Epigenetic Regulation of Toxicity of Environmental Toxicants or Stresses

Abstract

The epigenetic regulation mechanisms can provide us a possibility that a small set of molecules potentially govern many important molecular signaling pathways to regulate the biological processes. We here focused on the methylation, acetylation, microRNAs (miRNAs), and long noncoding RNAs (lncRNAs) to introduce and to discuss the contributions of epigenetic regulation to toxicity induction of environmental toxicants or stresses and the underlying mechanisms. These investigations may open a new window to understand the full story on the toxicity induction in nematodes exposed to environmental toxicants or stresses.

12.1 Introduction

Besides the important roles and functions of molecular signaling pathways encoded by genes, in recent years, the epigenetic control of toxicity of environmental toxicants or stresses has gradually received the attention. The epigenetic regulation can be formed and reflected by several forms, such as methylation, acetylation, and small RNA control. The epigenetic regulation mechanisms provide such a possibility that a small set of molecules can act upstream and govern many important molecular signaling pathways to regulate the toxicity of environmental toxicants or stresses.

In this chapter, we first introduced and discussed the contributions of methylation regulation and acetylation regulation to the toxicity induction of environmental toxicants or stresses and the underlying mechanisms. Moreover, we further introduced and discussed the roles of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in regulating the toxicity of environmental toxicants and stresses and the underlying mechanisms. The investigations on the epigenetic regulation mechanisms open a new window for us to understand the possible full story on the toxicity induction in nematodes exposed to environmental toxicants or stresses.

12.2 Methylation Regulation

12.2.1 Methylation of Histone H3K4

12.2.1.1 Function of ASH-2 Trithorax Complex

In nematodes, environmental pathogens, including the Pseudomonas aeruginosa, can potentially result in toxic effects on animals and even kill the animals [1,2,3,4,5,6,7]. E. coli OP50 is the normal food source for nematodes. Using P. aeruginosa PA4 and E. coli OP50, a preference choice model can be established [8]. In this preference choice model, it was found that the ADL sensory neurons regulated the preference choice by inhibiting function of a G protein-coupled receptor (GPCR)/SRH-220 [8]. Moreover, the ADL sensory neurons regulated the preference choice through peptidergic signals of FLP-4 and NLP-10, and the function of FLP-4 or NLP-10 in regulating preference choice was under the regulation of GPCR SRH-220 [8]. The FLP-4 released from the ADL sensory neurons further affected the preference choice through its receptor of NPR-4 in the AIB interneurons [8].

In nematodes, ASH, WDR-5, and SET-2 constitute an ASH-2 trithorax complex, which trimethylates the histone H3K4. ASH-2 is a trithorax group protein, WDR-5 is a WD40 repeat-containing protein, SET-2 is a histone H3 at lysine 4 (H3K4) methyltransferase, and RBR-2 is a H3K4 demethylase. In the AIB interneurons, NPR-4 regulated the preference choice by activating the function of SET-2 [8]. Mutation of set-2, ash-2, or wdr-5 caused the significant decrease in choice index; however, mutation of rbr-2 did not significantly affect the preference choice behavior (Fig. 12.1) [8], suggesting the involvement of ASH-2 trithorax complex in the control of preference choice behavior. In this ASH-2 trithorax complex, genetic interactions demonstrated that SET-2 functioned in the same pathway with ASH-2 and WDR-5 in regulating the preference choice (Fig. 12.1) [8]. Additionally, expression of set-2, ash-2, or wdr-5 in AIB interneurons could rescue the deficit in preference choice in corresponding mutant nematodes [8]. Therefore, the GPCR NPR-4 is involved in the control of preference choice by activating the functions of ASH-2 trithorax complex consisting of SET-2, ASH-2, and WDR-5. These results also highlight the important function of the ASH-2 trithorax complex-mediated molecular machinery for trimethylation of histone H3K4 in the regulation of toxicity of environmental toxicants or stresses in nematodes.

Fig. 12.1

SET-2 functioned together with ASH-2 and WDR-5 to regulate preference choice in nematodes [8]. (a) Preference choice phenotype in ash-2, wdr-5, or rbr-2 mutants. (b) Genetic interaction of SET-2 with ASH-2 or WDR-5 in regulating preference choice. (c) Leaving behavior from bacterial lawns and chemotaxis to OP50 in ash-2 or wdr-5 mutants. (d) The molecular signals regulating preference choice in the neuronal circuit of ADL sensory neuron-AIB interneuron. Sensory neurons are shown as triangles and interneurons as hexagons. Arrows indicate chemical synapses. Bars represent means ± S.E.M. **P < 0.01 vs N2

12.2.1.2 S-Adenosyl Methionine (SAM) and SET-16-Mediated Complex

SAM, the sole methyl donor, modifies the molecules including the histones, and its fluctuation is associated with the variations in histone methylation. In nematodes, it was observed that RNAi knockdown of sams-1 encoding a SAM synthase resulted in the activation of innate immune genes and the constitutively activation of p38 MAPK signaling pathway under the normal conditions [9], suggesting that the low SAM can activate or attenuate the innate immune response. Additionally, the innate immune activation was formed downstream of the phosphatidylcholine (PC) in the sams-1(RNAi) nematodes [9]. Moreover, it was found that the sams-1(lof) mutant nematodes died rapidly after infection with Pseudomonas, and the failure of transcriptional response to Pseudomonas was observed in the sams-1(lof) mutant nematodes [9]. The analysis on the H3K4 methylation further demonstrated that the infection response genes could not cause the increase in histone methylation marks in sams-1(lof) mutant nematodes after Pseudomonas infection, and the low SAM restricted the H3K4me3, thereby limiting their expression in sams-1 mutant nematodes (Fig. 12.2) [9].

Fig. 12.2

Infection response genes do not accumulate activating histone methylation marks in sams-1(lof) mutants exposed to Pseudomonas [9]. (a) H3K4me3 is diminished in nuclei of intestinal cells after sams-1(RNAi) and in choline-treated sams-1(RNAi). Yellow bar shows 2 μm. (b) Quantitation of immunofluorescence showing an average of pixel intensity over the area for 8–12 nuclei per sample. (c) Immunostaining comparing markers of active phosphorylated RNA polymerase II (Pol II PSer 5, PSer 2) with total Pol II (unP). (d) Other histone modifications associated with active transcription (H3K36me and H3K9ac) or with heterochromatin (H3K9me3) within intestinal nuclei in control or sams-1(RNAi) animals. (e–j) Chromatin immunoprecipitation comparing levels of H3K4me3 on infection response or control genes grown on E. coli (OP50) or Pseudomonas (PA14) in wild-type (WT) or sams-1(lof) mutants. Input levels were normalized to the WT E. coli value on the upstream primer pair. Numerical representation of primer location is based on translational start site. Legend in (j) refers to all images. Error bars show SD. Results from Student’s t-test shown by *<0.05, **<0.01, ***<0.005

The H3K4 modification occurs through the action of methyltransferases with the functions in COMPASS methyltransferase complex. The nematodes contain the orthologs of catalytic COMPASS complex components such as set-2/SETD1A/KMT2F/Set1 and set-16/MLL/KMT2A [10]. It was further found that the genes requiring set-16/MLL-dependent methylation were preferentially affected by SAM depletion during the transcriptional response to pathogen infection (Fig. 12.3) [9]. That is, the set-16/MLL was critical for the transcriptional response to pathogenic infection, and this response depended on the H3K4 methyltransferase set-16/MLL (Fig. 12.3) [9].

