RESUMO
Crassostrea angulata, a major shellfish cultivated in Southern China, has experienced a notable surge in commercial value in recent years. Understanding the molecular mechanisms governing their reproductive processes holds significant implications for advancing aquaculture practices. In this study, we cloned the orphan nuclear receptor gene, Fushi Tarazu transcription factor 1 (FTZ-F1), of C. angulata and investigated its functional role in the gonadal development. The full-length cDNA of FTZ-F1 spans 2357 bp and encodes a protein sequence of 530 amino acids. Notably, the amino acid sequence of FTZ-F1 in C. angulata shares remarkable similarity with its homologues in other species, particularly in the DNA-binding region (>90%) and ligand-binding region (>44%). In C. angulata, the highest expression level of FTZ-F1 was observed in the ovary, exhibiting more than a 200-fold increase during the maturation stage compared to the initiation stage (P < 0.001). Specifically, FTZ-F1 was mainly expressed in the follicular cells surrounding the oocytes of C. angulata. Upon inhibiting FTZ-F1 gene expression in C. angulata through RNA interference (RNAi), a substantial reduction in the expression of genes involved in the synthesis of sex steroids in the gonads, including 3ß-HSD, Cyp17, and follistatin, was observed. In addition, estradiol (E2) and testosterone (T) levels also showed a decrease upon FTZ-F1 silencing, resulting in a delayed gonadal development. These results indicate that FTZ-F1 acts as a steroidogenic factor, participating in the synthesis and regulation of steroid hormones and thus playing an important role in the reproductive and endocrine systems within oysters.
Assuntos
Crassostrea , Gônadas , Fatores de Transcrição , Animais , Crassostrea/genética , Crassostrea/crescimento & desenvolvimento , Crassostrea/metabolismo , Gônadas/metabolismo , Gônadas/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Feminino , Sequência de Aminoácidos , Regulação da Expressão Gênica no Desenvolvimento , Filogenia , Clonagem Molecular , Hormônios Esteroides Gonadais/metabolismo , Hormônios Esteroides Gonadais/biossíntese , Ovário/metabolismo , Ovário/crescimento & desenvolvimento , Esteroides/metabolismo , Esteroides/biossínteseRESUMO
Fushi-tarazu factor 1 (FTZ-F1) is a class of transcription factors belonging to the nuclear receptor superfamily and an important molting regulator in insects; however, its detailed function in the molting process of Locusta migratoria is still unclear. This study identified two FTZ-F1 transcripts (LmFTZ-F1-X1 and LmFTZ-F1-X2) in L. migratoria. The classical domains of FTZ-F1 were present in their protein sequences and distinguished based on their variable N-terminal domains. Reverse-transcription quantitative polymerase chain reaction analysis revealed that LmFTZ-F1-X1 and LmFTZ-F1-X2 were highly expressed in the integument. RNA interference (RNAi) was used to explore the function of LmFTZ-F1s in the molting of the third-instar nymph. Separate LmFTZ-F1-X1 or LmFTZ-F1-X2 silencing did not affect the normal development of third-instar nymphs; however, the simultaneous RNAi of LmFTZ-F1-X1 and LmFTZ-F1-X2 caused the nymphs to be trapped in the third instar stage and finally die. Furthermore, the hematoxylin-eosin and chitin staining of the cuticle showed that the new cuticles were thickened after silencing the LmFTZ-F1s compared to the controls. RNA-seq analysis showed that genes encoding four cuticle proteins, two chitin synthesis enzymes, and cytochrome P450 303a1 were differentially expressed between dsGFP- and dsLmFTZ-F1s-injected groups. Taken together, LmFTZ-F1-X1 and LmFTZ-F1-X2 are involved in the ecdysis of locusts, possibly by regulating the expression of genes involved in cuticle formation, chitin synthesis, and other key molting processes.
