Lipid accumulation from the HFD exposure can be transmitted across generations
Supplementation of different lipids, including oleic acid (OA) or palmitoleic acid (PA), in food, can significantly increase fat accumulation in C. elegans 21, 22. Similarly, using Oil-Red-O (ORO) staining, we found that the ORO level was obviously elevated when animals were fed with OA, PA, cholesterol, or egg yolk compared with the OP50 medium, and that supplementation with egg yolk showed the most significant lipid accumulation (Fig 1a). Therefore, in the subsequent assays, we used egg yolk as a high-fat diet (HFD) to feed C. elegans and establish a fat accumulation model. To test whether the HFD-induced lipid accumulation phenomenon can transmit to the descendants raised on OP50, we randomly selected one wild-type hermaphrodite worm and obtained a population with the same genetic background through its self-fertilization. Then we fed wild-type worms with the HFD and bleached the mothers to obtain F1, F2, and Fn generations (Fig 1b). We found that the ORO levels were significantly increased, in either the HFD-fed parents or their F1 and F2 progeny (fed with normal OP50) (Fig 1c and d), but did not increase ORO levels of their F3 descendants (data not shown), suggesting that the HFD can induce a multigenerational epigenetic inheritance phenotype on descendants from a single exposure of the P0 generation. Considering that the F1 generation may have been exposed to the HFD while still inside the mother, in which case the obesogenic effect in the F1 worms would be considered intergenerational inheritance, not trans-generational inheritance. Therefore, we fed P0 generation animals with HFD from L1 to L4 larvae, then transferred them to NGM plates with normal food to prevent F2 primordial germ cells (PGCs) from exposure. We found that pre-exposure of P0 worms to HFD until L4 larvae could also induce the lipid accumulation of their naive F1 and F2 progeny (Fig 1e), suggesting that HFD can induce a transgenerational epigenetic inheritance (TEI) obesity phenotype.
In our heritable obesity effect model, we found that F2 populations exhibited a minor lipid accumulation phenotype. One concern is that HFD-induced fat accumulation could trigger a maternally heritable response in a dose-dependent manner instead of TEI effect. In order to eliminate this concern, we exposed multiple consecutive generations of animals to HFD. We found that, the exposure of four consecutive generations of animals (P0, F1, F2, and F3), three (F1, F2, and F3), two (F2, and F3), or one (F3), did not affect lipid accumulation their recovered F4 or F5 descendants (Fig 1f and g). In addition, we observed that lipid accumulation induced by oleic acid (OA) or palmitoleic acid (PA) can also transmit to their naive progeny (Fig S1c-e). Altogether, these results demonstrated that exposure of HFD induces transgenerational obesity effect.
To further elucidate the TEI phenotype and possible mechanisms induced by the HFD, we performed RNA sequencing (RNA-seq) analysis. Consistent with a previous study, functional annotation analysis indicated that genes regulated after feeding with the HFD were enriched in the innate immune response, metabolism and lipid metabolism (Fig S1a). Notably, a series of genes which were regulated by the HFD in the P0 generation overlapped with those regulated in the F1 and F2 naive progeny derived from P0 parents given the sole exposure to the HFD (Fig S1b). Collectively, these results demonstrated that the HFD can induce a memory of fat accumulation that can be transmitted to descendants.
HFD-induced transgenerational inheritance is mediated by nuclear receptors NHR-49, NHR-80, and transcription factors SBP-1 and DAF-16
To determine the molecular mechanisms underlying the HFD-induced TEI, we evaluated the contribution of several nuclear receptors and transcription factors which play essential roles in lipid metabolism in C. elegans. SBP-1, a sterol regulatory element-binding protein (SREBP), is a crucial transcription factor governing fat metabolism 21. NHR-49, a functional homolog of mammalian peroxisome proliferator activated receptor (PPAR) alpha, and NHR-80, a homolog of mammalian hepatocyte nuclear factor 4 (HNF4), are both important nuclear hormone receptors involved in the control of fat consumption and fatty acid composition in C. elegans 23, 24, 25, 26. Forkhead transcriptional factor DAF-16/FOXO, a central downstream effector of the insulin/insulin-like growth factor (IGF) signaling pathway, is a critical and well-conserved metabolic regulator 27, 28. Our results demonstrated that the elevated fat level induced by HFD in P0 was not affected in a sbp-1 loss-of-function (lof) mutant (Fig 2a), an nhr-80 lof mutant (Fig 2b) or an nhr-49 lof mutant (Fig 2c). However, lipid accumulation was abrogated in F1 or F2 descendants of these mutants. By contrast, the elevated lipid phenotype induced by HFD was abolished in P0 parents as well as in their recovery F1 and F2 progeny in the daf-16 lof mutant (Fig 2d). These results indicated that SBP-1, NHR-49 and NHR-80 work in the F1 generation to mediate TEI of fat accumulation induced by the HFD; whereas DAF-16 not only act in the F1 generation, but also is required for the execution of the lipid metabolism response in the P0 generation.