Fig. 12.3

set-16/MLL is important for expression of infection response genes upon Pseudomonas exposure [9]. (a) Immunostaining of intestinal nuclei with antibodies to H3K4me3 after RNAi of set-2 or set-16. Yellow bar shows 2 mm. (b) Quantitation of immunofluorescence showing an average of pixel intensity over area for 8–12 nuclei per sample. Requirement for set-2/SET1 (b–d) or set-16/MLL (e–h) for induction of infection response genes upon a 6 h exposure to PA14 compared to E. coli (HT115). Error bars show SD. Results from Student’s t-test shown by *<0.05, **<0.01, ***<0.005

BLMP-1 is the BLIMP-1 ortholog. The BLMP-1 was necessary for controlling the histone methylation level in daf-7 mutant nematodes, and the BLMP-1 regulated the expression of SAMS-1 [11]. Therefore, BLMP-1 can further act upstream of SAMS-1 to regulate the histone methylation and the stress responses, such as the dauer formation.

12.2.1.3 The Possible Downstream Molecular Response After H3K4 Trimethylation

In nematodes, it was observed that the chronic exposure to MeHg (10 μM) could cause the epigenetic landscape modifications of histone H3K4 trimethylation (H3K4me3) marks based on the chromatin immunoprecipitation sequencing (ChIP-seq) analysis (Fig. 12.4) [12]. These modifications corresponded to the locations of 1467 upregulated genes and 508 downregulated genes [12]. The upregulated genes contain those encoding glutathione S-transferases, lipocalin-related protein, and a cuticular collagen (Fig. 12.4) [12]. Among these genes, RNAi knockdown of lipocalin-related protein gene lpr-5, which is involved in intercellular signaling, or cuticular collagen gene dpy-7, structural component of the cuticle, led to the increased lethality in nematodes exposed to MeHg [12].

Fig. 12.4

ChIP-seq and RNA-seq in control treated and MeHg-treated animals [12]. (a) ChIP-seq peaks were identified using H3K4Me3 antibody or input DNA as a control. MeHg treatment increased H3K4me3 signals in genes gst-5, gst-38. (b) Peaks from lpr-5 and dpy-7 genes. ChIP-Seq data (lines 1–3) and the corresponding genes were compared with RNA-Seq data (lines 4–5). Gene structure is shown at the bottom. Images were generated by Integrated Genome Viewer 2.3

12.2.2 Methylation of Histone H3K9

12.2.2.1 MET-2 and Nuclear Co-factor LIN-65

In nematodes, LIN-65 was identified as a new regulator for the activation of mitochondrial UPR response, because the neuronal polyglutamine expression could induce a lin-65 dependent activation of the mitochondrial UPR response [13]. Histone H3K9 methylation plays an important role in regulating the gene expression, and the histone H3K9 is methylated sequentially by methyltransferases MET-2/SETDB1 and SET-25 in nematodes [14]. MET-2 potentially mono- and di-methylate the H3K9 in the cytoplasm, and the nuclear SET-25 acts as a trimethylation methyltransferase for the H3K9 [14]. It was further observed that the MET-2 was required for the LIN-65 nuclear accumulation induced by mitochondrial UPR response [13].

In nematodes, the full activation of mitochondrial UPR required both the remodeling of chromatin and the di-methylation of histone H3K9 (Fig. 12.5) [13]. Additionally, the global chromatin reorganization induced by mitochondrial stress was dependent on both MET-2 and LIN-65 (Fig. 12.5) [13], suggesting that lin-65 and met-2 are required for the regulation of transcriptional response to mitochondrial stress and chromatin reorganization induced by mitochondrial stress in nematodes.

Fig. 12.5

Mitochondrial stress-induced chromatin reorganization [13]. (a) Representative maximal intensity projection images of H3K9me2 immunostaining of intestinal nuclei in Day 1adult WT, met-2 or lin-65 mutant animals grown on EV or cco-1 RNAi from hatch (as indicated). H3K9me2 (red); DAPI (gray). Scale bar represents 10 μm. (b) Quantification of H3K9me2 level. The genotypes and treatments are as in (a). H3K9me2 intensity is normalized to DAPI intensity. (*** denotes p < 0.0001; ns denotes p > 0.05 via t-test, error bars indicate SEM, n ≥ 15 nuclei). (c) Representative of three center images of DAPI staining with lmn-1p::emr-1::gfp of intestinal nuclei in Day 1 adult animals grown on EV or cco-1 RNAi from hatch as indicated. lmn-1p::emr-1::gfp (green); DAPI (gray). Scale bar represents 10 μm. (d) Quantification of the intestinal nuclear size at Day 1 adulthood using lmn-1p::emr-1::gfp as a marker. Animals grown on EV or cco-1 RNAi from hatch (*** denotes p < 0.0001 via t-test, error bars indicate SEM, n ≥ 20 nuclei). (e) Quantification of the distribution of DAPI staining signal in intestinal nuclei at Day 1 of adulthood in animals grown on EV or cco-1 RNAi from hatch. The distributions of fractions from the top four bins are shown as boxplots. (*** denotes p < 0.0001 via Mann–Whitney test, error bars indicate SEM, n ≥ 25 nuclei)

In nematodes, the full induction of mitochondrial UPR response requires the nuclear localization of transcription factor DVE-1 and co-factor UBL-5, as well as the activity of protease CLPP-1. The nuclear localization of LIN-65 was further found to be partially dependent on CLPP-1/DVE-1 and independent of ATFS-1 [13]. Meanwhile, the H3K9me2 was required for the mitochondrial stress-induced DVE-1 nuclear puncta formation [13]. Based on the so far obtained data, a model for the epigenetic regulation of mitochondrial UPR response was summarized in Fig. 12.6 [13].

Fig. 12.6

Model for mitochondrial stress signaling pathway [13]. (a) Under non-stressed conditions, MET-2 produces H3K9me1/2 histone subunits in the cytoplasm. ATFS-1 translocates to the mitochondria and is degraded. DVE-1 and LIN-65 do not accumulate in the nucleus, and the UPR^mt is not induced, animals are normal lived, and the nucleus is not compacted. (b) During mitochondrial stress, MET-2 continues to produce H3K9me2 histone subunits, ATFS-1 now translocates to the nucleus to induce UPR^mt. DVE-1 and LIN-65 accumulate in the nucleus. Animals are long-lived, the UPR^mt is induced, nuclei become compacted, and H3K9me2 levels remain unchanged. (c) Loss of met-2 during mitochondrial stress results in reduced nuclear H3K9me2 levels, nuclei that are less compacted, reduced DVE-1 and LIN-65 nuclear accumulation, reduced UPR^mt induction and partial suppression of increased lifespan. (d) Loss of lin-65 during mitochondrial stressresults in reduced nuclear H3K9me2 levels, nuclei that are less compacted, reduced DVE-1 nuclear accumulation, reduced UPR^mt induction, and partial suppression of increased lifespan. (e) Loss of atfs-1 during mitochondrial stress results in suppressed UPR^mt induction and partial suppression of lifespan extension. This is independent of compacted nuclei and nuclear accumulation of DVE-1 and LIN-65. (f) Loss of both met-2 and atfs-1 during mitochondrial stress results in nuclei that are less compacted, reduced nuclear accumulation of DVE-1 and LIN-65, no UPR^mt induction, and complete suppression of lifespan extension. (g) During mitochondrial stress, LIN-65 and DVE-1 accumulate in the nucleus, chromatin becomes remodeled, and DVE-1 is able to form puncta at the loose regions of the chromatin, activating transcription of UPR^mt targets. This remodeling works in parallel to the relocation of mitochondrial-specific transcription factor ATFS-1 to initiate UPR^mt and regulate longevity

12.2.2.2 JMJD-1.2 and JMJD-3.1

In nematodes, JMJD-1.2, a member of the KDM7 family, is a demethylase toward lysine residues on histone 3 (H3). The jmjd-1.2 potentially controlled the level of histone 3 lysine 9, lysine 23, and lysine 27 di-methylation (H3K9/K23/K27me2) [15]. Moreover, it was found that mutation of jmjd-1.2 induced a susceptibility to replication stress (Fig. 12.7) [15]. The progeny of jmjd-1.2 mutant nematodes exposed to hydroxyurea showed an increased embryonic lethality and an increased mutational rate compared to those in wild-type nematodes (Fig. 12.7) [15].

