Molecular Mechanism Underlying The Inhibition Of Transcription Factor Kr-h1 Of JH Signaling On 20E Biosynthesis And Effect Of 20E On Protein Synthesis In Insects

Insect growth and development is mainly regulated by juvenile hormone(JH)and 20-hydroxyecdysone(20E)as active form of the steroid hormone ecdysone through the periodic changes of their titers.The high peak of JH titer occurs at the beginning of each larval instar and is responsible for maintaining larval growth.The 20 E pulses appear at the end of each larval instar and trigger larval molting.High concentration of 20 E during the prepupal and the middle stage of the pupa,a period that JH is disappeared,caused larval–pupal and pupal-adult metamorphosis.Opposite fluctuation of JH and ecdysone titers and antagonistic effects of JH on 20 E during insects growth and development suggest that JH may have an inhibitory effect on 20 E biosynthesis.Exploring the regulatory mechanism underlying the regulation of JH and its signal transducer Krüppel-homolog 1(Kr-h1)on 20 E biosynthesis will help us to better understand cross-talk between these two endocrine hormones during insect growth and development.20E acts on target tissues and organs to induce apoptosis or tissue remodeling for metamorphosis during the larval-pupal transformation of insects.As one of the 20 E target tissue,fat body is the main place for protein synthesis,material metabolism and energy metabolism.As well known,fat body cells dissociate and provide nutrition and energy for larval molting and larval–pupal transition.However,it is still unclear the characteristics of protein synthesis in fat body during insect growth and development and whether it is regulated by 20 E.Deciphering the effect of 20 E on protein synthesis in fat body will be helpful to establish a new function of 20 E during insect metamorphosis.Our study aims to use Drosophila and Bombyx as research objects,and comprehensively use genetics,molecular biology,biochemistry and bioinformatics analysis to explore whether JH can directly act on the prothoracic gland(PG)where 20 E is biosynthesized and the molecular mechanism of JH inbibits 20 E biosynthesis through the key transcription factor Kr-h1,as well as the effect of 20 E on protein synthesis in fat body at cell,tissue and individual levels.The main findings obtained were as follows:1.JH and its signaling transcription factor Kr-h1 inhibit 20 E biosynthesis1)JH mimic treatment delays Drosophila and Bombyx pupariation and inhibits 20 E biosynthesis and the transcription of steroidogenic enzyme genesWe treated with JH mimic(JHM)resulted in a pupariation delay and significantly decreased ecdysone production in Drosophila and Bombyx.Using JHM to treat ex vivo-cultured brain–ring gland(RG)complex containing PG from Drosophila and PGs from Bombyx,all steroidogenic enzyme genes were also transcriptionally reduced after JHM treatment.This indicated that JH can directly act on the insect PG and inhibit the 20 E biosynthesis.2)JH signal transcription factor Kr-h1 is expressed in PG as 20 E biosynthetic organ in Drosophila and BombyxWe further found that JH signaling pathway gene BmKr-h1 was high in the early stage of larval instar and decreased in late stage,which was an opposite trend with 20 E titer during development stage.RNA in situ hybridization and immunofluorescence experiment in Drosophila PG revealed that the expression of DmKr-h1 was relatively high at the early stage of the third instar,then decreased,and it was almost undetectable before developmental transitions,which were completely opppsite to the expression pattern of the rate-limiting enzyme DmSpok in 20 E biosynthetic pathway.3)The change of DmKr-h1 expression in Drosophila PG disrupt pupariation,20 E biosynthesis and the expression of steroidogenic enzyme genesTo investigated the effects DmKr-h1 expression change on 20 E biosynthesis,we performed RNAi-mediated knockdown of DmKr-h1 expression in Drosophila PG by using PG-specific Phm-Gal4 driver,which resulted in precocious pupariation,increased ecdysone production,and elevated the expression of the 20 E response gene DmE75 B in the fat body as a 20 E targeting tissue,as well as the expression of steroidogenic enzyme genes in PG.Besides,the effect of DmKr-h1 mutant reduced DmKr-h1 levels in the pre-thymus of Drosophila resulting in the same result.However,we used Phm-Gal4 to carry out PG-specific DmKr-h1 overexpression and observed that PG-specific DmKr-h1 overexpression caused developmental arrest at the L1 stage.In addition,following PG-specific DmKr-h1 overexpression,20 E production,DmE75 B expression in fat body,the transcription of steroidogenic enzyme genes,and DmSpok expression were significantly suppressed at 48 h after egg laying(AEL).Strikingly,the developmental L1 arrest caused by PG-specific DmKr-h1 overexpression could be rescued by feeding ecdysone or 20 E as an active derivative of ecdysone,and ～50% of L1-arrested larvae could develop to pupariation.4)Kr-h1 has no effect on DNA replication in Drosophila PG cellsWe also found that specific interference or overexpression of DmKr-h1 and DmKr-h1 mutaant in the PG of Drosophila does not affect DNA replication.However,at 96 h AEL,the size of the PG from Drosophila larvae with PG-specific DmKr-h1 overexpression is smaller than the control.Feeding exogenous 20 E to Drosophila larvae with DmKr-h1 overexpression in the PG recovered normal developmental transition and body size,as well as PG size comparable to the control.Taken together,our results indicated that Kr-h1-mediated JH signaling in Drosophila PG affect 20 E biosynthesis by not affecting the endoreplication in PG cells,instead by inhibiting the transcription of steroidogenic enzyme genes.2.Molecular mechanism underlying Kr-h1 inhibition on the transcription of steroidogenic enzyme genes1)Kr-h1 negatively regulates the activities of the promoters for steroidogenic enzyme genes in Drosophila and BombyxTo explore the molecular mechanism underlying the zinc