Fzr regulates silk gland growth by promoting endoreplication and protein synthesis in the silkworm.

Silkworm silk gland cells undergo endoreplicating cycle and rapid growth during the larval period, and synthesize massive silk proteins for silk production. In this study, we demonstrated that a binary transgenic CRISPR/Cas9 approach-mediated Fzr mutation in silkworm posterior silk gland (PSG) cells caused an arrest of silk gland growth and a decrease in silk production. Mechanistically, PSG-specific Fzr mutation blocked endoreplication progression by inducing an expression dysregulation of several cyclin proteins and DNA replication-related regulators. Moreover, based on label-free quantitative proteome analysis, we showed in PSG cells that Fzr mutation-induced decrease in the levels of cyclin proteins and silk proteins was likely due to an inhibition of the ribosome biogenesis pathway associated with mRNA translation, and/or an enhance of the ubiquitin-mediated protein degradation pathway. Rbin-1 inhibitor-mediated blocking of ribosomal biogenesis pathway decreased DNA replication in PSG cells and silk production. Altogether, our results reveal that Fzr positively regulates PSG growth and silk production in silkworm by promoting endoreplication and protein synthesis in PSG cells.

Histone H3K27 methylation-mediated repression of Hairy regulates insect developmental transition by modulating ecdysone biosynthesis.

Insect development is cooperatively orchestrated by the steroid hormone ecdysone and juvenile hormone (JH). The polycomb repressive complex 2 (PRC2)-mediated histone H3K27 trimethylation (H3K27me3) epigenetically silences gene transcription and is essential for a range of biological processes, but the functions of H3K27 methylation in insect hormone action are poorly understood. Here, we demonstrate that H3K27 methylation-mediated repression of Hairy transcription in the larval prothoracic gland (PG) is required for ecdysone biosynthesis in Bombyx and Drosophila H3K27me3 levels in the PG are dynamically increased during the last larval instar. H3K27me3 reduction induced by the down-regulation of PRC2 activity via inhibitor treatment in Bombyx or PG-specific knockdown of the PRC2 component Su(z)12 in Drosophila diminishes ecdysone biosynthesis and disturbs the larval-pupal transition. Mechanistically, H3K27 methylation targets the JH signal transducer Hairy to repress its transcription in the PG; PG-specific knockdown or overexpression of the Hairy gene disrupts ecdysone biosynthesis and developmental transition; and developmental defects caused by PG-specific Su(z)12 knockdown can be partially rescued by Hairy down-regulation. The application of JH mimic to the PG decreases both H3K27me3 levels and Su(z)12 expression. Altogether, our study reveals that PRC2-mediated H3K27 methylation at Hairy in the PG during the larval period is required for ecdysone biosynthesis and the larval-pupal transition and provides insights into epigenetic regulation of the crosstalk between JH and ecdysone during insect development.

Wds-Mediated H3K4me3 Modification Regulates Lipid Synthesis and Transport in Drosophila.

Lipid homeostasis is essential for insect growth and development. The complex of proteins associated with Set 1 (COMPASS)-catalyzed Histone 3 lysine 4 trimethylation (H3K4me3) epigenetically activates gene transcription and is involved in various biological processes, but the role and molecular mechanism of H3K4me3 modification in lipid homeostasis remains largely unknown. In the present study, we showed in Drosophila that fat body-specific knockdown of will die slowly (Wds) as one of the COMPASS complex components caused a decrease in lipid droplet (LD) size and triglyceride (TG) levels. Mechanistically, Wds-mediated H3K4me3 modification in the fat body targeted several lipogenic genes involved in lipid synthesis and the Lpp gene associated with lipid transport to promote their expressions; the transcription factor heat shock factor (Hsf) could interact with Wds to modulate H3K4me3 modification within the promoters of these targets; and fat body-specific knockdown of Hsf phenocopied the effects of Wds knockdown on lipid homeostasis in the fat body. Moreover, fat body-specific knockdown of Wds or Hsf reduced high-fat diet (HFD)-induced oversized LDs and high TG levels. Altogether, our study reveals that Wds-mediated H3K4me3 modification is required for lipid homeostasis during Drosophila development and provides novel insights into the epigenetic regulation of insect lipid metabolism.