Fig. 12.7

jmjd-1.2 mutants have increased mutational rate after HU [15]. (a) Quantification of the percentage of abnormalities identified in F2 progeny derived from animals of indicated genotypes exposed to HU (25 mM, 16 h). The percentage of F2 abnormalities was determined based on the number of plates that had at least one F2 abnormal animal. (b) Representative images of jmjd-1.2 F2 progeny derived from animals exposed to HU (25 mM, 16 h). Dpy, dumpy; ste, sterile; sma, small; vab, organism morphology variant; egl d, egg-laying defective. A wild-type animal (N2) is shown, for comparison. (c) Quantification of the percentage of lacZ-positive animals in F1 generation after parental HU treatment (25 mM, 16 h). N2 and jmjd-1.2(tm3713) carrying the pkIs1604 transgene are used. (d) Representative images of the F1 generation after parental HU treatment, (25 mM, 16 h) stained by X-gal. N2 and jmjd-1.2(tm3713), carrying the pkIs1604 transgene, are shown. A magnification of a lacZ-positive animal is shown. In (a and c) the graphics show the average of two independent experiments, and data are presented as mean ± SD. *p ≤ 0.05, ****p ≤ 0.0001, with χ ² test. Scale bars, 100 μm

Moreover, both JMJD-1.2 and JMJD-3.1, two histone lysine demethylases, were identified to be required for the induction of mitochondrial UPR response [16]. Reduction of function of JMJD-1.2 or JMJD-3.1 suppressed the longevity and mitochondrial UPR induction, whereas the overexpression of jmjd-1.2 or jmjd-3.1 was sufficient for the lifespan extension and the mitochondrial UPR induction in nematodes [16].

12.2.3 Methylation of HIS-24K14

HIS-24 is a linker histone (H1) variant, and HPL-1 and HPL-2 are heterochromatin protein 1 (HP1)-like proteins. These proteins are essential components of heterochromatin and contribute to the transcriptional repression of genes. In nematodes, it was observed that pathogen infection could increase the cellular levels of HIS-24K14me1 [17]. Meanwhile, the HIS-24K14me1 localization was changed from being mostly nuclear to both nuclear and cytoplasmic in intestinal cells by pathogen infection [17]. Moreover, HIS-24 recruited the HPL proteins to the promoters of genes involved in the defense response against the pathogen infection [17]. The HPL-1 was further found to interact with the HIS-24 monomethylated at the lysine 14 (HIS-24K14me1) [17].

12.2.4 Methylated Glycans

The tectonin 2 of the mushroom Laccaria bicolor (Lb-Tec2) has been shown to agglutinate the Gram-negative bacteria and to exert the toxicity toward nematodes [18]. Additionally, both the bacterial agglutination and the Lb-Tec2 depended on the recognition of methylated glycans (O-methylated mannose and fucose residues) as part of bacterial lipopolysaccharide (LPS) and cell-surface glycans in nematodes (Fig. 12.8) [18]. Moreover, the Lb-Tec2 was toxic for nematodes, and this toxicity formation depended on the binding to N-glycans (Fig. 12.8) [18]. SAMT-1, a membrane transport protein for presumptive donor substrate of glycan methylation from cytoplasm to Golgi, was identified to be required for the regulation of Lb-Tec2 toxicity, and mutation of samt-1 induced a Lb-Tec2 resistance in nematodes [18].

Fig. 12.8

Carbohydrate-binding-dependent toxicity of Lb-Tec2 toward C. elegans [18]. (a) Development of C. elegans wild-type (N2) and various glycosylation mutants feeding on Lb-Tec2-expressing E. coli (dark gray) or empty pET24 vector containing E. coli (light gray) (n = 5). Error bars indicate SD. Comparisons between Lb-Tec2 and vector control for each C. elegans strain were performed using the Mann–Whitney U test (**P < 0.01). (b) Development of C. elegans pmk-1(km25) strain (wild-type) and various glycosylation mutants with pmk-1(km25) background feeding on Lb-Tec2-expressing E. coli (dark gray) or empty pET24 vector containing E. coli (light gray) (n = 5). Error bars indicate SD. Comparisons between Lb-Tec2 and vector control for each C. elegans strain were performed using the Mann–Whitney U test (**P < 0.01). (c) Binding of TAMRA–Lb-Tec2 to the cuticle of C. elegans pmk-1(km25) (WT) and samt-1(op532)pmk-1(km25) (samt-1) at lower (upper) and higher magnification (lower). (Scale bars, 60 μm)

12.3 Histone Acetylation Regulation

12.3.1 MYST Family Histone Acetyltransferase Complex

The MYST family histone acetyltransferase complex contains MYS-1 and TRR-1. Both MYS-1 and TRR-1 were found to promote rather than inhibit the stress resistance and the longevity in nematodes (Fig. 12.9) [19]. Both single mutants and double mutants of mys-1 and trr-1 were resistant to environmental stress (Fig. 12.9) [19]. Mutation of mys-1 or trr-1 suppressed the induction of sod-3 induced by environmental stress (Fig. 12.9) [19]. Under the stress conditions, mutation of mys-1 or trr-1 suppressed the resistance of daf-2 mutant nematodes to environmental stress (Fig. 12.9) [19], suggesting that MYS-1 and TRR-1 act downstream of DAF-2 to regulate the toxicity of environmental toxicants or stresses.

