Molecular Mechanism Underlying The Regulation Of H3K27me3 Modification On Ecdysone Biosynthesis In Insects

Insect developmental transitions are cooperatively mediated by two major endocrine hormones,the steroid hormone ecdysone and juvenile hormone(JH).Ecdysone,as the precursors of 20-hydroxyecdysone(20E),is highly produced in the prothoracic gland(PG)and is secreted into hemolymph to converse into the biologically active form 20E.20E major emerges at the end of each larval instar and directly triggers larval molting or the larval-pupal transition.JH is massively synthesized in corpora allata at the early stage of each larval instar and functions as an antimetamorphic hormone to maintain insect larval growth and to prevent larvae from undergoing a developmental transition.The cross-talk between 20E and JH shows that they mutually repress their biosynthesis by inhibiting the transcription of the enzymes involved in biosynthetic pathways.20E prevents JH biosynthesis in the corpus allata by inhibiting the transcription of JHAMT,a key enzyme of JH biosynthesis.JH directly acts on the PG to induce the expression of Kr-h1,then Kr-h1 further inhibits the transcription of steroidogenic enzymes by directly binding to the KBS of their promoter regions and finally represses ecdysone biosynthesis.Although the antagonistic actions of 20E and JH in insect developmental transitions has been reported,the detailed molecular mechanism is need to been further illustrated.Histone methylation modification,as an importantly epigenetic way,regulates gene expression by influencing the chromatin structure and finally participates in the regulation of diverse biological processes in animals and plants.H3K27 methylation is catalyzed by the polycomb repressive complex 2(PRC2).PRC2 contains four core components,namely Enhancer of zeste(E(z)),Suppressor of zeste 12(Su(z)12),Extra sex combs(Esc),and Nurf55(also known as chromatin assembly factor 1 p55 subunit,Caf1-55),and isconserved in terms of both sequence and function in eukaryotes.PRC2-mediated H3K27 methylation mechanically causes the compaction of the related chromatin regions at target gene loci including promoter and enhancer regions and subsequently represses target gene expression.It is not clear whether H3K27 methylation is involved in insect metamorphosis and the regulation of endocrine hormone signaling.Exploring the molecular mechanism of underlying the regulation of H3K27me3 modification on ecdysone biosynthesis is helpful for improving the epigenetic regulation network of insect development transition.Our study aims to regard Bombyx and Drosophila as research subjects and explores whether and how H3K27me3 regulate insect metamorphosis separately from individual,tissue and cell levels by comprehensively using genetics,molecular biology,biochemistry,chromatin immunoprecipitation sequencing and bioinformatics.The major results obtained were as following:1.Dynamic changes of H3K27me3 levels in the PGs of Bombyx and Drosophila larvaeTo determine the role of H3K27 methylation in insect larval development,we first examined dynamic changes of H3K27me3 levels in the PG of both Bombyx and Drosophila individuals during larval-pupal transition.An immunostaining analysis and a Western blot experiment showed that H3K27me3 levels were slightly low in Bombyx PG at the early stage of the last larval instar and progressively increased until reaching a high level at the just wandering stage.In addition,during Drosophila larval-pupal transition,low and high H3K27me3 levels were detected in the PG of the brain-ring gland(RG)complex at 84 h after egg laying(AEL)and at 132 h AEL respectively.We further investigated the expression patterns of PRC2 core components in Bombyx PG.RT-q PCR analysis revealed that the messenger RNA(m RNA)expression of two PRC2 components,Su(z)12 and E(z),exhibited a progressive increase during the last larval instar,and the other two components were highly expressed at the early stage.In addition,Drosophila Su(z)12 expression in the brain-RG complex containing the PG exhibited an obvious increase before pupariation.Collectively,our results showed that the dynamics of both H3K27me3 levels and the PRC2 component expressions were similar to