| Organism aging is a deleterious process characterized by the progressive decline of cellular and tissue functions in the late life stage.However,the rate of aging is often determined by an organism’s historic experiences that occur in early life.For example,some mild stresses experienced in early life can extend animal lifespan,implying that early stresses might impact the health of aged animals,the mechanisms of which remain largely unknown.Temperature is an important environmental factor with substantial impacts on animal physiology and aging.Higher ambient temperature,representing mild heat stress,is harmful to animals and shortens lifespan.The nematode C.elegans is a powerful genetic model for studying temperature effects at the organismal level.A recent study has discovered that high environmental temperature has a hormetic effect on aging,as C.elegans exposed to high temperature only during development exhibited an extended lifespan.Here,by using the model organism C.elegans,we reveal a mechanism of how early life heat stress impacts the health of aged animals and promotes longevity.The main results are as follows: 1.High temperature activates innate immune and detoxification responsesAs stress resistance is often believed to confer longevity in response to multiple hormetic stresses,we first sought to understand the activation of stress responses at high temperature by comparing the transcriptome of 25℃-cultivated animals to that of 15℃-cultivated control animals through RNA-seq analysis.Functional annotation analysis revealed that many genes associated with stress responses,especially innate immune and detoxification responses,were induced by high temperature.We further confirmed the expression of several innate immune and detoxification genes by qPCR.Next,we examined the expression of well-established transcriptional immune reporters for T24B8.5,the target gene of p38/ATF-7,in live animals.Phosphorylation of p38/ATF-7 by PMK-1 will activated the innate immune responses.We found that cultivation at 25℃ from L1 to day 1 adults induced T24B8.5p::GFP expression.Besides,the endogenous T24B8.5 mRNA levels were elevated over 100-fold by high temperature.C.elegans cultivated at 25℃ from L1 larvae to day 1 adults indeed exhibited enhanced pathogen and oxidative resistance compared to worms cultured at 15℃,suggested that high temperature activates innate immune and detoxification responses to promote animal stress resistance.2.PMK-1 is required for stress resistance and longevity at high temperatureConserved PMK-1/p38 mitogen-activated protein kinase is a major component of the C.elegans immune defense system,which regulates the expression of innate immune and detoxification genes.We found that PMK-1 phosphorylation levels were significantly increased by high temperature.In addition,the activation of the innate immune reporter T24B8.5p::GFP was largely dependent on PMK-1,as pmk-1 RNAi inhibited its expression at 25℃.The mRNA levels of endogenous T24B8.5 and two other defense response genes were further confirmed by qPCR analysis.Moreover,mutation of pmk-1 significantly compromised animal lifespan at 25℃.Thus,PMK-1 is essential for longevity at 25℃ high temperature,likely through the activation of innate immune and detoxification responses.3.Early life heat stress induces persistent stress responsesCultivation at mild heat of 25℃ during development promotes longevity in C.elegans,implying long-term effects of early life heat on the health of older animals.Considering the significant impacts of stress resistance on longevity regulation,we speculated that early exposure to 25℃ might have long-lasting effects on somatic stress responses.We observed that animals exposed to 25℃ until day 1 adults exhibited persistently elevated expression of the immune reporter T24B8.5p::GFP on day 4,represents midlife,and day 7,represents agedness,when compared to controls kept at 15℃ from the L1 larval stage.In addition,qPCR analysis confirmed the persistent mRNA induction of defense genes,including T24B8.5,F49F1.7 and cyp-37B1.Moreover,animal resistance to pathogen and oxidative stresses were also maintained.we conclude that early exposure to high temperature is sufficient to promote long-lasting stress resistance.4.CBP-1 mediates the persistent activation of defense responses to heat stressAs epigenetic regulations are usually involved in long-term effects of environmental factors on animal physiology.So we performed a small-scale RNAi screening targeting putative C.elegans epigenetic genes,and found that RNAi of cbp-1,encoding a histone acetyltransferase,almost completely abolished the activation of T24B8.5p::GFP,as well as the expression of endogenous T24B8.5,F49F7.1 and C32H11.4.The enhancement of pathogen and oxidative resistance at 25℃ was also abrogated by cbp-1 RNAi.More importantly,cbp-1 knockdown after heat exposure abolished the maintenance of T24B8.5p::GFP activation.The expression pattern of endogenous T24B8.5 was also verified by qPCR.We thus conclude that high