Fig. 12.9

The MYS-1 HAT complex regulates longevity and oxidative stress resistance [19]. (a) Survival curves of the indicated RNAi-treated N2 worms under AL and IF are shown [control RNAi, n = 117 (AL), 98 (IF); trr-1 RNAi, n = 117 (AL), 107 (IF); mys-1 RNAi, n = 118 (AL), 104 (IF); trr-1;mys-1 RNAi, n = 111 (AL), 106 (IF)]. The bars represent the mean lifespan of three independent experiments. n, total number of worms in three independent experiments. Error bars, SD. ***P < 0.001, n.s. not significant; one-way ANOVA followed by Tukey’s test. (b) Immunoprecipitates with anti-Tip60/MYS-1 antibody from extracts of the indicated RNAi-treatedN2 worms at day 2 adulthood were subjected to immunoblot analysis using anti-TRR-1 antibody. Representative images of two independent experiments are shown. (c) Survival curves of the indicated RNAi-treated daf-2(e1370) mutants (left) or daf-16(mu86) mutants (right) under AL are shown [daf-2(e1370) mutants n = 180 (control RNAi), 180 (trr-1RNAi), 180 (mys-1RNAi); daf-16(mu86) mutants n = 178 (control RNAi), 180 (trr-1RNAi), 180 (mys-1RNAi)]. The bars represent the mean lifespan from three independent experiments. n, total number of worms in three independent experiments. Error bars, SD ***P < 0.001, one-way ANOVA followed by Tukey’s test. (d) Survival curves of the indicated RNAi-treated N2 worms or daf-2(e1370) mutants, which were exposed to 5 mM hydrogen peroxide (H₂O₂) [N2 worms n = 180 (control RNAi), 180 (trr-1RNAi), 180 (mys-1RNAi); daf-2(e1370) mutants n = 180 (control RNAi), 180 (trr-1RNAi), 180 (mys-1RNAi)] or 250 mM paraquat [N2 worms n = 128 (control RNAi), 128 (trr-1RNAi), 128 (mys-1RNAi); daf-2(e1370) mutants n = 90 (control RNAi), 90 (trr-1RNAi), 90 (mys-1RNAi)] at the young adult stage, are shown. The number of surviving worms was counted every hour. Error bars represent the SD derived from three independent experiments. n, total number of worms in three independent experiments. (e) Indicated RNAi-treated N2 worms at the young adult stage were exposed to 5 mM H₂O₂ for 30 min, and sod-3 mRNA expression levels were determined by qRT–PCR. The value of the control RNAi-treated N2 worms (0 mM H₂O₂) was set to 1. Error bars represent the SD derived from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by Tukey’s test

The beneficial effects of MYS-1 and TRR-1 were largely mediated through the transcriptional upregulation of FOXO transcription factor DAF-16 and its targets [19]. More importantly, it was found that MYS-1 and TRR-1 could be recruited to the promoter regions of daf-16, where they functioned in the histone acetylation, including the H4K16 acetylation (Fig. 12.10) [19].

Fig. 12.10

The MYS-1 HAT complex is recruited to the daf-16 promoter region and contributes to histone acetylation [19]. (a) Whole-worm lysates isolated from the indicated RNAi-treated N2 worms at day 2 adulthood were subjected to immunoblot analysis using the indicated antibodies. Representative images of three independent experiments are shown. (b) MYS-1 binding was examined by ChIP–PCR using cross-linked DNA–protein complexes isolated from the indicated RNAi-treated N2 worms at day 2 adulthood with anti-Tip60/MYS-1 and control IgG antibody. PCR amplification was done with specific primers for the daf-16 promoter region and the daf-16 3′UTR. Representative images of two independent experiments are shown. Genomic DNA in the input samples was used as a positive control. (c) MYS-1 binding and histone H4K16 acetylation status were examined by ChIP–qPCR using cross-linked DNA–protein complexes isolated from the indicated RNAi-treated N2 worms at day 2 adulthood with anti-Tip60/MYS-1, anti-AcH4K16, and control IgG antibody. The bars represent the percentage of total input DNA for each ChIP sample, and error bars represent the SD derived from three independent experiments. **P < 0.01, ***P < 0.001, one-way ANOVA followed by Tukey’s test

12.3.2 N-Terminal Acetyltransferase C (NAT) Complex

In nematodes, natc-1 encodes a N-a-acetyltransferase 35, an auxiliary subunit of the N-terminal acetyltransferase C (NAT) complex. NATC-1 is expressed in many cells and tissues and localizes to the cytoplasm [20]. The natc-1(am138) mutant nematodes showed a zinc resistance in multiple genetic backgrounds [20]. Additionally, loss-of-function mutations in natc-1 caused the resistance to a broad spectrum of stressors, including heat stress and oxidation stress [20]. DAF-16 was predicted to directly bind the natc-1 promoter, and the natc-1 mRNA levels were suppressed by DAF-16 activity (Fig. 12.11) [20], indicating that NATC-1 is a physiological target of DAF-16. natc-1 was epistatic to daf-16 in resistance to both the heat stress and the zinc stress [20]. That is, NATC-1 functions downstream of insulin/IGF-1 signaling pathway to regulate the toxicity of environmental toxicants or stresses in nematodes.

Fig. 12.11

natc-1 is regulated by daf-16 and functions in dauer formation [20]. (a) A model of the natc-1 promoter and open reading frame showing DAF-16 protein binding an evolutionarily conserved DAF-16 binding site positioned 90 base pairs upstream of the natc-1 start codon. (b) Wild-type, daf-2(e1370), and daf-16(mu86);daf-2(e1370) animals were synchronized at the L1 stage of development and cultured for 2 days at 20 °C on NGM. For each genotype, mRNA was extracted, analyzed by qRT–PCR, and natc-1 mRNA levels were normalized to the control gene rps-23. Bars show mRNA fold-change values calculated by comparing daf-2 and daf-16; daf-2 to WT by the comparative CT method (N = 4–6 biological replicates). Error bars indicate standard deviation. natc-1 mRNA levels were significantly reduced in daf-2(e1370) animals compared to WT but were not significantly different from WT in daf-16;daf-2 animals (*, p < 0.05). The mtl-1 gene was utilized as a positive control, since it is an established target of DAF-16, and mtl-1 mRNA levels were significantly increased in daf-2(e1370) compared to WT (p < 05), and this effect was daf-16 dependent (p < 0.05). (C) daf-2(e1370), natc-1(am138), and daf-2(e1370);natc-1(am138) embryos were cultured for 4 days on NGM at the indicated temperature and scored as either dauer or non-dauer (N = 200–341). Values for daf-2;natc-1 were significantly higher than daf-2 at 17.5 °C, 20 °C, and 22.5 °C (*p < 0.05). (d) daf-2(e1370) embryos were cultured on NGM at 20 °C, fed E. coli expressing dsRNA targeting natc-1, natc-2, or an empty vector control and scored as dauer or non-dauer after 4 days (N = 590–627). natc-1 and natc-2 RNAi significantly increased dauer formation compared to control (*p < 0.05)

12.3.3 CBP-1

In nematodes, the sirtuin SIR-2.4 can promote the DAF-16–dependent transcription and the stress-induced DAF-16 nuclear localization. It was further observed that the DAF-16 was hyperacetylated in the sir-2.4 mutants [21]. SIR-2.4 regulated both the acetylation and the localization of DAF-16 independently of its catalytic function (Fig. 12.12) [21]. Additionally, SIR-2.4 blocked the acetyltransferase CBP1-dependent DAF-16 acetylation, since DAF-16 was acetylated by CBP-1 and DAF-16 was hypoacetylated and constitutively nuclear in response to cbp-1 inhibition (Fig. 12.12) [21]. The in vitro assay further suggested that SIR-2.4 regulated the DAF-16 acetylation indirectly by preventing the CBP-1-mediated acetylation under the stress conditions [21]. Therefore, a novel role for acetylation in modulating DAF-16 localization and function was revealed, and this acetylation was modulated by the antagonistic activities of CBP-1 and SIR-2.4 in nematodes.