A novel transcriptional cascade is involved in Fzr-mediated endoreplication.

Endoreplication, known as endocycle, is a variant of the cell cycle that differs from mitosis and occurs in specific tissues of different organisms. Endoreplicating cells generally undergo multiple rounds of genome replication without chromosome segregation. Previous studies demonstrated that Drosophila fizzy-related protein (Fzr) and its mammalian homolog Cdh1 function as key regulators of endoreplication entrance by activating the anaphase-promoting complex/cyclosome to initiate the ubiquitination and subsequent degradation of cell cycle factors such as Cyclin B (CycB). However, the molecular mechanism underlying Fzr-mediated endoreplication is not completely understood. In this study, we demonstrated that the transcription factor Myc acts downstream of Fzr during endoreplication in Drosophila salivary gland. Mechanistically, Fzr interacts with chromatin-associated histone H2B to enhance H2B ubiquitination in the Myc promoter and promotes Myc transcription. In addition to negatively regulating CycB transcription, the Fzr-ubiquitinated H2B (H2Bub)-Myc signaling cascade also positively regulates the transcription of the MCM6 gene that is involved in DNA replication by directly binding to specific motifs within their promoters. We further found that the Fzr-H2Bub-Myc signaling cascade regulating endoreplication progression is conserved between insects and mammalian cells. Altogether, our work uncovers a novel transcriptional cascade that is involved in Fzr-mediated endoreplication.

Krüppel homolog 1 represses insect ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes.

In insects, juvenile hormone (JH) and the steroid hormone ecdysone have opposing effects on regulation of the larval-pupal transition. Although increasing evidence suggests that JH represses ecdysone biosynthesis during larval development, the mechanism underlying this repression is not well understood. Here, we demonstrate that the expression of the Krüppel homolog 1 (Kr-h1), a gene encoding a transcription factor that mediates JH signaling, in ecdysone-producing organ prothoracic gland (PG) represses ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes in both Drosophila and Bombyx Application of a JH mimic on ex vivo cultured PGs from Drosophila and Bombyx larvae induces Kr-h1 expression and inhibits the transcription of steroidogenic enzymes. In addition, PG-specific knockdown of Drosophila Kr-h1 promotes-while overexpression hampers-ecdysone production and pupariation. We further find that Kr-h1 inhibits the transcription of steroidogenic enzymes by directly binding to their promoters to induce promoter DNA methylation. Finally, we show that Kr-h1 does not affect DNA replication in Drosophila PG cells and that the reduction of PG size mediated by Kr-h1 overexpression can be rescued by feeding ecdysone. Taken together, our data indicate direct and conserved Kr-h1 repression of insect ecdysone biosynthesis in response to JH stimulation, providing insights into mechanisms underlying the antagonistic roles of JH and ecdysone.

Endocrine Regulation of Energy Balance by Drosophila TGF-ß/Activins.

The Transforming growth factor beta (TGF-ß) family of secreted proteins regulates a variety of key events in normal development and physiology. In mammals, this family, represented by 33 ligands, including TGF-ß, activins, nodal, bone morphogenetic proteins (BMPs), and growth and differentiation factors (GDFs), regulate biological processes as diverse as cell proliferation, differentiation, apoptosis, metabolism, homeostasis, immune response, wound repair, and endocrine functions. In Drosophila, only 7 members of this family are present, with 4 TGF-ß/BMP and 3 TGF-ß/activin ligands. Studies in the fly have illustrated the role of TGF-ß/BMP ligands during embryogenesis and organ patterning, while the TGF-ß/activin ligands have been implicated in the control of wing growth and neuronal functions. In this review, we focus on the emerging roles of Drosophila TGF-ß/activins in inter-organ communication via long-distance regulation, especially in systemic lipid and carbohydrate homeostasis, and discuss findings relevant to metabolic diseases in humans.

Activin signaling mediates muscle-to-adipose communication in a mitochondria dysfunction-associated obesity model.