RESUMO
The nuclear receptors HR3 and FTZ-F1 are highly conserved and function to regulate molting and reproduction in both hemimetabolous and holometabolous insects. However, their roles in Nilaparvata lugens are largely unknown. In the present study, we discover that NlHR3 and NlFTZ-F1 are activated in the nymph stages by ecdysone signaling. Transcription disruption of NlHR3 and NlFTZ-F1 expression prevents nymph ecdysis and metamorphosis, which leads to abnormal appearance, malformed ovaries, and lethal phenotypes. In addition, we demonstrate that NlHR3 and NlFTZ-F1 regulate molting and reproduction by interacting with the intrinsic 20E and JH signaling pathways. Our work offers a deep insight into the action mechanisms of HR3 and FTZ-F1 in insects. Moreover, NlHR3 and NlFTZ-F1 could properly be exploited as potential target genes for developing RNAi-based pesticides to control N. lugens.
RESUMO
The PRDM family transcription repressor Blimp-1 is present in almost all multicellular organisms and plays important roles in various developmental processes. This factor has several conserved motifs among different species, but the function of each motif is unclear. Drosophila Blimp-1 plays an important role in determining pupation timing by acting as an unstable transcriptional repressor of the ßftz-f1 gene. Thus, Drosophila provides a good system for analyzing the molecular and biological functions of each region in Blimp-1. Various Blimp-1 mutants carrying deletions at the conserved motifs were induced under the control of the heat shock promoter in prepupae, and the expression patterns of ßFTZ-F1 and Blimp-1 and pupation timing were observed. The results showed that the regions with strong and weak repressor functions exist within the proline-rich middle section of the factor and near the N-terminal conserved motif, respectively. Rapid degradation was supported by multiple regions that were mainly located in a large proline-rich region. Results revealed that pupation timing was affected by the repression ability and stability of Blimp-1. This suggests that both the repression function and instability of Blimp-1 are indispensable for the precise determination of pupation timing.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismoRESUMO
Vitellogenesis in crustaceans is controlled by several steroid hormones. In humans, the expression of SF-1, a gene that regulates gonadal development and the synthesis of steroid hormones, is affected by DDX20. However, how the homologous gene FTZ-F1 is regulated by DDX20 and its association with vitellogenesis remains unknown in the mud crab Scylla paramamosain. In this study, SpDDX20 and SpFTZ-F1 were identified in the transcriptome of mature ovarian tissue from the mud crab. qRT-PCR results revealed that the expression levels of SpFTZ-F1 and SpVTG in the ovaries of crab in the experimental group injected with dsDDX20 (EO) were significantly higher (P < 0.05) than those in the negative control group injected with dsEGFP (NO) and the blank control group injected with SPSS (BO). The differentially expressed genes (DEGs) identified by comparative transcriptome analysis of the EO group and NO group were enriched into five pathways related to ovarian steroidogenesis. The expression of CYP17, CYP3A4, CYP1A1 and 3ß-HSD were up-regulated in pathways related to steroid hormone production and biosynthesis. The expression of the INSR, IRS and PI3K genes in the insulin signaling pathway were significantly increased (P < 0.05). The expression level of the TGF-ß gene was up-regulated (P < 0.05) in the transforming growth factor pathway, whereas the expression level of the Smad2 gene was down-regulated (P < 0.05). The expression of GnRHR, GS, AC and PKA genes in the gonadotropin-releasing hormone signaling pathway were up-regulated. Our data provide a foundation for investigating the relationship between DDX20 and FTZ-F1 in the regulation of vitellogenin expression in S. paramamosain.
Assuntos
Braquiúros , Animais , Feminino , Proteína DEAD-box 20/genética , Proteína DEAD-box 20/metabolismo , Perfilação da Expressão Gênica , Hormônios/metabolismo , Interferência de RNA , RNA-Seq , Vitelogeninas/genética , Vitelogeninas/metabolismoRESUMO
Fushi Tarazu Factor 1 (FTZ-F1), a member of the nuclear receptor superfamily, is the downstream factor of 20-hydroxyecdysone signaling. In Drosophila melanogaster, alternative transcription start and splicing in the FTZ-F1 gene generate αFTZ-F1 and ßFTZ-F1 isoforms, which are vital for pair-rule segmentation in early embryogenesis and post-embryonic development, respectively. However, whether the same mRNA isoforms are present and exert the conservative roles remains to be clarified in other insects. In the present paper, we first mined the genomic data of representative insect species and unveiled that the same post-transcriptional processing in FTZ-F1 occurred in coleopterans, lepidopterans, dipterans and hymenopterans. Our expression data in Henosepilachna vigintioctopunctata, a serious polyphagous defoliator damaging a wide range of crops in Solanaceae and Cucurbitaceae, showed that both αFTZ-F1 and ßFTZ-F1 were actively transcribed throughout the development, from embryo to adult. The RNA interference-aided knockdown of both isoforms completely arrested larval ecdysis from the third to the fourth instar, in contrast to the depletion of either isoform. In contrast, silencing ßFTZ-F1, rather than αFTZ-F1, severely impaired the larval-pupal transformation. We accordingly propose that both FTZ-F1 isoforms are essential but mutually interchangeable for larval-larval molting, while ßFTZ-F1 is necessary for the larval-pupal transition and sufficient to exert the role of both FTZ-F1s during larval-pupal metamorphosis in H. vigintioctopunctata.