Fig. 12.12

SIR-2.4 interacts with DAF-16 and promotes DAF-16 deacetylation and function independently of catalytic activity [21]. (a) sir-2.4 deletion promotes DAF-16 hyperacetylation. DAF-16 acetylation was assessed in control or sir-2.4 KO worms by acetyl-lysine immunoprecipitation followed by GFP immunoblot. (b) SIR-2.4 and DAF-16 interact. Plasmids encoding FLAG-tagged SIR-2.4 and HA-tagged DAF-16 were transfected into 293T cells as indicated (GFP, negative control). Immunoprecipitation and immunoblotting were performed as shown. (c) Rescue of DAF-16 nuclear localization with a catalysis-defective sir-2.4 mutant. Stable transgenic strains of sir-2.4(n5137) were generated expressing either wild-type SIR-2.4 or the sir-2.4 N124A mutant. Worms were scored for GFP accumulation within the head hypodermic nuclei as day 1 adult (n = 50 or greater) after 20 min of heat-shock at 35 °C. P-values were calculated by Pearson’s χ ² test

12.4 miRNA Regulation

12.4.1 Dysregulated miRNAs by Environmental Toxicants or Stresses

It has been recognized that some engineered nanomaterials (ENMs) can potentially cause the toxicity on organisms, including the nematodes [22,23,24,25,26,27,28,29,30]. The dysregulated miRNAs have been systematically identified in nematodes exposed to certain ENMs [31, 32]. The dysregulated miRNAs have also been examined and identified in nematodes exposed to other toxicants or stresses [33,34,35]. With graphene oxide (GO), a carbon-based ENM, as an example, it was observed that GO (10 mg/L) exposure induced 31 dysregulated expressed miRNAs (23 upregulated miRNAs and 8 downregulated miRNAs) (Fig. 12.13) [31].

Fig. 12.13

Results of SOLiD sequencing for GO exposure [31]. (a) miRNA gene expression analysis by hierarchical clustering assay to reveal a characteristic molecular signature for GO (10 mg/L)-exposed nematodes. (b) Scatter diagram of miRNA coverage of the control group and GO treatment group. (c) Fold changes of up- and downregulated miRNAs in GO (10 mg/L)-exposed nematodes. (d) Expression pattern of mature miRNAs detected by real-time PCR after exposure to different concentrations of GO. Bars represent means ± S.E.M

Based on gene ontology analysis, these dysregulated miRNAs were associated with some important biological processes (such as cell metabolism, immune response, response to stimulus, cell communication, cell cycle, cell adhesion, reproduction, and development) in GO-exposed nematodes (Fig. 12.14) [31]. Based on KEGG pathway analysis, these dysregulated miRNAs were associated with some important signaling pathways (such as those related to cell metabolism, immune response, vesicle transportation, DNA damage and repair, oxidative stress response, transcription regulation, neuronal degeneration, cell death, cell cycle, and development) in GO-exposed nematodes (Fig. 12.14) [31]. These bioinformatics analysis data provide important clues for the further elucidation of the underlying mechanisms for GO toxicity induction in nematodes.

Fig. 12.14

Assessment of gene ontology terms and signal pathways [31]. (a) Gene ontology terms with gene counts based on dysregulated miRNAs in GO-exposed nematodes. (b) The predicted KEGG signal pathways based on dysregulated miRNAs in GO-exposed nematodes

Among these dysregulated miRNAs, mir-244 of mir-235 mutation could induce a susceptibility to GO toxicity, whereas mir-247/797, mir-73/74, or mir-231 mutation could induce a resistance to GO toxicity [31]. In contrast, mutation of mir-360, mir-81/82, mir-246, or mir-259 did not obviously affect the GO toxicity in reducing lifespan in nematodes [31].

12.4.2 Functions of miRNAs in Response to Environmental Toxicants or Stresses and the Underlying Molecular Mechanisms

The important function of let-7 in the regulation of toxicity of environmental toxicants or stresses has been introduced and discussed in Chap. 6. We further introduced and discussed the important functions of other miRNAs in response to environmental toxicants or stresses and the underlying molecular mechanisms.

12.4.2.1 Biological Function of mir-73

In C. elegans, SEK-1, a mitogen-activated protein kinase kinase (MAPKK), is an important member of the p38 MAPK signaling pathway. Using locomotion behavior as the toxicity assessment endpoint, mutation of mir-73 induced a resistance to GO toxicity, and mutation of sek-1 induced a susceptibility to GO toxicity (Fig. 12.15) [36]. Moreover, it was found that sek-1 mutation could inhibit the resistance of mir-73 mutant to GO toxicity (Fig. 12.15) [36], implying the role of SEK-1 as target of mir-73 in regulating GO toxicity. mir-73 expression could be decreased by GO exposure [36], suggesting the alteration in mir-73 expression mediates a protective response to GO exposure.

Fig. 12.15

Genetic interaction of mir-73 and sek-1 in regulating GO toxicity on locomotion behavior in nematodes [36]. Bars represent the mean ± S.E.M. **P < 0.01 vs wild-type N2. GO (10 mg/L) was exposed from L1-larvae to adult day 1

12.4.2.2 Biological Function of mir-84 and mir-241

In nematodes, the nuclear receptor DAF-12 negatively regulated the defense against pathogens, because mutation of daf-12 induced a resistance to pathogen infection and enhanced the expressions of antimicrobial gene expression [37]. Mutation of nsy-1 or pmk-1 suppressed the resistance of daf-12 mutant nematodes to pathogen infection [37], suggesting that the DAF-12 acts upstream of p38 MAPK signaling pathway to regulate the innate immune response to pathogen infection. mir-84 and mir-241 are members of let-7 family. Mutation of daf-12 decreased the expression of mir-84 and mir-241 after pathogen infection, and mutation of mir-84 or mir-241 induced a resistance to pathogen infection [37]. Moreover, SKN-1 was identified as a direct functional target of let-7s miRNAs (mir-84 and mir-241), and mutation of skn-1 suppressed the resistance of mir-84 or mir-241 mutant nematodes to pathogen infection (Fig. 12.16) [37], indicating that mir-84 and mir-241 regulate the innate immunity by suppressing the expression and function of their target SKN-1 in nematodes.

Fig. 12.16

SKN-1 is a direct functional target of let-7s miRNAs [37]. (a) Bioinformatics alignment of let-7s miRNAs and the 3′UTR of SKN-1. (b) Luciferase assays using the skn-1 3′UTR or the skn-1 3′UTR (mut) with let-7s mimics in HEK293T cells. The data shown are the mean ± SEM of three independent experiments, each of which was performed in triplicate, *P < 0.05. (c) Immunoblot analysis of the lysates from N2, daf-12(RNAi), mir-84(n4037), and mir-241(n4316) worms on E. coli or P. aeruginosa using anti-SKN-1 antibody and anti-tubulin antibody (loading control)

12.4.2.3 Biological Function of mir-231

In nematodes, mir-231 acted in the intestine to regulate GO toxicity [38]. Additionally, nematodes overexpressing intestinal mir-231 showed a susceptibility to GO toxicity [37]. smk-1, a predict targeted gene of mir-231, encodes a homolog of SMEK (suppressor of MEK null) protein. After GO exposure, mir-231 mutation increased smk-1 expression [38]. mir-231 mutants showed a susceptibility to GO toxicity [38]. Genetic analysis has indicated that smk-1 mutation could inhibit the resistance of mir-231(n4571) mutants to GO toxicity (Fig. 12.17) [38].

Fig. 12.17

Genetic interaction between mir-231 and SMK-1 in regulating GO toxicity in nematodes [38]. (a) Genetic interaction between mir-231 and SMK-1 in regulating GO toxicity in reducing lifespan. (b) Genetic interaction between mir-231 and SMK-1 in regulating GO toxicity in decreasing locomotion behavior. (c) Genetic interaction between mir-231 and SMK-1 in regulating GO toxicity in inducing intestinal ROS production. GO exposure concentration was 100 mg/L. Prolonged exposure was performed from L1-larvae to young adults. Bars represent means ± SD. **P < 0.01 vs N2 (if not specially indicated)

SMK-1 acted in the intestine to regulate GO toxicity, and DAF-16 further acted as a downstream target of SMK-1 during the regulation of GO toxicity [38]. That is, an intestinal signaling cascade (mir-231-SMK-1-DAF-16) was formed during the control of GO toxicity. mir-231 expression could be deceased by GO exposure [31], suggesting that the alteration in mir-231 expression mediates a protective response to GO exposure in nematodes.

