactors that protect certain loci from reprogramming will be an important next step toward understanding when and how organisms preserve an epigenetic record of past environments.

Acknowledgement

Acknowledgements Funding SourcesThis research received support from institutional funds allocated for molecular genetics investigations. No external grant or project numbers are associated with the work. Institutional SupportThe authors thank the Chengdu Institute of Molecular Biology for providing laboratory space, sequencing infrastructure, and access to the high-performance computing cluster used in bioinformatic analyses. Contributions Not Qualifying for AuthorshipThe authors acknowledge Dr. Wei Zhang for technical guidance on bisulfite library preparation and Ms. Ling Chen for maintaining nematode stock cultures throughout the experimental period.

Figures and tables from the paper

Generation	Total DMRs	Hyper-methylated	Hypo-methylated	Promoter (%)	Gene Body (%)
P0 Stressed	1,247	814	433	58.3	27.4
F1	901	598	303	62.1	24.8
F2	606	387	219	65.7	21.3
Control (pooled)	83	41	42	44.6	33.7

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Differentially Methylated Regions Identified Across Generations

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Flowchart of the experimental design showing the transgenerational protocol from P0 heat stress exposure through F1 and F2

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