RESUMO
The Tachinidae are natural enemies of many lepidopteran and coleopteran pests of crops, forests, and fruits. However, host-tachinid parasitoid interactions have been largely unexplored. In this study, we investigated the effects of tachinids on host biological traits, using Exorista japonica, a generalist parasitoid, and the silkworm Bombyx mori, its lepidopteran host, as models. We observed that E. japonica parasitoidism did not affect silkworm larval body weight gain and cocooning rate, whereas they caused shortened duration of molting from the final instar to the pupal stage, abnormal molting from larval to pupal stages, and a subsequent decrease in host emergence rate. Moreover, a decrease in juvenile hormone (JH) titer and an increase in 20-hydroxyecdysone (20E) titer in the hemolymph of parasitized silkworms occurred. The transcription of JH and 20E responsive genes was downregulated in mature parasitized hosts, but upregulated in parasitized prepupae while Fushi tarazu factor 1 (Ftz-f1), a nuclear receptor essential in larval ecdysis, showed dramatically reduced expression in parasitized hosts at both the mature and prepupal stages. Moreover, the transcriptional levels of BmFtz-f1 and its downstream target genes encoding cuticle proteins were downregulated in epidermis of parasitized hosts. Meanwhile, the content of trehalose was decreased in the hemolymph, while chitin content in the epidermis was increased in parasitized silkworm prepupae. These data reveal that the host may fine-tune JH and 20E synthesis to shorten developmental duration to combat established E. japonica infestation, while E. japonica silences BmFtz-f1 transcription to inhibit host pupation. This discovery highlights the novel target mechanism of tachinid parasitoids and provides new clues to host/tachinid parasitoid relationships.
RESUMO
MLE helicase is an evolutionarily conserved eukaryotic protein involved in a wide range of processes in the regulation of gene expression. Previously, we studied the properties of MLE on the Drosophila melanogaster model. In the present work we continue studying the functions of MLE and show that MLE interacts with the components of the SWI/SNF chromatin remodelling complex. To clarify the work of MLE, the profile of MLE binding to the regulatory elements of the SWI/SNF-dependent ftz-f1 gene was analyzed. The effect of MLE on the expression of this gene, the transcription of which occurs by the RNA polymerase II pausing mechanism, was investigated. The data obtained indicate the important role of MLE in ensuring timely activation and high level of expression of the ftz-f1 gene in vivo.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transcrição GênicaRESUMO
The Drosophila melanogaster Germ cell-expressed protein (GCE) is a paralog of the juvenile hormone (JH) receptor - Methoprene tolerant protein (MET). Both proteins mediate JH function, preventing precocious differentiation during D. melanogaster development. Despite that GCE and MET are often referred to as equivalent JH receptors, their functions are not fully redundant and show tissue specificity. Both proteins belong to the family of bHLH-PAS transcription factors. The similarity of their primary structure is limited to defined bHLH and PAS domains, while their long C-terminal fragments (GCEC, METC) show significant differences and are expected to determine differences in GCE and MET protein activities. In this paper we present the structural characterization of GCEC as a coil-like intrinsically disordered protein (IDP) with highly elongated and asymmetric conformation. In comparison to previously characterized METC, GCEC is less compacted, contains more molecular recognition elements (MoREs) and exhibits a higher propensity for induced folding. The NMR shifts perturbation experiment and pull-down assay clearly demonstrated that the GCEC fragment is sufficient to form an interaction interface with the ligand binding domain (LBD) of the nuclear receptor Fushi Tarazu factor-1 (FTZ-F1). Significantly, these interactions can force GCEC to adopt more fixed structure that can modulate the activity, structure and functions of the full-length receptor. The discussed relation of protein functionality with the structural data of inherently disordered GCEC fragment is a novel look at this protein and contributes to a better understanding of the molecular basis of the functions of the C-terminal fragments of the bHLH-PAS family. Video abstract.