12.4.2.4 Biological Function of mir-233

In nematodes, P. aeruginosa infection upregulated the expression of mir-233 [39]. Meanwhile, the mir-233 mutant nematodes were more sensitive to P. aeruginosa infection [39]. SCA-1, a homolog of sarco-/endoplasmic reticulum Ca²⁺-ATPase, was identified as a target of mir-233 during the control of innate immune response to pathogen infection (Fig. 12.18) [39]. During the P. aeruginosa PA14 infection, mir-233 repressed the protein levels of SCA-1, which might in turn lead to the activation of XBP-1-mediated ER UPR response, since the mir-233 was required for the activation of ER UPR (Fig. 12.18) [39]. RNAi knockdown of sca-1 caused the enhanced resistance to the killing by P. aeruginosa and suppressed the susceptibility of mir-233 mutant nematodes to pathogen infection [39]. The mir-233/SCA-1 signaling cascade was further found to act downstream of p38 MAPK signaling to activate the ER UPR and to regulate the innate immunity in nematodes [39].

Fig. 12.18

sca-1 is a target gene of mir-233 [39]. (a) Complementarity between the 3′ UTR of sca-1 gene and mir-233. mir-233 is presented in the form of DNA from 39 to 59 end. WT seeds are marked in red, mutated seeds in blue, Watson–Crick base pairing with a straight line and U-G wobbles with a dotted line. For constructing of sca-1-3′UTR (mut) reporter, the putative mir-233 binding site was replaced with an oligonucleotide containing the exact complementary sequence of mir-233. (b) The protein levels of SCA-1 were determined by Western blotting at 8 h after PA14 infection. The blot is typical of three independent experiments. P < 0.05, mir-233(+) (wild-type worms)+PA14 versus mir-233(+)+OP50; P < 0.01, mir-233(−) (mir-233(n4761) mutant worms)+PA14 versus mir-233(+)+PA14; mir-233(+)+OP50+sca-1 RNAi versus mir-233(+)+OP50. (c) Deletion of mir-233 did not affect the mRNA levels of sca-1. (d) Fluorescence images of the sca-1-3′ UTR GFP reporter in worms grown on E. coli OP50 or P. aeruginosa PA14. Mutagenesis of the putative binding site for mir-233 in the sca-1-3′UTR led to depression of the GFP expression in mir-233(+) worms after PA14 infection. Meanwhile, the GFP expression was significantly higher in mir-233 worms than that in mir-233(+) worms after PA14 infection. The lower panel shows quantification of GFP/mCherry ratio. The data are expressed as percent of control (the “wt” construct on OP50). ^a P < 0.01 versus mir-233(+)+wt on OP50; ^b P < 0.01 versus mir-233(+)+wt on PA14

12.4.2.5 Biological Function of mir-247

In nematodes, GO exposure could increase the expression of mir-247, and the mir-247/797 mutant nematodes showed a resistance to GO toxicity on lifespan and aging-related phenotypes [31]. Moreover, it was found that the mir-247 acted in neurons, but not in pharynx, to regulate the GO toxicity in reducing lifespan and in inducing intestinal ROS production (Fig. 12.19) [40]. In contrast, the neuronal overexpression of mir-247 induced a susceptibility to GO toxicity in reducing lifespan and in inducing intestinal ROS production [40]. Furthermore, the significant increase in mir-247 could be detected in wild-type nematodes exposed to GO at concentrations of more than 10 mg/L, and the GO toxicity in a transgenic strain overexpressing neuronal mir-247 after exposure to GO at concentrations of more than 10 μg/L was observed [40]. This implies the potential of mir-247 in warning of the GO toxicity in the range of μg/L.

Fig. 12.19

Tissue-specific activity of mir-247 in the regulation of GO toxicity [40]. (a) Tissue-specific activity of mir-247 in the regulation of GO toxicity on lifespan. (b) Tissue-specific activity of mir-247 in the regulation of GO toxicity in inducing intestinal ROS production. Prolonged exposure was performed from L1-larvae to adult day 1. Bars represent means ± SD. **P < 0.01 vs wild-type (if not specially indicated)

12.4.2.6 Biological Function of mir-259

Multiwalled carbon nanotubes (MWCNTs) exposure could increase the expression of mir-259 [32], and MWCNTs exposure induced an increase in mir-259::GFP in pharyngeal/intestinal valve and reproductive tract [41]. Meanwhile, mutation of mir-259 induced a susceptibility to MWCNTs toxicity [32], implying that mir-259 mediates a protection mechanism for nematodes against the MWCNTs toxicity. The expression of mir-259 in pharynx/intestinal valve could suppress the susceptibility of mir-259(n4106) mutant nematodes to MWCNTs toxicity on lifespan, suggesting that mir-259 acts in the pharynx/intestinal valve to regulate the MWCNTs toxicity [41]. RSKS-1, a putative ribosomal protein S6 kinase, was identified as a target of mir-259 in regulating the MWCNTs toxicity, and the rsks-1(ok1255) mutant nematodes had a resistance to the MWCNTs toxicity (Fig. 12.20) [41]. In nematodes, mutation of rsks-1 suppressed the susceptibility of mir-259 mutant nematodes to the MWCNTs toxicity (Fig. 12.20) [41]. RSKS-1 functioned in the pharynx to regulate the MWCNTs toxicity [41]. Moreover, the resistance of rsks-1(ok1255) mutant nematodes to MWCNTs toxicity on lifespan and aging-related properties could be inhibited by aak-2 mutation [41], indicating that RSKS-1 acts upstream of AAK-2 to regulate the MWCNTs toxicity. Additionally, AAK-2 acted together with DAF-16 in the same genetic pathway to regulate the MWCNTs toxicity [41]. Therefore, RSKS-1 regulated the MWCNTs toxicity by suppressing the function of AAK-2-DAF-16 signaling cascade in nematodes.

Fig. 12.20

Genetic interaction between mir-259 and rsks-1 in regulating MWCNTs toxicity in nematodes [41]. (a) Genetic interaction between mir-259 and rsks-1 in regulating MWCNTs toxicity in reducing lifespan in nematodes. (b) Genetic interaction between mir-259 and rsks-1 in regulating MWCNTs toxicity in decreasing locomotion behavior in nematodes. Exposure concentration of MWCNTs was 1 mg/L. Prolonged exposure was performed from L1-larvae to young adults. Bars represent means ± SD. **P < 0.01 vs N2 (if not specially indicated)