the temporal changes in ecdysone titer during the larval-pupal transition.2.H3K27me3 positively regulates ecdysone biosynthesis and insect metamorphosis1)H3K27me3 positively regulates the larval-pupal transition and ecdysone production in Bombyx and Drosophila larvaeWe next analyzed the effects of PRC2-mediated H3K27 methylation in the PG on insect larval development.First,we injected Bombyx larvae on the fifth day of the last larval instar with GSK126,an inhibitor of PRC2 activity,and found that compared with DMSO treatment as a control,GSK126 treatment decreased H3K27me3 levels in the PG but had no effect on the levels of the other two histone methylation modifications,H3K36me3 and H3K4me3.GSK126 treatment obviously delayed Bombyx larval-pupal transition,when the control grew into W0 and pupal stage,Bombyx larval treated by GSK126 were still feeding and prepupal.Furthermore,we performed RNAi mediated knockdown of four core PRC2 components,including E(z),Su(z)12,Esc,and Nurf55,in the PG of Drosophila larvae using the PG-specific driver Phm-Gal4.The results revealed that knockdown of the Su(z)12 gene decreased H3K27me3 levels and caused a developmental defect in the larval-pupal transition and formed a large permanent L3(the third instar).Other than Su(z)12,knockdown of other PRC2 components in the PG did not affect larval-pupal transition.These data demonstrate that PRC2-mediated H3K27methylation in the PG of Bombyx and Drosophila larvae is required for the larval-pupal transition.We next investigated whether ecdysone production could be affected by changes of H3K27 methylation levels.The results showed that both the application of GSK126 to Bombyx larvae and PG-specific knockdown of Su(z)12 in Drosophila larvae impaired ecdysone production,down-regulated the transcription of the ecdysone-responsive E75B gene.Feeding 20-hydroxyecdysone(20E),an active derivative of ecdysone,partially rescued the defect in the larval-pupal transition caused by PG-specific Su(z)12knockdown in Drosophila or by GSK126 treatment in Bombyx.2)H3K27me3 positively regulates the transcription of steroidogenic enzyme genes in Bombyx and Drosophila larvaeWe further examined the expression of steroidogenic enzyme genes using RT-q PCR,and founded that both the application of GSK126 to Bombyx larvae and PG-specific knockdown of Su(z)12 in Drosophila larvae reduced the expression of steroidogenic enzyme genes,including Nvd,Sro,Spo/Spok,and Sad in the PG,compared with the control.In addition,the application of GSK126 to ex vivo-cultured Bombyx PG or Drosophila brain-RG complex also led to a decrease in both H3K27me3 levels and steroidogenic enzyme transcription.Taken together,our results suggest that H3K27me3in the PG positively regulated ecdysone production and the larval-pupal transition in both Bombyx and Drosophila larvae3.H3K27me3 inhibits the transcription of JH signal transducer Hairy1)The ChIP-seq analysis of H3K27me3 in Bombyx PGTo identify H3K27me3 targets related to ecdysone biosynthesis in the PG,we performed a chromatin immunoprecipitation sequencing(ChIP-seq)analysis in Bombyx PG at the first day of the fifth larval instar(L5D1)without ecdysone production and the just wandering(W0)with high ecdysone titer.A comparative analysis identified 2,089differential peaks between L5D1 and W0,1,506 of which were highly present at the W0stage and associated with 650 genes.Further analysis revealed that 465 genes were specifically marked by W0-biased H3K27me3 peaks and most of them enriched in the Gene Ontology(GO)categories of nucleic acid binding and transcription regulator activity.62 genes of 465 genes belongs to transcription factors.2)H3K27me3 inhibits the transcription of JH signal transducer HairyWe observed that the Hairy gene,which encodes a basic helix-loop-helix(b HLH)transcription factor that functions as a key transducer in the JH repression hierarchy,was included in the collection of 62 transcription factors as potential H3K27me3 targets in Bombyx PG at the W0 stage.ChIP-seq data and ChIP-q PCR assay confirmed that H3K27me3 levels at the Bm Hairy locus were dynamic at different developmental stages in Bombyx PG,H3K27me3 levels at the Bm Hairy locus were higher at the W0 stage.Simultaneously,ChIP-seq