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Área Sob a Curva , Células COS , Chlorocebus aethiops , Simulação por Computador , Fluorescência , Hidrodinâmica , Espectroscopia de Ressonância Magnética , Ligação Proteica , Domínios Proteicos , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Insect metamorphosis originated around the middle Devonian, associated with the innovation of the final molt; this occurs after histolysis of the prothoracic gland (PG; which produces the molting hormone) in the first days of adulthood. We previously hypothesized that transcription factor E93 is crucial in the emergence of metamorphosis, because it triggers metamorphosis in extant insects. This work on the cockroach Blattella germanica reveals that E93 also plays a crucial role in the histolysis of PG, which fits the above hypothesis. Previous studies have shown that the transcription factor FTZ-F1 is essential for PG histolysis. We have found that FTZ-F1 depletion towards the end of the final nymphal instar downregulates the expression of E93, whereas E93-depleted nymphs molt to adults that retain a functional PG. Interestingly, these adults are able to molt again, which is exceptional in insects. The study of insects able to molt again in the adult stage may reveal clues about how nymphal epidermal cells definitively become adult cells, and whether it is possible to reverse this process.
Assuntos
Blattellidae/metabolismo , Proteínas de Insetos/deficiência , Metamorfose Biológica , Muda , Fatores de Transcrição/deficiência , Animais , Blattellidae/genética , Proteínas de Insetos/metabolismo , Ninfa/genética , Ninfa/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Culex pipiens pallens poses a serious threat to human health because of its widespread distribution, high carrier capacity for several arboviruses, frequent human-biting, and growth in urban environments. Pyrethroid insecticides have been mainly used to control adult Cx. pipiens pallens during outbreaks of mosquito-borne diseases. Unfortunately, mosquitoes have developed resistance, rendering the insecticides ineffective. Cuticular resistance is the primary mechanism of pyrethroid resistance. Previously, we revealed that cuticular protein of low complexity CPLCG5 is a major cuticular protein associated with deltamethrin resistance in Cx. pipiens pallens, which is enriched in the cuticle of mosquitoes' legs and participates in pyrethroid resistance by forming a rigid matrix. However, the regulatory mechanisms of its transcription remain unknown. RESULTS: First, qRT-PCR analysis revealed that the expression of FTZ-F1 (encoding Fushi tarazu-Factor 1) was ~ 1.8-fold higher in the deltamethrin-resistant (DR) than deltamethrin-susceptible (DS) strains at 24 h post-eclosion (PE) and ~ 2.2-fold higher in the DR strain than in the DS strain at 48 h PE. CPLCG5 and FTZ-F1 were co-expressed in the legs, indicating that they might play an essential role in the legs. Dual luciferase reporter assays and EMSA (electrophoretic mobility shift experiments) revealed that FTZ-F1 regulates the transcription of CPLCG5 by binding to the FTZ-F1 response element (- 870/- 864). Lastly, knockdown of FTZ-F1 not only affected CPLCG5 expression but also altered the cuticle thickness and structure of the legs, increasing the susceptibility of the mosquitoes to deltamethrin in vivo. CONCLUSIONS: The results revealed that FTZ-F1 regulates the expression of CPLCG5 by binding to the CPLCG5 promoter region, altering cuticle thickness and structure, and increasing the susceptibility of mosquitoes to deltamethrin in vivo. This study revealed part of the mechanism of cuticular resistance, providing a deeper understanding of insecticide resistance.