12.4.2.7 Biological Function of mir-355

In nematodes, infection with Pseudomonas aeruginosa could dysregulate some miRNAs [42]. Among these miRNAs, loss-of-function mutation of mir-45, mir-75, mir-246, mir-256, or mir-355 induced a resistance to P. aeruginosa infection, whereas loss-of-function mutation of mir-63 or mir-360 induced a susceptibility to P. aeruginosa infection [42]. Based on the prediction, SMA-3 in the TGF-β signaling pathway might function as a potential target for mir-246 in the regulation of innate immunity [42]. Moreover, the effects of intestinal overexpression of daf-2 lacking 3′ UTR or containing 3′ UTR on innate immune response of nematodes overexpressing intestinal mir-355 to P. aeruginosa PA14 infection, as well as the in vivo 3′-UTR binding assay, demonstrated that the DAF-2 in the insulin signaling pathway acted as a target for intestinal mir-355 to regulate the innate immunity (Fig. 12.21) [42]. Mutation of daf-2 resulted in a resistance to pathogen infection and suppressed the susceptibility of mir-355(n4618) mutant nematodes to pathogen infection [42]. In nematodes, mir-355 acted downstream of PMK-1 to regulate the innate immune response to P. aeruginosa PA14 infection [42]. Additionally, mir-355 acted upstream of DAF-16 and SKN-1 to regulate the innate immune response to P. aeruginosa PA14 infection [42]. Therefore, mir-355 functions as an important link between p38 MAPK signaling pathway and insulin signaling pathway in the regulation of innate immunity in nematodes. Similarly, DAF-2 was also found to act as a target of mir-355 in regulating the MWCNTs toxicity in nematodes [43].

Fig. 12.21

Effects of intestinal overexpression of daf-2 lacking 3′ UTR on innate immune response to P. aeruginosa PA14 infection in nematodes overexpressing intestinal mir-355 [42]. (a) Effects of intestinal overexpression of daf-2 lacking 3′ UTR on survival of nematodes overexpressing intestinal mir-355 after P. aeruginosa PA14 infection. Statistical comparisons of the survival plots indicate that, after the P. aeruginosa PA14 infection, the survival of transgenic strain Is(Pges-1-mir-355);Ex(Pges-1-daf-2-3′UTR) was significantly different from that of transgenic strain Is(Pges-1-mir-355) (P < 0.001). Bars represent mean ± SD. (b) Effects of intestinal overexpression of daf-2 lacking 3′ UTR on P. aeruginosa PA14 CFU in the body of nematodes overexpressing intestinal mir-355. Bars represent mean ± SD. **P < 0.01 vs wild-type (if not specially indicated). (c) Effects of intestinal overexpression of daf-2 lacking 3′ UTR on expression patterns of putative antimicrobial genes of nematodes overexpressing intestinal mir-355 after P. aeruginosa PA14 infection. Normalized expression is presented relative to wild-type expression. Bars represent mean ± SD. **P < 0.01

12.4.2.8 Biological Function of mir-360

Among the dysregulated miRNAs induced by GO, mir-360 mutation enhanced reproductive toxicity of GO; however, mir-360 overexpression inhibited reproductive toxicity of GO (Fig. 12.22) [44]. CEP-1 was identified as the target of mir-360 during the control of reproductive toxicity of GO in inducing germline apoptosis (Fig. 12.22) [44]. mir-360 expression could be increased by GO exposure [31], suggesting that the alteration in mir-360 expression mediates a protective response to GO exposure in nematodes.

Fig. 12.22

mir-360 negatively regulated the functions of signaling pathways of DNA damage checkpoints and apoptosis in the control of reproductive toxicity in GO-exposed nematodes [44]. (a) Germline apoptosis in mir-259, mir-81/82, or mir-360 mutant exposed to GO. The used mutants were wild-type N2, mir-360(n4635), mir-259(n4106), and mir-81&82(nDf54). (b) Genetic interaction of mir-360 with cep-1 in regulating germline apoptosis in nematodes exposed to GO. The used strains were wild-type N2, mir-360(n4635), cep-1(RNAi), and cep-1(RNAi);mir-360(n4635). (c) A summary model for the molecular control of reproductive toxicity of GO in inducing germline apoptosis in nematodes. GO exposure concentration was 10 mg/L. Prolonged exposure to GO was performed from L1-larvae to young adults. Bars represent means ± S.E.M. **P < 0.01

12.4.3 The mRNAs–miRNA Network Involved in the Regulation of Toxicity of Environmental Toxicants or Stresses

The mRNAs–miRNA network has been raised to be involved in the regulation of GO toxicity [36]. Based on the prediction, mir-259, mir-1820, mir-36, mir-82, mir-239, mir-246, mir-392, mir-2217, mir-360, mir-4810, mir-4807, mir-4805, mir-800, mir-1830, mir-236, mir-4806, mir-244, mir-235, mir-4812, mir-43, mir-231, mir-42, mir-2210, and mir-73 might be involved in the control of GO toxicity with some of the dysregulated genes as their targets in GO-exposed nematodes [36]. Among these potential targeted genes, gas-1 might act as a molecular target for mir-4810 to regulate the activation of oxidative stress in GO-exposed nematodes, and par-6 might act as a molecular target for mir-1820 to regulate intestinal development and function in GO-exposed nematodes [36]. Additionally, mir-231, mir-236, mir-259, mir-36, mir-42, mir-43, mir-73, mir-82, and mir-4805 might act through the functions of unc-44, mig-2, itr-1, ced-10, unc-93, fat-2, smp-1, and/or cab-1 to regulate the defecation behavior in GO-exposed nematodes [36]. Therefore, certain miRNA–mRNA networks may exist to regulate the toxicity of environmental toxicants or stresses by influencing some important biological processes, such as oxidative stress, intestinal development and function, and defecation behavior, in nematodes.

12.5 lncRNA Regulation

Long noncoding RNAs (lncRNAs) are normally defined as the noncoding RNAs having at least 200 nucleotides and no (or weak) protein coding ability, which have been shown to potentially regulate various biological processes [45,46,47,48].

12.5.1 Dysregulation of lncRNAs by Environmental Toxicants or Stresses

With the GO as an example, the Illumina HiSeq 2000 sequencing was performed for nematodes after prolonged exposure (L1-larvae stage to young adults) to GO (100 mg/L). Among the dysregulated 39 lncRNAs, 10 known lncRNAs were 1 upregulated lncRNA (linc-3) and 9 downregulated lncRNAs (linc-14, anr-24, linc-68, linc-103, linc-83, linc-24, anr-36, linc-5, and linc-37) (Fig. 12.23) [49]. linc-14, linc-68, linc-103, linc-83, linc-24, linc-5, linc-37, and linc-3 are intergenic lncRNAs, and anr-36 and anr-24 are antisense lncRNAs (Fig. 12.23) [49]. Besides this, 29 novel lncRNAs were also identified in GO-exposed nematodes (Fig. 12.23) [49]. Among these lncRNAs, 34 differentially expressed lncRNAs could be confirmed by qRT–PCR analysis in GO-exposed nematodes [49].

Fig. 12.23

Dysregulated lncRNAs induced by GO exposure in nematodes [49]. (a) Hierarchical clustering assay to show the characteristic molecular signature of 39 dysregulated lncRNAs in GO-exposed nematodes. (b) Dysregulated known lncRNAs induced by GO exposure in nematodes. logFC >2 and p < 0.05. (c) Dysregulated novel lncRNAs induced by GO exposure in nematodes. logFC >2 and p < 0.05

12.5.2 Effect of Ascorbate or Paraquat Treatment on lncRNA Profiling in Nematodes Exposed to Environmental Toxicants or Stresses

After pretreatment with paraquat (2 mM), a ROS generator, the expression of candidate 34 lncRNAs was more severely affected in GO-exposed nematodes (Fig. 12.24) [49]. Different from this, ascorbate (10 mM) treatment could recover the dysregulation in expressions of some lncRNAs caused by GO exposure (Fig. 12.24) [49].