Mitochondrial dysfunction has been associated with obesity and metabolic disorders. However, whether mitochondrial perturbation in a single tissue influences mitochondrial function and metabolic status of another distal tissue remains largely unknown. We analyzed the nonautonomous role of muscular mitochondrial dysfunction in Drosophila Surprisingly, impaired muscle mitochondrial function via complex I perturbation results in simultaneous mitochondrial dysfunction in the fat body (the fly adipose tissue) and subsequent triglyceride accumulation, the major characteristic of obesity. RNA-sequencing (RNA-seq) analysis, in the context of muscle mitochondrial dysfunction, revealed that target genes of the TGF-ß signaling pathway were induced in the fat body. Strikingly, expression of the TGF-ß family ligand, Activin-ß (Actß), was dramatically increased in the muscles by NF-κB/Relish (Rel) signaling in response to mitochondrial perturbation, and decreasing Actß expression in mitochondrial-perturbed muscles rescued both the fat body mitochondrial dysfunction and obesity phenotypes. Thus, perturbation of muscle mitochondrial activity regulates mitochondrial function in the fat body nonautonomously via modulation of Activin signaling.

Transcription factor E93 regulates wing development by directly promoting Dpp signaling in Drosophila.

Transcription factor E93 is a steroid hormone ecdysone early response gene and plays crucial roles in both the degradation of larval tissues and the formation of adult organs during insect metamorphosis with the prepupal-pupal-adult transition. However, the molecular mechanism underlying E93 regulation is poorly understood. In this study, we found that specific knockdown of the E93 gene in the Drosophila wing disrupted wing development. Analyzing ChIP-seq signals for E93 in Drosophila wing identified that the decapentaplegic (Dpp) gene was a potential downstream target of E93. ChIP-PCR analysis and dual-luciferase reporter assay confirmed that E93 could bind to the Dpp promoter and enhanced its activity. Furthermore, the expressions of Dpp and other components in the Dpp signaling pathway were upregulated following E93 overexpression in Drosophila S2 cells but were decreased after E93 knockdown in the wing. Moreover, the impairment of the Dpp signaling pathway phenocopied the defects of E93 knockdown on wing development. Taken together, our results suggest that E93 modulates the Dpp signaling pathway to regulate wing development during Drosophila metamorphosis.

Protein kinase A-mediated phosphorylation of the Broad-Complex transcription factor in silkworm suppresses its transcriptional activity.

The insect-specific transcription factor Broad-Complex (BR-C) is transcriptionally activated by the steroid 20-hydroxyecdysone (20E) and regulates the expression of many target genes involved in insect growth and development. However, although the transcriptional regulation of BR-C proteins has been well studied, how BR-C is regulated at post-transcription and -translation levels is poorly understood. To this end, using liquid chromatography-tandem mass spectrometry analysis, we identified residue Ser-186 as a phosphorylation site of BR-C in silkworm. Site-directed mutagenesis and treatment with specific kinase activators and inhibitors indicated that the Ser-186 residue in silkworm BR-C is phosphorylated by protein kinase A (PKA). Immunostaining assays disclosed that PKA-mediated phosphorylation of silkworm BR-C has no effect on its nuclear import. However, luciferase reporter analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation revealed that the PKA phosphorylation event suppresses the transcriptional activation of silkworm BR-C target genes and that this inhibition was caused by repression of BR-C binding to its DNA targets. Of note, both in vitro and ex vivo experiments disclosed that a continuous 20E signal inhibits the PKA-mediated BR-C phosphorylation and also the cAMP/PKA pathway, indicating that 20E's inhibitory effect on PKA-mediated phosphorylation of silkworm BR-C contributes to maintaining BR-C transcriptional activity. In conclusion, our findings indicate that PKA-mediated phosphorylation inhibits silkworm BR-C activity by interfering with its binding to DNA and that 20E signaling relieves PKA-mediated phosphorylation of BR-C, thereby maintaining its transcriptional activity.

Comparative Transcriptome Analysis Provides Novel Insight into Morphologic and Metabolic Changes in the Fat Body during Silkworm Metamorphosis.