finger transcription factor Kr-h1 inhibition on the transcription of steroidogenic enzyme genes,we cloned the potential promoter region of the steroidogenic enzyme genes in Drosophila and Bombyx.Further analysis revealed that the promoters for all the steroidogenic enzyme genes contained core motif GACCTNNNAA within potential KBS.The luciferase reporter assays results showed that JHM treatment or Kr-h1 overexpression significantly reduced the activities of most of the promoters for the steroidogenic enzymes genes.2)Kr-h1 inhibits DmSpok/BmSpo promoter activity via KBSFurther analysis revealed that DmKr-h1 overexpression significantly inhibited the activity of DmSpok promoter truncations that include the distal and proximal KBSs,and this inhibition disappeared when the proximal KBS was truncated.Besides,either deletion or mutation of the proximal KBS in the DmSpok promoter resulted in a loss of DmKr-h1 inhibition on promoter activity.In Bombyx,BmKr-h1 inhibition on Bombyx BmSpo promoter activity was also eliminated following deletion or mutation of the proximal KBS in the BmSpo promoter.These results indicated that the KBS of the DmSpok/BmSpo promoter region is a key motif for Kr-h1 inhibition on their activities.3)Kr-h1 directly binds to the KBS site on the DmSpok/BmSpo promoterWe performed a chromatin immunoprecipitation PCR(ChIP-PCR)experiment,which demonstrated that DmKr-h1 could specifically immunoprecipitate DNA regions covering the proximal KBS of the DmSpok promoter.Also,BmKr-h1 could directly bind to the proximal KBS motif in the BmSpo promoter by ChIP-PCR.In additon,we expressed and purified the bioactive supernatant protein of recombinant DmKr-h1 and BmKr-h1 in prokaryotes,respectively.Subsequently,we carried out electrophoretic mobility shift assays(EMSA)experiments by using a specific biotinylated probe covering the DNA sequence of the KBS.The results showed that the purified recombinant DmKr-h1 protein can bind to the biotinylated probe.Furthermore,EMSA examination in Bombyx also revealed that BmKr-h1 could directly bind to the proximal KBS motif in the BmSpo promoter.These findings collectively suggest that Kr-h1 can directly bind to the KBS of DmSpok / BmSpo promoters.4)Kr-h1 induces DNA methylation in the DmSpok/BmSpo promoter regionWe further found that cytosines located near the proximal KBS in the DmSpok promoter were methylated following DmKr-h1 overexpression in Drosophila S2 cells by using the bisulfite sequencing PCR(BSP)approach.But,the methylation of these cytosines was eliminated by as addition of specific DNA methylation inhibitor 5-Aza-2,9-deoxycytidine(Aza).Further luciferase reporter assay confirmed that DmKr-h1/BmKr-h1 inhibition on transcriptional activity of DmSpok/BmSpo promoter was potently abolished by the treatment with Aza.Taken together,these results indicated that Kr-h1 inhibits the transcription of the steroidogenic enzymes gene by directly binding to the KBS of the promoter region of the steroidogenic enzymes gene and subsequently inducing DNA methylation of the promoter region.3.20 E inhibits protein synthesis in insects fat body1)20E inhibits protein synthesis levels in insects fat bodyInsect fat body functions as an important place for protein synthesis and energy metabolism.To elucidate the roles of 20 E on protein synthesis in insect fat body,we established a methodological system based on the puromycin(one of the protein synthesis inhibitor)to analyze protein synthesis levels in Drosophila and Bombyx fat body and cells.Further western blot and immunofluorescence analysis showed that the level of protein synthesis in fat body gradually decreased during larval-pupal metamorphosis in Drosophila and was not detectable during prepupal period.The protein synthesis level was also decreased in the Bombyx fat body during metamorphosis.In addition,the experiments in Drosophila S2 cells or Bombyx BmE cells as well as the fat body of Drosophila or Bombxy larvae at the early age of the last instar demonstrated that the level of nascent proteins were decreased after 20 E treatment.The overexpression of dominate negative form of the 20 E receptor EcR gene in Drosophila fat body by using fat body-specific CG-Gal4 driver increased the level of protein synthesis in Drosophila fat body.These data indicated that 20 E inhibits protein synthesis in insect fat body.2)20E inhibits ribosome levels in insects fat bodyThe number of ribosomes is an indicator of the protein synthesis level,and ribosomal RNA(rRNA)accounted for more than one third of the total RNA.Therefore,we used Q-PCR experiments to analyze the number of 18 S rRNA,an important component of ribosomes in Drosophila and Bombyx fat body.The results showed that the number of 18 S rRNA in Drosophila and Bombyx fat body significantly decreased during the metamorphosis.Similarly,20 E treatment reduced the expression of 18 S rRNA in Drosophila S2 cells or Bombyx BmE cells as well as the fat body of either Drosophila or Bomby larvae at the early age of the end instar.Conversely,the dominant negative regulation of DmEcR expression in Drosophila fat body significantly increased 18 S rRNA expression and total RNA levels.3)20E treatment reduces the nucleolus size in insect fat body cellsNucleolus is a key site for ribosome formation,and nucleolar size is closely related to the biosynthesis of both rRNA and protein.The nucleolus is obvious in the Drosophila fat body,so we analyzed nucleolar size by using nucleolar-labeled Fibrillarin staining.We have found that the expression of 20 E receptor EcR increased gradually while the size of nucleolus decreased significantly during metamorphosis in Drosophila fat body.In addition,20 E treatment on ex vitro cultured Drosophila fat body also reduced the nucleolar size.But dominant negative regulation of DmEcR expression caused the bigger nucleolus in fat body cells.In summary,20 E could inhibit protein synthesis by affecting the level of ribosome RNA and the size of the nucleolus in insect fat body.