temperature promotes long-lasting activation of stress responses via CBP-1.5.Histone acetylation regulates the expression of defense response genes at high temperatureIncreased histone acetylation is typically associated with enhanced gene expression.The crucial role for CBP-1 in temperature-induced activation of defense response genes implies the involvement of histone acetylation.Consistent with the enhanced expression of stress response genes,we found that histone acetylation was dramatically increased at 25℃,which was abrogated by cbp-1 RNAi and pmk-1 mutation.Besides,we observed persistent elevation of histone acetylation levels after 25℃.We further examined the occupancy of acetylated histone on the promoter regions of defense response genes by ChIP-qPCR analysis,and confirmed that temperature regulates the expression of defense genes directly through histone acetylation.Together,these data suggest that high temperature might promote the activation of defense response genes by increasing histone acetylation at their promoters.6.SWSN-1 modulates stress resistance at high temperatureSWSN-1 is another epigenetic regulator of T24B8.5p::GFP identified at 25℃.swsn-1 encodes the subunit of the SWI/SNF complex,an important chromatin remodeling apparatus that displaces or exchanges nucleosomes to open chromatin.By knocking down the subunit genes,we found that several of them participated in T24B8.5p::GFP regulation at 25℃,further supporting that the SWI/SNF complex play important roles in mediating high temperature-induced stress responses.Besides,we also found that knockdown of swsn-1 completely abolished 25℃-induced resistance to pathogen and oxidative stress.7.Somatic epigenetic memory promotes longevity in response to hormetic high temperatureOur findings imply an intriguing explanation for this phenotype,that the induction of persistent defense responses protects animals against aging-related damage such as endogenous ROS and infiltrated bacteria.To test the hypothesis,we used RNAi or mutation to silence cbp-1,swsn-1 and pmk-1 individually in worms and examined their lifespan in response to early life heat stress.Remarkably,knockdown of cbp-1 completely abrogated the beneficial effect of hormetic heat stress on lifespan,and swsn-1 RNAi partially abrogated the lifespan extension.The mutation of pmk-1,the crucial regulator of defense response and histone acetylation at 25℃,also completely abolished the lifespan extending effects of hormetic heat stress.These data support a model in which persistent defense responses,promoted by PMK-1,CBP-1 and SWSN-1,confer health benefits and longevity to aged animals,which were exposed to mild heat stress during development.8.FAT-6/7 and oleic acid regulate stress responses at high temperatureRNA-seq data revealed that genes of fatty acid metabolism were enriched,implying their important roles in temperature adaptation.We examined the involvement of fatty acid metabolism by performing a RNAi screen targeting fatty acid metabolic genes,and found that knockdown of fat-6/7,encoding delta-9 desaturases,completely abrogated 25℃-induced T24B8.5p::GFP expression,abolished the effect of warm temperature on pathogen and oxidative resistance.More importantly,the elevation of acetylated levels of histone was dramatically inhibited by ablation of FAT-6/7 at warm temperature,suggesting that these enzymes regulate stress response via histone acetylation.Do FAT-6/7 regulate stress responses through specific fatty acid species? We therefore examined the roles of other fat genes using RNAi,but found none of them affected T24B8.5p::GFP activity at 25℃.Supplementation of OA,the product of FAT-6/7,to fat-6/7 RNAi-treated worms restored the T24B8.5p::GFP activation in a dose-dependent manner.OA also largely rescued the pathogen resistance of fat-6/7 RNAi-treated worms pre-cultivated at 25℃.These data imply that OA mediates high temperature-induced stress responses.9.FAT-6/7 regulate histone acetylation and stress responses via HSF-1HSF-1 is the major heat-responsive transcription factor that regulates stress responses and lifespan in adaptation to temperature rise.We observed modest activation of HSF-1 reporter hsp-16.2p::GFP at 25oC,which could be abolished by fat-6/7 RNAi,indicating FAT-6/7 act upstream of HSF-1.Furthermore,hsf-1 RNAi largely suppressed the T24B8.5p::GFP expression and pathogen resistance at 25oC.Moreover,hsf-1 RNAi largely abolished 25oC-induced histone acetylation,suggesting HSF-1 regulates epigenetic modification and innate immunity downstream of FAT-6/7.Knockdown of pmk-1 did not affect the expression of HSF-1 reporter hsp-16.2p::GFP,while hsf-1 RNAi abrogated PMK-1 phosphorylation at 25oC,indicating HSF-1 acts upstream of PMK-1.In line with these findings,pmk-1 mutation abolished the induction of histone acetylation at 25oC.Together,our data reveal a FAT-6/7(OA)—HSF-1—PMK-1 pathway in regulation of histone acetylation and stress responses to mild heat stress... |
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