Molecular Mechanism Of Histone Methyltransferase Related Signals In Regulating The Toxicity Of Polystyrene Nanoparticles In Caenorhabditis Elegans

In recent years,the role of epigenetic regulation in response to the toxicity of environmental toxicants or stresses has received the wide attention.Histone methylation is one of the important epigenetic regulation mechanisms for the control of gene expressions in organisms.The histone methylation level can be balanced by methyltransferases and demethylases.So far,some methylation related signals have been identified to be required for the control of toxicity induced by environmental exposures.Polystyrene nanoparticles(PS-NPs)is one form of nanoplastics.The PS-NPs could be detected in marine,soil and air ecosystems,and their potential toxic effects on both environmental organisms and even human beings have been gradually paid attention to.Caenorhabditis elegans is very sensitive to the toxicity of environmental toxicants,and is an important model for the study of molecular toxicology.A series of studies have examined the PS-NPs toxicity at various aspects and the underlying molecular mechanisms in nematodes.However,it is still unclear whether the histone methyltransferase related signals are involved in the regulation of PS-NPs toxicity.In this study,we focused on the elucidation of molecular mechanisms for the potential function of histone methyltransferase related signals in regulating the toxicity of PS-NPs in nematodes.1.Molecular mechanism of methyltransferase MET-2 in regulating the toxicity of PS-NPsIn nematodes,prolonged exposure to 1-100 μg/L PS-NPs significantly decreased the expression of MET-2,a H3K9 methyltransferase.Meanwhile,RNAi knockdown of met-2 suppressed the PS-NPs toxicity in inducing production of reactive oxygen species(ROS)and in decreasing locomotion behavior,which suggesting that the decrease of MET-2 expression reflected a protective response to PS-NPs exposure.MET-2 could act in both intestine and germline to regulate the toxicity of PS-NPs.In PS-NPs exposed nematodes,intestinal RNAi knockdown of met-2 significantly increased the expressions of daf-16,bar-1,and elt-2.Meanwhile,intestinal RNAi knockdown of daf-16,bar-1 or elt-2 suppressed the resistance of met-2(RNAi)worms to PS-NPs toxicity,suggesting that MET-2 functioned upstream of ELT-2,BAR-1,and DAF-16 in intestinal cells to control PS-NPs toxicity.In addition,intestinal RNAi knockdown of lin-65 significantly suppressed the resistance of met-2(RNAi)worms to PS-NPs toxicity,and RNAi knockdown of daf-16,bar-1 or elt-2 further inhibited the susceptibility of worms overexpressing intestine LIN-65 to PS-NPs toxicity,That is,the LIN-65 acted downstream of MET-2 and further functioned upstream of DAF-16,BAR-1,and ELT-2 in intestinal cells to regulate the PS-NPs toxicity.Moreover,in PS-NPs exposed worms,germline RNAi knockdown of met-2 significantly decreased the expressions of wrt-3 and pat-12.RNAi knockdown of wrt-3 or pat-12 further inhibited the susceptibility of worms overexpressing germline MET-2 to PS-NPs toxicity,suggesting that MET-2 functioned upstream of PAT-12 and WRT-3 in germline cells to control PS-NPs toxicity.Additionally,germline RNAi knockdown of lin-65 also inhibited the resistance of met-2(RNAi)worms to PS-NPs toxicity,and the susceptibility of lin-65(RNAi)worms to PS-NPs toxicity was inhibited by germline RNAi knockdown of pat-12 or wrt-3.That is,the germline LIN-65 acted downstream of MET-2 and further functioned downstream targets PAT-12 and WRT-3 to regulate the PS-NPs toxicity.Therefore,MET-2-mediated methylation regulation provided an important molecular basis for induction of protective response to nanoplastics in nematodes.2.Molecular mechanism of methyltransferases in regulating transgenerational toxicity of PS-NPsAt the P0 generation of nematodes,exposure to 20 nm PS-NPs(≥ 1 μg/L)resulted in inhibition of locomotion behavior and decrease of brood size,and this toxicity could be transferred transgenerationally.The expressions of 7 histone methyltransferases(HMTs)were altered which exposure to PS-NPs at the predicted environmental concentration(1 μg/L).Among these HMT genes,the expressions of met-2,ttll-12,and set-6 were significantly increased,and the expressions of set-5,set-12,set-26,and set-23 were significantly decreased.In PS-NPs exposed worms,germline RNAi knockdown of met-2 or set-6 significantly suppressed the transgenerational toxicity of PS-NPs in inhibiting locomotion behavior and in decreasing brood size,suggesting their resistance to transgenerational PS-NPs toxicity.In contrast,germline RNAi knockout of set-26 significantly enhanced the transgenerational toxicity of PS-NPs in inhibiting locomotion behavior and in decreasing brood size,suggesting its susceptibility to transgenerational PS-NPs toxicity.In PS-NPs exposed worms,gremline RNAi knockdown of met-2 significantly increased expressions of pat-12 and wrt-3.In adition,germline RNAi knockdown of pat-12 or wrt-3 suppressed the transgenerational toxicity of PS-NPs,indicating that germline PAT-12 and WRT-3 were also involved in the regulation of transgenerational toxicity of PS-NPs.Moreover,RNAi knockdown of pat-12 or wrt-3 significantly suppressed the susceptibility of worms overexpressing germline MET-2 to transgenerational PS-NPs toxicity,indicating that PAT-12 and WRT-3 could act as downstream targets of germline MET-2 to regulate the transgenerational toxicity of PS-NPs.Furthermore,in germline cells,insulin polypeptides INS-3 and INS-39 act as downstream targets of PAT-12 and WRT-3 to regulate the transgenerational toxicity of PS-NPs by transgenerationally targeting insulin receptor DAF-2.Therefore,a germline signal cascade of MET-2-PAT-12/WRT-3-INS-3/INS-39-DAF-2 was raised to be involved in the regulation of transgenerational PS-NPs toxicity.In conclusion,we determined the molecular mechanisms of histone methyltransferase related signals in regulating the PS-NPs toxicity.During the control of protective response to PS-NPs at the P0 generation,intestine MET-2 suppressed the downstream signaling cascade of LIN-65-ELT-2/BAR-1/DAF-16,and germline MET-2 inhibited the downstream signaling cascade of LIN-65-PAT-12/WRT-3,which provided an important methylation regulation related molecular mechanism.During the control of transgenerational PS-NPs toxicity,histone methyltransferases of MET-2,SET-6 and SET-26 played important functions.Moreover,germline MET-2 activated the downstream signaling cascade of PAT-12/WRT-3-INS-3/INS-39-DAF-2 to regulate transgenerational toxicity of PS-NPs.Our results will be helpful for further deeply understanding the mechanisms for epigenetic control of PS-NPs toxicity.