Regulation Of DAF-16 By SKN-1 In Ceanorbabditis Elegans

Caenorhabditis elegans(C.elegans)is a free-living roundworm found in bacteria-rich habitats such as humus.Because of its transparent body,short life cycle,convenience of culturing and genetically friendly to manufacture,it has been used by Sydney Brenner as a model organism in cellular and developmental biology.Over the past decades,C.elegans has emerged as a major model organism for aging research.DAF-16 and SKN-1 then emerged and have been described in many aging related studies.DAF-16/FOXO is a key transcription factor regulated by the insulin and insulin-like factors?(IIS)pathway,and is essential for longevity of many long-lived mutants in C.elegans.The main function of DAF-16 is to regulate the metabolism of glucose and lipid,and the resistance response to many environmental and physical stresses such as bacterial infection,heat shock(HS)and ROS.Another important factor in the aging research of C.elegans is SKN-1,which is the ortholog of mammalian Nrf/CNC proteins(Nrf1,Nrf2,Nrf3,p45 NF-E2).SKN-1 was found to regulate oxidative stress and xenobiotic defense responses and also participate in proteostasis and metabolic regulation.SKN-1 was also required for lifespan extension in many long-lived worm mutants.SKN-1 also regulates lipid metabolism and mediates the transfer of yolk from intestine cells to eggs.Although DAF-16 and SKN-1 are both vital factors in longevity responnses,and in many longevity mutants either DAF-16 or SKN-1 deficient can result in the dispear of long-lifespan phynotype,the connections between these two important transcription factors were largely unknown.Here we found that the skn-1 gain-of-function mutant strain skn-1(lax120)had a weaker HS stress resistance ability compare to the wild type N2,which triggered the hypothesis that SKN-1 may be able to influence DAF-16 directly.To confirm this,we used skn-1(lax120);sod-3p:: GFP and found out that it had a dimmer density of GFP light than sod-3p:: GFP.Along with a Q-PCR result that the DAF-16 upregulated gene mtl-1 was downregulated while the DAF-16 downregulated gene dod-24 was upregulated in the strain skn-1(lax120)compared with wild type N2,from which we concluded that SKN-1 weakened the function of DAF-16.We then used a GFP-labeled DAF-16 protein containing strain daf-16 a/b:: GFP to comfirmed the result that SKN-1 repressed the function of DAF-16 by influencing the nuclear localization of DAF-16.Because SKN-1 is reported to be able to help transfer yolk to eggs in C.elegans through VITs(vit-1,vit-2,vit-3,vit-4,vit-5,vit-6).We then confirmed that vit-2 deficient mutant strain vit-2(ok3211)had a weaker HS resistance along with a brighter GFP in sod-3p:: GFP;vit-2(ok3211).Next,we crossed vit-2:: GFP and and vit-2(ok3211)with skn-1(lax120)and then tested the HS resistance ability.We found out that on later stage of adult worms(D5),the DAF-16 regulation effect from SKN-1 was diminished by VIT-2 dificient.This means that SKN-1 and VIT-2 may in the same regulation pathway of DAF-16.Because the vitellogenin can combine the lipid compounds in somatic cells to form the yolk and then be transferred to germline.We wondered that SKN-1 and VIT-2 may influence the metabolism of lipid to affect the function of DAF-16.To support this,we constructed a GC-MS/MS analysis of fatty acid between skn-1(lax120)and N2.After analysis of the results,we discovered a significant difference in the compound palmitoleic acid,which seldom be described in previous studies.We used fat-5 dificient strain fat-5(tm420)and palmitic acid to confirm this.We found out that when lack fat-5,which is responsible for the synthesis of palmitoleic acid from palmitic acid.We found that fat-5(tm420)has the similar effects as skn-1(lax120)to DAF-16.And neither skn-1(lax120);fat-5(tm420)or daf-16(mu86);fat-5(tm420)double mutants showed a strengened weaker HS resistance ability than single mutants.These together indicated that palmitoleic acid or its derivative may be the mediator through which SKN-1 inhibited the function of DAF-16.In this study,we found out that SKN-1 can influence the function of DAF-16 through affecting the DAF-16 nuclear localization process.Because both SKN-1 and DAF-16 are important transcription factors in regulation of aging,metabolism and stress resistance,the relationship of these two factors have an understandable meaning for the studying of mechanism about how these two transcription factor progressed to co-regulate the resistance to different stresses and together increased the lifespan in many long-lived C.elegans mutants.Indeed,SKN-1 and DAF-16 both participated in the metabolism of lipid and are both responses to the stress resistance like ROS and pathogens.There may be a regulation network exists to coordinate the function of these two transcription factors.In this paper,we found out the direct influence on DAF-16 from SKN-1,which indicated that these two factors may have a cross-regulated relationship to coordinate the healthy balance within C.elegans.