Here, considering that daf-16 contributed to regulation of lipid metabolism in P0 generation, to more definitively identify the role for daf-16 in F1 generation, we used RNAi to knock down daf-16 exclusively in the F1 generation derived from the P0 parents fed the HFD. We found that specific knockdown of daf-16 in the F1 generation also abrogates the elevated fat level (Fig 2e & f). Moreover, we also performed similar analyses using nhr-49, nhr-80 and sbp-1 RNAi. We found that silencing nhr-49, nhr-80 and sbp-1 in F1 progeny also abolished their lipid accumulation (Fig S2a-e), suggesting that daf-16, nhr-49, nhr-80 and sbp-1 act in the progeny to increase lipid level.
DAF-16 responses to environmental stress by activating a series of target genes 28. We found that the mRNA level of the target genes of DAF-16 (sod-3 and dod-3) was significantly upregulated in P0 fed with the HFD or their naive F1 progeny (Fig S3c). The expression of SOD-3::GFP was consistent with the mRNA level of sod-3 (Fig S3a and b). Altogether, these results indicated that DAF-16 functioned in response to altered lipid metabolism induced by HFD in P0 and their recovered progeny.
To more definitively dissect whether the role of daf-16, nhr-49, nhr-80 and sbp-1 is to implement regulation of lipid level (an “executor”), to transmit the heritable memories (a “transmitter”), or both, we used RNAi to silence daf-16, nhr-49, nhr-80 and sbp-1 exclusively in the P0 generation, and then analyzed the fat level of F1 generation raised on OP50 (Fig 2g). As reported in our recent study 19 (in press), if a gene is a transmitter, the silencing of that gene in the P0 generation will prevent the transmission of trans-generational memory, which will result in the elimination of the lipid accumulation of the F1 generation. Alternatively, if a gene only functions as an executor, the silencing of that gene in the P0 generation should only influence the lipid level of P0 generation, so that we would still detect the elevation of fat level in F1 generation. The results showed that silencing of nhr-49 and nhr-80 did not abrogate the lipid accumulation of F1 progeny (Fig 2i and j), suggesting that nhr-49 and nhr-80 are purely executors but not transmitters; the silencing of daf-16 or sbp-1 in P0 generation caused the loss of lipid accumulation of F1 (Fig 2h and k), indicating their roles as transmitters.
Taken together, our results demonstrated that daf-16, nhr-49, nhr-80 and sbp-1 were required for HFD-induced TEI of lipid accumulation. Among them, nhr-49 and nhr-80 functioned solely as executors; sbp-1 was responsible for transmitting the heritable memories, though we could not rule out its role as an executor; for daf-16, it not only functions as an executor to regulate lipid accumulation, but also as a transmitter to pass down heritable memory to progeny.
Delta-9 desaturase mediated the heritable memories
In C. elegans, delta-9 desaturases are well-characterized targets downstream of highly conserved transcriptional factors SBP-1, NHR-49, NHR-80 and DAF-16 (Fig 3a) 11, 25, 29, 30. Because SBP-1, NHR-49, NHR-80 and DAF-16 have been detected to mediate TEI of lipid accumulation in our settings, we wondered whether delta-9 desaturases participated in the TEI of lipid accumulation. We used the delta-9 desaturases-related mutants including fat-5, fat-6, and fat-7 lof mutants, and fat-5; fat-6 and fat-5; fat-7 double mutants to test our hypothesis. Our results showed that the fat elevated phenotype remained largely unaffected in the P0 generation of these mutants; however, these mutations abrogated the elevated fat level in F1 progeny (Fig 3b-f). These results revealed that delta-9 desaturases function in TEI induced by the HFD. To more definitively specify the role of delta-9 desaturases in HFD-induced TEI, we chose fat-5 as the representative of desaturase to conduct two sets of RNAi experiments similar to that performed in the above-mentioned treatments (daf-16, nhr-49, nhr-80 and sbp-1). We found that silencing fat-5 in F1 abolished the lipid accumulation (Fig 3g), however, silencing fat-5 in P0 generation did not affect the phenotype of fat accumulation in their recovered F1 progeny (Fig 3h), suggesting that delta-9 desaturases just execute regulation of lipid metabolism in HFD-induced TEI, but not transmit heritable memory.