The fat body plays key roles in energy storage and utilization as well as biosynthetic and metabolic activities in insects. During metamorphosis from larva to pupa, the fat body undergoes dramatic changes in morphology and metabolic processes. However, the genetic basis underlying these changes has not been completely understood. In this study, the authors performed a time-course transcriptome analysis of the fat body during silkworm metamorphosis using RNA-sequencing. A total of 5217 differentially expressed genes (DEGs) were identified in the fat body at different developmental time points. DEGs involved in lipid synthesis and degradation were highly expressed at the third day of the last larval instar and during the prepupal-pupal transition, respectively. DEGs involved in the ecdysone signaling and bone morphogenetic protein (BMP) signaling pathways that modulate organ development exhibited a high expression level during the fat body remodeling process from prepupa to pupa. Intriguingly, the RNA interference-mediated knockdown of either decapentaplegic (Dpp) or protein 60A (Gbb), two DEGs involved in the BMP signaling pathway, inhibited fat body dissociation but promoted lipid mobilization, suggesting that the BMP signaling pathway not only is required for fat body remodeling, but also moderately inhibits lipid mobilization to ensure an appropriate lipid supply during the pupal-adult transition. In conclusion, the comparative transcriptome analysis provides novel insight into morphologic and metabolic changes in the fat body during silkworm metamorphosis.

The homeodomain transcription factors antennapedia and POU-M2 regulate the transcription of the steroidogenic enzyme gene Phantom in the silkworm.

The steroid hormone ecdysone, which controls insect molting and metamorphosis, is synthesized in the prothoracic gland (PG), and several steroidogenic enzymes that are expressed specifically in the PG are involved in ecdysteroidogenesis. In this study, we identified new regulators that are involved in the transcriptional control of the silkworm steroidogenic enzyme genes. In silico analysis predicted several potential cis-regulatory elements (CREs) for the homeodomain transcription factors Antennapedia (Antp) and POU-M2 in the proximal promoters of steroidogenic enzyme genes. Antp and POU-M2 are expressed dynamically in the PG during larval development, and their overexpression in silkworm embryo-derived (BmE) cells induced the expression of steroidogenic enzyme genes. Importantly, luciferase reporter analyses, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays revealed that Antp and POU-M2 promote the transcription of the silkworm steroidogenic enzyme gene Phantom (Phm) by binding directly to specific motifs within overlapping CREs in the Phm promoter. Mutations of these CREs in the Phm promoter suppressed the transcriptional activities of both Antp and POU-M2 in BmE cells and decreased the activities of mutated Phm promoters in the silkworm PG. In addition, pulldown and co-immunoprecipitation assays demonstrated that Antp can interact with POU-M2. Moreover, RNA interference-mediated down-regulation of either Antp or POU-M2 during silkworm wandering not only decreased the ecdysone titer but also led to the failure of metamorphosis. In summary, our results suggest that Antp and POU-M2 coordinate the transcription of the silkworm Phm gene directly, indicating new roles for homeodomain proteins in regulating insect ecdysteroidogenesis.

Ecdysone receptor (EcR) is involved in the transcription of cell cycle genes in the silkworm.

EcR (ecdysone receptor)-mediated ecdysone signaling pathway contributes to regulate the transcription of genes involved in various processes during insect development. In this work, we detected the expression of EcR gene in silkworm ovary-derived BmN4 cells and found that EcR RNAi result in an alteration of cell shape, indicating that EcR may orchestrate cell cycle progression. EcR RNAi and EcR overexpression analysis revealed that in the cultured BmN4 cells, EcR respectively promoted and suppressed the transcription of E2F-1 and CycE, two genes controlling cell cycle progression. Further examination demonstrated that ecdysone application in BmN4 cells not only changed the transcription of these two cell cycle genes like that under EcR overexpression, but also induced cell cycle arrest at G2/M phase. In vivo analysis confirmed that E2F-1 expression was elevated in silk gland of silkworm larvae after ecdysone application, which is same as its response to ecdysone in BmN4 cells. However, ecdysone also promotes CycE transcription in silk gland, and this is converse with the observation in BmN4 cells. These results provide new insights into understanding the roles of EcR-mediated ecdysone signaling in the regulation of cell cycle.

Homeodomain Protein Scr Regulates the Transcription of Genes Involved in Juvenile Hormone Biosynthesis in the Silkworm.