Fig. 12.24

Effects of ascorbate or paraquat treatment on lncRNA expression in nematodes [49]. L4-larval nematodes were treated with 2 mM paraquat for 12 h in the 12-well sterile tissue culture plates. Nematodes were exposed to GO (100 mg/L) first from L1-larvae to L4-larvae and then treated with 10 mM ascorbate for 24 h. Bars represent means ± S.E.M. **P < 0.01

12.5.3 Effects of PEG Modification or FBS Surface Coating on Graphene Oxide-Induced lncRNA Profiling

After the comparison of 34 candidate long noncoding RNAs (lncRNAs) in control, GO-, or GO–PEG-exposed nematodes, expression patterns of some dysregulated lncRNAs induced by GO exposure could be reversed by PEG surface modification (Fig. 12.25) [49]. PEG surface modification could increase the expressions of linc-37, linc-5, linc-24, linc-14, XLOC_013642, XLOC_010849, and XLOC_004416 and decrease the expression of XLOC_007959 in GO-exposed nematodes (Fig. 12.25) [49]. Similarly, FBS surface coating could suppress the increase in the expressions of linc-37, linc-24, linc-14, XLOC_004416, XLOC_013698, and XLOC_012820, and the decrease in the expression of XLOC_007959 induced by GO exposure (Fig. 12.25) [49]. More importantly, it was found that both the FBS surface coating and the PEG surface modification could decrease the expressions of linc-37, linc-24, linc-14, and XLOC_004416 and increase the expression of XLOC_007959 in GO-exposed nematodes (Fig. 12.25) [49], which implies that FBS surface coating or PEG surface modification may potentially reduce the GO toxicity by influencing the functions of some certain conserved molecular basis mediated by same lncRNAs in nematodes.

Fig. 12.25

Surface modification altered the expression patterns of some lncRNAs in GO-exposed wild-type nematodes [49]. (a) lncRNA expression patterns in GO-, GO–FBS-, or GO–PEG-exposed nematodes. GO-, GO–FBS-, or GO–PEG exposure concentration was 100 mg/L. Prolonged exposure to GO was performed from L1-larvae to young adults. Bars represent means ± S.E.M. **P < 0.01. (b) Summary on the expression patterns of lncRNAs in GO-, GO–FBS-, or GO–PEG-exposed nematodes. Dark blue color indicates the decreased lncRNAs by GO, red color indicates the increased lncRNA by GO, yellow color indicate the increased lncRNAs by modification, and light blue color indicate the decreased lncRNA by modification. (c) Diagram for molecular mechanism of FBS coating or PEG surface modification to reduce the GO toxicity through the regulation of functions of lncRNAs in nematodes

12.5.4 The lncRNA–miRNA Network Involved in the Regulation of Toxicity of Environmental Toxicants or Stresses

Based on the identified dysregulated miRNAs and lncRNAs, a lncRNA–miRNA network that responds to GO exposure was raised in nematodes using a bioinformatics analysis algorithm. In this lncRNA–miRNA network, lncRNAs may regulate the GO toxicity by affecting or altering the function of certain targeted miRNA(s) [49]. These identified 34 lncRNAs might potentially only bind to a very limited number of miRNAs to regulate the GO toxicity in nematodes [49]. The lncRNA–miRNA networks provide important clues for further elucidation of underlying mechanisms for candidate lncRNAs in the regulation of toxicity of environmental toxicants or stresses in nematodes.

12.5.5 Functional Analysis of linc-37 and linc-14 in Regulating the Toxicity of Environmental Toxicants or Stresses

In nematodes, RNAi knockdown of linc-37 or linc-14 could induce a resistance to GO toxicity in reducing lifespan, in inducing intestinal ROS production, and in decreasing locomotion behavior [49]. In contrast, nematodes overexpressing linc-37 or linc-14 showed a susceptibility to GO toxicity [49]. Based on the screen of candidate binding transcriptional factors (TFs) for linc-37, it was found that linc-37 RNAi knockdown increased the expressions of ama-1, daf-16, nhr-28, elt-3, fos-1, and gmeb-1 in GO-exposed nematodes (Fig. 12.26) [49]. DAF-16 is the FOXO TF in the insulin signaling pathway. linc-37 RNAi knockdown also enhanced expression and translocation of DAF-16::GFP into intestinal nucleus (Fig. 12.26) [49]. Genetic analysis has indicated that linc-37 regulates GO toxicity by modulating DAF-16 function in nematodes.

Fig. 12.26

Interaction of DAF-16/TF with linc-37 in the regulation of GO toxicity [49]. (a) TFs expression patterns in linc-37 RNAi knockdown nematodes. (b) Subcellular localization of DAF-16::GFP fusion protein in GO-exposed linc-37 RNAi knockdown nematodes. (c) Lifespan of wild-type N2, daf-16(mu86), linc-37 RNAi, and daf-16(mu86);linc-37 RNAi nematodes exposed to GO. (d) Locomotion behavior in wild-type N2, daf-16(mu86), linc-37 RNAi, and daf-16(mu86);linc-37 RNAi nematodes exposed to GO. GO exposure concentration was 100 mg/L. Prolonged exposure to GO was performed from L1-larvae to young adults. Bars represent means ± S.E.M. **P < 0.01

linc-37 RNAi knockdown altered expressions of sod-1, sod-2, sod-3, sod-4, isp-1, clk-1, nhx-2, pkc-3, jkk-1, jnk-1, ced-3, ced-4, cep-1, and daf-16 [49], demonstrating the possible important roles of linc-37 in affecting oxidative stress, stress response, intestinal development, and cell apoptosis and DNA damage in GO-exposed nematodes. linc-14 RNAi knockdown also altered expressions of sod-3, sod-4, isp-1, nhx-2, par-6, pkc-3, jnk-1, mek-1, ced-3, ced-4, clk-2, cep-1, daf-16, akt-1, and daf-18 [49], suggesting the similar functions of linc-14 in affecting oxidative stress, stress response, intestinal development, and cell apoptosis and DNA damage in GO-exposed nematodes.

12.6 Perspectives

In nematodes, in this chapter, we only focused on the methylation, acetylation, microRNAs (miRNAs), and long noncoding RNAs (lncRNAs) to discuss the involvement of epigenetic mechanisms in the regulation of toxicity of environmental toxicants or stresses. Actually, there are still some other forms of epigenetic regulation, such as the circular RNAs. Therefore, more efforts are needed to elucidate the molecular basis for epigenetic control of toxicity of environmental toxicants or stresses.

Nevertheless, so far, the researchers still do not pay more attention to the epigenetic control of biological processes, as done on genes encoding molecular signaling pathways, that is, whether the epigenetic signals can play a pivotal role in regulating the biological processes, including the stress response or, in other words, whether the contribution of epigenetic signals is equally important to the genes encoding molecular signaling pathways in regulating the biological processes. A further possibility also exist that certain forms of epigenetic control are only very important at certain aspects in the regulation of biological processes. More convincing data are still needed to prove the possible or potential pivotal roles of different forms of epigenetic signals in regulating different biological processes at least in nematodes.

In nematodes, it is assumed that a small set of epigenetic signals can govern many important molecular signaling pathways to regulate the toxicity of environmental toxicants or stresses. If this assumption is correct, the study on the epigenetic regulation at least can help us identify more important signaling pathways involved in the regulation of toxicity of environmental toxicants or stresses. But, this should not be the real focus of work on the epigenetic regulation itself.