Assuntos
Culex/genética , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Inseticidas/farmacologia , Piretrinas/farmacologia , Fatores de Transcrição/metabolismo , Animais , Culex/efeitos dos fármacos , Feminino , Inativação Gênica , Proteínas de Insetos/genética , Nitrilas/farmacologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genéticaRESUMO
The NR5A-family nuclear receptors are highly conserved and function within the somatic follicle cells of the ovary to regulate folliculogenesis and ovulation in mammals; however, their roles in Drosophila ovaries are largely unknown. Here, we discover that Ftz-f1, one of the NR5A nuclear receptors in Drosophila, is transiently induced in follicle cells in late stages of oogenesis via ecdysteroid signaling. Genetic disruption of Ftz-f1 expression prevents follicle cell differentiation into the final maturation stage, which leads to anovulation. In addition, we demonstrate that the bHLH/PAS transcription factor Single-minded (Sim) acts as a direct target of Ftz-f1 to promote follicle cell differentiation/maturation and that Ftz-f1's role in regulating Sim expression and follicle cell differentiation can be replaced by its mouse homolog steroidogenic factor 1 (mSF-1). Our work provides new insight into the regulation of follicle maturation in Drosophila and the conserved role of NR5A nuclear receptors in regulating folliculogenesis and ovulation.
When animals reproduce, females release eggs from their ovaries which then get fertilized by sperm from males. Each egg needs to properly mature within a collection of cells known as follicle cells before it can be let go. As the egg matures, so do the follicle cells surrounding it, until both are primed and ready to discharge the egg from the ovary. Mammals rely on a protein called SF-1 to mature their follicle cells, but it is unclear how this process works. Most animals from humans to fruit flies release their eggs in a very similar way, using many of the same proteins and genes. For example, the gene for SF-1 in mammals is similar to a gene in fruit flies which codes for another protein called Ftz-f1. Since it is more straightforward to study ovaries in fruit flies than in humans and other mammals, investigating this protein could shed light on how follicle cells mature. However, it remained unclear whether Ftz-f1 plays a similar role to its mammalian counterpart. Here, Knapp et al. show that Ftz-f1 is present in the follicle cells of fruit flies and is required for them to properly mature. Ftz-f1 controlled this process by regulating the activity of another protein called Sim. Further experiments found that the gene that codes for the SF-1 protein in mice was able to compensate for the loss of Ftz-f1 and drive follicle cells to mature. Studying how ovaries release eggs is an essential part of understanding female fertility. This work highlights the similarities between these processes in mammals and fruit flies and may help us understand how ovaries work in humans and other mammals. In the future, the findings of Knapp et al. may lead to new therapies for infertility in females and other disorders that affect ovaries.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Proteínas Nucleares/genética , Folículo Ovariano/fisiologia , Ovulação , Fatores de Transcrição/genética , Animais , Drosophila melanogaster/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Transcrição GênicaRESUMO
BACKGROUND: Chlorantraniliprole (CAP) is widely used in agriculture and forestry to prevent and control pests. The effects of environmental CAP residue on non-target insect metamorphosis have not been reported. Our research aimed to investigate the sublethal effect of CAP on larva-pupa transformation in silkworm, and explore the mechanism of sublethal CAP exposure-mediated pupation metamorphosis defects. RESULT: Sublethal CAP exposure affected the growth and development of silkworm larvae and caused defects in pupation metamorphosis. After CAP exposure, formation the of prepupa procuticle, ecdysial membrane and new epidermis was inhibited. Also, the level of 20-hydroxyecdysone (20E) and mRNA levels of the 20E signaling pathway-related genes EcR, USP, E74, E75 and Ftz-f1 were significantly reduced. Moreover, genes involved in chitin synthesis, such as ChsA, CDA1 and CDA2, were downregulated. Injection of 20E led to the upregulation of chitin synthesis-related genes and increased formation of new epidermis in CAP-treated silkworm. However, injection of 20E failed to prevent downregulation of Ftz-f1 and the defects in pupation metamorphosis. CONCLUSION: Our results suggested that 20E is a target hormone of CAP exposure-mediated epidermis formation phenotype. Ftz-f1 was silenced by CAP and might be a direct target gene of sublethal CAP exposure. Our study provided new evidence of the effects of sublethal CAP exposure on insect development and metamorphosis. © 2020 Society of Chemical Industry.