The silkworm Dominant trimolting (Moltinism, M³) mutant undergoes three larval molts and exhibits precocious metamorphosis. In this study, we found that compared with the wild-type (WT) that undergoes four larval molts, both the juvenile hormone (JH) concentration and the expression of the JH-responsive gene Krüppel homolog 1 (Kr-h1) began to be greater in the second instar of the M³ mutant. A positional cloning analysis revealed that only the homeodomain transcription factor gene Sex combs reduced (Scr) is located in the genomic region that is tightly linked to the M³ locus. The expression level of the Scr gene in the brain-corpora cardiaca-corpora allata (Br-CC-CA) complex, which controls the synthesis of JH, was very low in the final larval instar of both the M³ and WT larvae, and exhibited a positive correlation with JH titer changes. Importantly, luciferase reporter analysis and electrophoretic mobility shift assay (EMSA) demonstrated that the Scr protein could promote the transcription of genes involved in JH biosynthesis by directly binding to the cis-regulatory elements (CREs) of homeodomain protein on their promoters. These results conclude that the homeodomain protein Scr is transcriptionally involved in the regulation of JH biosynthesis in the silkworm.

Ecdysone signaling at metamorphosis triggers apoptosis of Drosophila abdominal muscles.

One of the most dramatic examples of programmed cell death occurs during Drosophila metamorphosis, when most of the larval tissues are destroyed in a process termed histolysis. Much of our understanding of this process comes from analyses of salivary gland and midgut cell death. In contrast, relatively little is known about the degradation of the larval musculature. Here, we analyze the programmed destruction of the abdominal dorsal exterior oblique muscle (DEOM) which occurs during the first 24h of metamorphosis. We find that ecdysone signaling through Ecdysone receptor isoform B1 is required cell autonomously for the muscle death. Furthermore, we show that the orphan nuclear receptor FTZ-F1, opposed by another nuclear receptor, HR39, plays a critical role in the timing of DEOM histolysis. Finally, we show that unlike the histolysis of salivary gland and midgut, abdominal muscle death occurs by apoptosis, and does not require autophagy. Thus, there is no set rule as to the role of autophagy and apoptosis during Drosophila histolysis.

RNAi silencing of the SoxE gene suppresses cell proliferation in silkworm BmN4 cells.

The transcription factor SoxE is mainly expressed in the gonad and involved in the regulation of gonad development and sex determination in animals. Here, we used the silkworm ovary-derived BmN4-SID1 cell line to survey the roles of the silkworm SoxE protein (BmSoxE) and predict its candidate binding targets. RNAi-mediated silencing of BmSoxE expression suppressed cell proliferation in BmN4-SID1 cells. A further cell cycle analysis revealed that this inhibition of cell proliferation was largely due to cell cycle arrest in G1 phase when BmSoxE expression was blocked in BmN4-SID1 cells. Genome-wide microarray expression analyses demonstrated that the expression levels of a set of genes were significantly altered following BmSoxE RNAi. More than half of these genes contained conserved binding sites for HMG box domain of the Sox proteins and were predicted to be candidate binding targets for BmSoxE. Importantly, some of the candidate targets may be associated with the effect of BmSoxE on cell proliferation. Several candidate target genes showed gonad-specific expression in silkworm larvae. Taken together, these data demonstrate that BmSoxE is required for cell proliferation in silkworm BmN4-SID1 cells and provide valuable information for further investigations of the molecular control exerted by the BmSoxE protein over cell proliferation and gonad development in the silkworm.

Genome-wide comparison of genes involved in the biosynthesis, metabolism, and signaling of juvenile hormone between silkworm and other insects.

Juvenile hormone (JH) contributes to the regulation of larval molting and metamorphosis in insects. Herein, we comprehensively identified 55 genes involved in JH biosynthesis, metabolism and signaling in the silkworm (Bombyx mori) as well as 35 in Drosophila melanogaster, 35 in Anopheles gambiae, 36 in Apis mellifera, 47 in Tribolium castaneum, and 44 in Danaus plexippus. Comparative analysis showed that each gene involved in the early steps of the mevalonate (MVA) pathway, in the neuropeptide regulation of JH biosynthesis, or in JH signaling is a single copy in B. mori and other surveyed insects, indicating that these JH-related pathways or steps are likely conserved in all surveyed insects. However, each gene participating in the isoprenoid branch of JH biosynthesis and JH metabolism, together with the FPPS genes for catalyzing the final step of the MVA pathway of JH biosynthesis, exhibited an obvious duplication in Lepidoptera, including B. mori and D. plexippus. Microarray and real-time RT-PCR analysis revealed that different copies of several JH-related genes presented expression changes that correlated with the dynamics of JH titer during larval growth and metamorphosis. Taken together, the findings suggest that duplication-derived copy variation of JH-related genes might be evolutionarily associated with the variation of JH types between Lepidoptera and other insect orders. In conclusion, our results provide useful clues for further functional analysis of JH-related genes in B. mori and other insects.