Memory of HFD-induced lipid metabolism was conferred by histone H3K4me3 modification
HFD-induced lipid accumulation in parents could just pass down to F2 generation, and F3 is back to normal lipid level, suggesting that HFD might induce epigenetic changes rather than genetic mutations in descendants. Reportedly, a set of chromatin-modifying enzymes, including deacetylases and H3K4me3 methyltransferase, influence lipid metabolism 31, 32. To understand whether methyltransferase mediated the HFD-induced TEI of obesogenic effect, we performed a targeted mutant screen by selecting genes which participated histone methylation (Fig S4a and b). We found that loss of wdr-5.1, a H3K4me3 complex component, abolished the lipid accumulation in progeny (Fig 4a). Furthermore, wdr-5.1 did not execute in responding to lipid metabolism induced by the HFD, because ORO signaling of the wdr-5.1 mutant remained mostly increased in the P0 parents (Fig 4a). Moreover, we detected that the mRNA level of wdr-5.1 was slightly (but statistically significant) upregulated when animals were fed with the HFD compared with the control (Fig S4c). By contrast, our results showed that other histone modifications including H3K27me3 and H3K36me3 did not contribute to the TEI induced by the HFD, because lipid accumulation induced by the HFD was not abrogated in the P0, F1 and F2 generations in the loss of function H3K27 demethylase mutant jmjd-3.1 33 and in the loss of function H3K36 methyltransferase mutant met-1 14 (Fig S4a and b). Furthermore, we observed that H3K4me3 methylation was significantly increased in the P0 generation fed with HFD (Fig 4b) and in their recovered progeny (Fig 4b, c and d). Moreover, upregulation of H3K4me3 methylation induced by feeding with HFD was abolished in a wdr-5.1 mutant (Fig S4d).
To more definitively characterize the role of wdr-5.1 in HFD-induced TEI, we also conducted two sets of RNAi experiments similar to that performed in the above-mentioned treatments (daf-16, nhr-49, nhr-80 and sbp-1). We found that silencing wdr-5.1 in F1 abrogated the lipid accumulation (Fig S4e), and silencing wdr-5.1 in P0 generation also abolished the phenotype of elevation of fat level in their recovered F1 progeny (Fig S4f), suggesting the role of wdr-5.1 as a transmitter instead of an executor. Collectively, these results suggested that histone H3K4me3 modification plays a specific role in transmitting the heritable memory induced by the HFD.
To test whether lipid accumulation induced by the HFD is associated with heritable changes of H3K4me3 at specific loci, we used CHIP-qPCR and found that H3K4me3 occupancy was significantly increased at the promoter of several genes related to lipid metabolism (Fig 4f). These genes are also significantly upregulated, as shown by the RNA-seq analysis of the P0, F1 and F2 generations. In addition, the mRNA level of these genes was confirmed to be elevated by RT-qPCR (Fig 4g).
We further investigated whether the transcription factors (sbp-1 and daf-16) and nuclear receptors (nhr-49 and nhr-80), which have been identified to mediate HFD-induced TEI, also regulated the levels of histone H3K4me3 modification. We found that all mutations (daf-16, nhr-49 and nhr-80) except sbp-1 had no effect on H3K4me3 modification in animals fed with the HFD when compared with controls (Fig 4e, S5b, S5c and S5d). In addition, the elevated transcription level of wdr-5.1 induced by the HFD was abrogated in the sbp-1 mutant (Fig S4C). These findings suggested that sbp-1 might mediate the HFD-induced memory transmission of lipid accumulation through upregulating wdr-5.1 expression to increase the H3K4me3 level.
Germ-to-soma communication in the HFD-induced transgenerational inheritance
To detect tissues in which daf-16 and sbp-1 function in response to lipid metabolism and in regulating histone modifications, respectively, we conducted tissue-specific gene knockdown assays by using strains which can process RNAi efficiently only in specific tissues, such as germline, muscle, or neuron (Fig 5a). Firstly, we performed the tissue-specific RNAi of daf-16 to detect the ORO staining. We found that muscle-specific RNAi of daf-16 in the P0 generation led to the suppression of the lipid accumulation (Fig 5g). However, other tissue-specific RNAi, including in the intestine, neuron or germline, could not compromise the elevated fat level (Fig 5h-k).
We then performed tissue-specific knockdown of sbp-1 to assess the variation of histone H3K4me3 induced by the HFD. We found that intestine-specific, muscle-specific, germline-specific, or intestine- and germline-specific RNAi of sbp-1 abolishes the elevated H3K4me3 modification levels (Fig 5b-f). These results indicated that the function of sbp-1 to regulate the variation of H3K4me3 modification induced by the HFD in several tissues may be redundant. Collectively, tissue-specific RNAi studies demonstrated that among tissues, communication across generations may coordinately regulate TEI of lipid accumulation induced by the HFD.