Assuntos
Bombyx , Animais , Ecdisterona , Proteínas de Insetos , Larva , Metamorfose Biológica , ortoaminobenzoatosRESUMO
The insect steroid hormone ecdysone is a key regulator of oogenesis in Drosophila melanogaster and many other species. Despite the diversity of cellular functions of ecdysone in oogenesis, the molecular regulation of most ecdysone-responsive genes in ovarian cells remains largely unexplored. We performed a functional screen using the UAS/Gal4 system to identify non-coding cis-regulatory elements within well-characterized ecdysone-response genes capable of driving transcription of an indelible reporter in ovarian cells. Using two publicly available transgenic collections (the FlyLight and Vienna Tiles resources), we tested 62 Gal4 drivers corresponding to ecdysone-response genes EcR, usp, E75, br, ftz-f1 and Hr3. We observed 31 lines that were sufficient to drive a UAS-lacZ reporter in discrete cell populations in the ovary. Reporter expression was reproducibly observed in both somatic and germ cells at distinct stages of oogenesis, including those previously characterized as critical points of ecdysone regulation. Our studies identified several useful new reagents, adding to the UAS/Gal4 toolkit available for genetic analysis of oogenesis in Drosophila. Further, our study provides novel insight into the molecular regulation of ecdysone signaling in oogenesis.
Assuntos
Ecdisona/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Oogênese/genética , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Expressão Gênica/genética , Células Germinativas/metabolismo , Ovário/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
The takeout family genes encode relatively small proteins that are related to olfaction and are regulated by juvenile hormone (JH). The takeout genes modulate various physiological processes, such as behavioral plasticity in the migratory locust Locusta migraloria and feeding and courtship behaviors in Drosophila. Therefore, to understand the regulatory mechanism of these physiological processes, it is important to study the expressions of the takeout genes that are regulated by JH signaling. We used quantitative real-time PCR (qRTPCR) to study the role of JH signaling in the regulation of the takeout family genes in the brown planthopper Nilaparvata lugens (N. lugens) through the application of Juvenile hormone III (JHIII) and the down-regulation of key genes in the JH signaling pathway. The topical application of JHIII induced the expressions of most of the takeout family genes, and their expressions decreased 2 and 3 days after the JHIII application. Down-regulating the brown planthopper JH receptor NlMethoprene-tolerant (NlMet) and its interacting partners, NlTaiman (NlTai) and Nlß-Ftz-F1 (Nlß-Ftz), through RNAi, exhibited distinct effects on the expressions of the takeout family genes. The down-regulation of NlMet and NlKrüppel-homolog 1 (NlKr-h1) increased the expressions of the takeout family genes, while the down-regulation of the Met interacting partners NlTai and Nlß-Ftz decreased the expressions of most of the takeout family genes. This work advanced our understanding of the molecular function and the regulatory mechanism of JH signaling.
RESUMO
BACKGROUND: The synthesis of silk protein is controlled by hormones. The expression of the nuclear hormone Bmftz-f1 in the posterior silk gland (PSG) is induced by 20-hydroxyecdysone in vivo and in vitro. However, whether Bmftz-f1 regulates silk protein expression is unknown. METHODS: In our study, western blotting and quantitative polymerase chain reactions were conducted to detect the expression of FTZ-F1 in the PSG. Electrophoretic mobility shift, chromatin immunoprecipitation, far-western blotting, bimolecular fluorescence complementation, and dual luciferase reporter assays were performed to investigate the effect of FTZ-F1 on the fibH promoter. RESULTS: (1) The expression of the hormone receptor BmFTZ-F1 was opposite to that of fibH. It was highly expressed in the PSG during the fourth molting stage and the beginning of the fifth instar, and then its expression decreased gradually until it disappeared at the end of the fifth instar and the wandering stage. (2) We identified a FTZ-F1 response element 390bp upstream of the transcription initiation site of the fibH promoter. (3) BmFTZ-F1 interacted with the basic helix-loop-helix transcription factor Bmdimm. (4) BmFTZ-F1 down-regulated fibH promoter activity and counteracted the effect of Bmdimm on fibH expression. CONCLUSIONS: Integrating these results, we conclude that BmFTZ-F1 regulates the transcription of fibH by binding to the FTZ-F1 response element in the fibH promoter and counteracts the effect of Bmdimm on fibH expression. GENERAL SIGNIFICANCE: These findings provide new insights into the mechanism of regulation of the silk protein gene.
