Polycomb Repressive Complex PRC2 Shortens Lifespan By Reducing DAF-16 Activity In C.Elegans

FOXO/DAF-16 is one of the key transcription factors currently known to be highly conserved and involved in various signaling pathways regulating biological aging,including the insulin/insulin like growth factor-1 signaling（IIS）and TOR signaling pathways.IIS signaling pathway is the earliest known aging signaling pathway.When insulin receptor combines with extracellular ligand,it can activate downstream kinase AKT to phosphorylate FOXO/DAF-16,resulting in FOXO/DAF-16 not entering the nucleus,thus inhibiting the expression of multiple anti-aging and anti-stress target genes of this transcription factor.Phosphatase PTEN/DAF-18 is a negative regulator of the IIS signaling pathway,which inhibits AKT kinase activity by hydrolyzing PIP3（3,4,5-triphosphate phosphatidylinositol）to PIP2（4,5-diphosphate phosphatidylinositol）,thereby promoting FOXO/DAF-16 entry into the nucleus and delaying aging.Changes in epigenetic information are one of the main characteristics of aging,including changes in various histone methylation modifications.Because of its short life span,Caenorhabditis elegans is a classical model organism for studying organism aging.There are literature reports that histones H3K4me3 and H3K36me3 methyltransferase in C.elegans can regulate the aging process.Knocking out the histone H3K27me3 demethylase UTX-1 in C.elegans resulted in an increase in the overall modification level of H3K27me3,thereby prolonging the lifespan of C.elegans.This study suggests that histone H3K27me3methyltransferase may also be involved in aging regulation.Polycomb repressive complex 2（PRC2）is a complex with histone H3K27me3methyltransferase activity that inhibits gene expression.It is mainly composed of three core components:SUZ12,EZH2,and EED.EZH2 contains the SET domain,which catalyzes the modification of histone H3K27me3;And SUZ12 and Esc/EED have a stabilizing effect on EZH2.The PRC2 complex exhibits high conservatism from C.elegans to mammals and plays an important regulatory role in embryonic development and tumorigenesis.The PRC2complex of C.elegans consists of MES-2/EZH2,MES-6/EED,and MES-3（MES-3 does not have mammalian homologs）.Recent studies have found that RNAi treatment of each member of the PRC2 complex after larval development（L4 stage）leads to an extension of C.elegans lifespan,and the extension of C.elegans lifespan by MES-2 requires the participation of DAF-16/FOXO.But can the PRC2 complex regulate the activity of DAF-16/FOXO?Which signaling pathway is its regulatory effect on the activity of the transcription factor?It is still unclear.In this study,we first performed mes-2/mes-3/mes-6 RNAi on the eggs and L4（larval development completion）stages of C.elegans,and found that the PRC2 complex mainly shortens the lifespan of after larval development is completed.Therefore,we will choose L4as the period for RNAi processing.Using the daf-16 mutant,we found that the process of extending lifespan and antioxidant capacity by the mes-2/mes-3/mes-6 RNAi requires the involvement of DAF-16.Using DAF-16::GFP transgenic C.elegans,laser confocal observation revealed that the mes-2/mes-3/mes-6 RNAi promoted the nuclear localization of DAF-16;Moreover,using transgenic C.elegans SOD-3::GFP,we found that mes-2/mes-3/mes-6 RNAi enhanced the protein expression level of SOD-3,the classical target gene of DAF-16;The RT-q PCR results showed that mes-2/mes-3/mes-6 RNAi increased the m RNA expression level of DAF-16 anti-aging target genes（such as ctl-3,lys-7,mtl-1）.The above results indicate that the PRC2 complex can inhibit the transcription activity of DAF-16.In order to understand which signaling pathway is involved in the inhibition of DAF-16/FOXO activity by the PRC2 complex,we used mutants of the IIS signaling pathway key gene daf-2 and the TOR signaling pathway key gene rsks-1 in,and found that the extension of C.elegans lifespan by the mes-2/mes-3/mes-6 RNAi was inhibited by the daf-2deficiency,but not by the rsks-1 deficiency.In addition,fluorescence quantitative analysis and RT-q PCR results showed that the mes-2/mes-3/mes-6 RNAi did not affect the target gene expression of another transcription factor SKN-1/Nrf2 in the IIS signaling pathway,indicating that the PRC2 complex reduces DAF-16/FOXO（rather than SKN-1/Nrf2）activity and regulates C.elegans lifespan through the IIS signaling pathway.Since we found that the mes-2/mes-3/mes-6 RNAi did not upregulate the protein expression level of DAF-16::GFP,it is speculated that this complex may inhibit DAF-16activity by altering the expression of other regulatory factors in the IIS signaling pathway.We used the negative regulatory factor DAF-18/PTEN of the IIS signaling pathway,the phosphatase PPTR-1 of AKT,and the arginine methyltransferase PRMT-1 and kinase JNK-1that regulate DAF-16 activity as candidate factors.Using RT-PCR,we found that mes-2/mes-3/mes-6 RNAi increased the m RNA levels of daf-18 and prmt-1.However,the prolongation of lifespan by mes-2 RNAi can only be inhibited by daf-18 RNAi,and is not affected by prmt-1 deficiency.The laser confocal results showed that compared to interfering with mes-2 alone,simultaneous RNAi treatment of mes-2 and daf-18 resulted in a significant decrease in the nuclear accumulation of DAF-16 and the expression level of SOD-3.The above results indicate that the PRC2 complex reduces the transcription activity of DAF-16 by inhibiting the expression of DAF-18/PTEN genes.It is known that IIS signaling pathway can also regulate endoplasmic reticulum unfolded protein response(UPR^ER)through DAF-16,but by observing the expression of HSP-4::GFP,the key marker protein of UPR^ER,we found that although PRC2 complex inhibits heat shock induced UPR^ER response,this process is independent of DAF-16 and DAF-18.In summary,we found that the PRC2 complex reduced the activity of key transcription factors DAF-16/FOXO in the IIS signaling pathway by inhibiting DAF-18 expression,thereby promoting C.elegans aging.And this process is not related to the UPR^ER.This discovery will propose a new molecular mechanism for the regulation of C.elegans senescence by the PRC2 complex,adding new content to the epigenetic regulation of senescence.