Peroxisomal ERK mediates Akh/glucagon action and glycemic control.

The enhanced response of glucagon and its Drosophila homolog, adipokinetic hormone (Akh), leads to high-caloric-diet-induced hyperglycemia across species. While previous studies have characterized regulatory components transducing linear Akh signaling promoting carbohydrate production, the spatial elucidation of Akh action at the organelle level still remains largely unclear. In this study, we find that Akh phosphorylates extracellular signal-regulated kinase (ERK) and translocates it to peroxisome via calcium/calmodulin-dependent protein kinase II (CaMKII) cascade to increase carbohydrate production in the fat body, leading to hyperglycemia. The mechanisms include that ERK mediates fat body peroxisomal conversion of amino acids into carbohydrates for gluconeogenesis in response to Akh. Importantly, Akh receptor (AkhR) or ERK deficiency, importin-associated ERK retention from peroxisome, or peroxisome inactivation in the fat body sufficiently alleviates high-sugar-diet-induced hyperglycemia. We also observe mammalian glucagon-induced hepatic ERK peroxisomal translocation in diabetic subjects. Therefore, our results conclude that the Akh/glucagon-peroxisomal-ERK axis is a key spatial regulator of glycemic control.

Decapentaplegic retards lipolysis during metamorphosis in Bombyx mori and Drosophila melanogaster.

Insect morphogen decapentaplegic (Dpp) functions as one of the key extracellular ligands of the Bone Morphogenetic Protein (BMP) signaling pathway. Previous studies in insects mainly focused on the roles of Dpp during embryonic development and the formation of adult wings. In this study, we demonstrate a new role for Dpp in retarding lipolysis during metamorphosis in both Bombyx mori and Drosophila melanogaster. CRISPR/Cas9-mediated mutation of Bombyx dpp causes pupal lethality, induces an excessive and premature breakdown of lipids in the fat body, and upregulates the expressions of several lipolytic enzyme genes, including brummer (bmm), lipase 3 (lip3), and hormone-sensitive lipase (hsl), and lipid storage droplet 1 (lsd1), a lipid droplets (LD)-associated protein gene. Further investigation in Drosophila reveals that salivary gland-specific knockdown of the dpp gene and fat body-specific knockdown of Mad involved in Dpp signaling phenocopy the effects of Bombyx dpp mutation on pupal development and lipolysis. Taken together, our data indicate that the Dpp-mediated BMP signaling in the fat body maintains lipid homeostasis by retarding lipolysis, which is necessary for pupa-adult transition during insect metamorphosis.

SilkDB v2.0: a platform for silkworm (Bombyx mori ) genome biology.

The SilkDB is an open-access database for genome biology of the silkworm (Bombyx mori). Since the draft sequence was completed and the SilkDB was first released 5 years ago, we have collaborated with other groups to make much remarkable progress on silkworm genome research, such as the completion of a new high-quality assembly of the silkworm genome sequence as well as the construction of a genome-wide microarray to survey gene expression profiles. To accommodate these new genomic data and house more comprehensive genomic information, we have reconstructed SilkDB database with new web interfaces. In the new version (v2.0) of SilkDB, we updated the genomic data, including genome assembly, gene annotation, chromosomal mapping, orthologous relationship and experiment data, such as microarray expression data, Expressed Sequence Tags (ESTs) and corresponding references. Several new tools, including SilkMap, Silkworm Chromosome Browser (SCB) and BmArray, are developed to access silkworm genomic data conveniently. SilkDB is publicly available at the new URL of http://www.silkdb.org.