data from whole bodies of Drosophila larvae at the third instar stage revealed the presence of H3K27 methylation within the potential promoter of Dm Hairy.H3K27me3 enrichment within the Dm Hairy promoter could be significantly diminished by PG-specific Su(z)12 knockdown.We further investigated the effects of PRC2-mediated H3K27 methylation on the expression of the Hairy gene in the PG of Bombyx and Drosophila larvae.An RT-q PCR analysis revealed that Hairy expression was high at the early stage of the last larval instar and progressively decreased until it reached a low level at the W0 stage in Bombyx or at132 h AEL in Drosophila,which was consistent with the temporal change in JH titer in the hemolymph but was contrary to the dynamic of H3K27me3 levels in the PG.In addition,the reduction in H3K27me3 levels in the PG caused by either GSK126 treatment in Bombyx larvae or PG-specific Su(z)12 knockdown in Drosophila larvae led to an elevation of Hairy transcription in the PG.Collectively,our results indicate that H3K27methylation in the PG of insect larvae targets the Hairy gene to repress its transcription.4.The founction of Hairy in ecdysone biosynthesis and metamorphosis in Drosophila1)The effects of Hairy in Drosophila PG on developmental transition,ecdysone production and the transcriptions of steroidogenic enzymesTo examine whether Hairy expression in the PG is involved in ecdysone-mediated larval-pupal transition,we first performed RNAi-mediated knockdown of the Dm Hairy gene in Drosophila PG.As a result,PG-specific Hairy knockdown caused precocious larval-pupal transition,induced a rapid increase of ecdysone titer in the hemolymph,and up-regulated the expression of both the ecdysone responsive gene E75B in the fat body and steroidogenic enzymes in the PG.In addition,the heterozygous mutation in Hairy also caused precocious pupariation and an increase in ecdysone production.We further used the Tub-gal80^ts;Phm-Gal4 driver to drive Hairy overexpression in the PG of Drosophila larvae at the beginning of the third instar.Compared with the control,Dm Hairy overexpression in the PG resulted in developmental arrest at the third larval instar.Ecdysone production and the expression of both steroidogenic enzymes in the PG and E75B in fat body were reduced following PG-specific Dm Hairy overexpression.Further luciferase reporter assay and ChIP-PCR experiments in Bombyx Bm E cells and Drosophila S2 cells confirmed that Hairy negatively regulated the promoter activity of steroidogenic enzyme Bm Spo/Dm Spok gene,by directly binding to its promoter.Further,the phenotype of developmental arrest caused by Dm Hairy overexpression in Drosophila PG could be partially rescued by feeding with 20E,and the animals could successfully complete the larval-pupal transition.2)The determination of the epistatic relationship between Su(z)12 and Hairy in the PGWe surveyed the epistatic relationship between Su(z)12 and Hairy in the PG.Compared with only Su(z)12 knockdown,the knockdown of both Hairy and Su(z)12 in the PG increased the number of larvae undergoing the larval-pupal transition and ecdysone production.At 144 h AEL,when the control larvae completed the larval-pupal transition,most Su(z)12 knockdown animals remained fed,while approximately half of the animals with knockdown of both Hairy and Su(z)12 entered pupariation.Taken together,the results indicate that the H3K27me3-mediated repression of Hairy transcription in the PG of insect larvae is required for ecdysone biosynthesis.5.JH represses H3K27me3 modificationTo further investigate whether JH regulates H3K27me3 methylation,we first used the JH mimic(JHM)methoprene to treat ex vivo-cultured PG from Bombyx larvae on the sixth day of the last larval instar and found that JHM treatment reduced H3K27me3 levels in the PG.An RT-q PCR analysis showed that m RNA expression of the Su(z)12 gene was significantly down-regulated after JHM application.Similarly,JHM treatment of the ex vivo-cultured brain-RG complex from Drosophila larvae at 108 h AEL also decreased H3K27me3 levels and impaired Su(z)12 transcription.Altogether,these data indicate that JH inhibits PRC2-mediated H3K27 methylation to maintain Hairy transcription in the PG of insect larvae.