Assuntos
Bombyx/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibroínas/metabolismo , Proteínas de Insetos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Bombyx/genética , Proteínas de Ligação a DNA/genética , Ecdisterona/farmacologia , Glândulas Exócrinas/efeitos dos fármacos , Glândulas Exócrinas/metabolismo , Fibroínas/genética , Proteínas de Insetos/genética , Ligação Proteica , Elementos de Resposta , Fatores de Transcrição/genéticaRESUMO
Cancer cells have abnormal gene expression profiles; however, to what degree these are chaotic or driven by structured gene regulatory networks is often not known. Here we studied a model of Ras-driven invasive tumorigenesis in Drosophila epithelial tissues and combined in vivo genetics with next-generation sequencing and computational modeling to decipher the regulatory logic of tumor cells. Surprisingly, we discovered that the bulk of the tumor-specific gene expression is controlled by an ectopic network of a few transcription factors that are overexpressed and/or hyperactivated in tumor cells. These factors are Stat, AP-1, the bHLH proteins Myc and AP-4, the nuclear hormone receptor Ftz-f1, the nuclear receptor coactivator Taiman/SRC3, and Mef2. Notably, many of these transcription factors also are hyperactivated in human tumors. Bioinformatic analysis predicted that these factors directly regulate the majority of the tumor-specific gene expression, that they are interconnected by extensive cross-regulation, and that they show a high degree of co-regulation of target genes. Indeed, the factors of this network were required in multiple epithelia for tumor growth and invasiveness, and knockdown of several factors caused a reversion of the tumor-specific expression profile but had no observable effect on normal tissues. We further found that the Hippo pathway effector Yorkie was strongly activated in tumor cells and initiated cellular reprogramming by activating several transcription factors of this network. Thus, modeling regulatory networks identified an ectopic and ordered network of master regulators that control a large part of tumor cell-specific gene expression.
Assuntos
Carcinogênese , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição/genética , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Redes Reguladoras de Genes , Transdução de Sinais , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Ovarian development in crustaceans is characterized by rapid production of egg yolk protein in a process called vitellogenesis. In the present study, we investigated the involvement of a DEAD (Asp-Glu-Ala-Asp) box RNA helicase 20 (DDX20), forkhead transcription factor (FOXL)2 and fushi tarazu factor (FTZ-F)1 in the regulation of vitellogenesis. Based on ESTs from the testis and accessory gland of Eriocheir sinensis, we cloned the full-length cDNAs of foxl2 and fushitarazu factor 1 (ftz-f1), which include the conserved structural features of the forkhead family and nuclear receptor 5A (NR5A) family respectively. The expression of foxl2 mRNA surged at the mature stage of the ovary, when vtg mRNA swooped, suggesting that foxl2 negatively affects the vitellogenin (VTG) synthesis at this developmental stage. Etoposide (inducing germ cell apoptosis) treatment up-regulated FOXL2 and DDX20 at both the mRNA and the protein levels, primarily in the follicular cells as shown by immunofluorescence analysis. Furthermore, foxl2, ddx20 and ftz-f1 mRNA levels increased significantly with right-eyestalk ablation. Interactions between FOXL2 and DDX20 or FTZ-F1 were confirmed by co-immunoprecipitation and the forkhead domain of FOXL2 was identified as the specific structure interacting with FTZ-F1. In conclusion, FOXL2 down-regulates VTG expression by binding with DDX20 in regulation of follicular cell apoptosis and with FTZ-F1 to repress the synthesis of VTG at the mature stage. This report is the first to describe the molecular mechanism of VTG synthesis in E. sinensis and may shed new light on the regulation of cytochrome P450 enzyme by FOXL2 and FTZ-F1 in vitellogenesis.
Assuntos
Proteína DEAD-box 20/genética , Fatores de Transcrição Forkhead/biossíntese , Fator Esteroidogênico 1/genética , Vitelogeninas/biossíntese , Animais , Braquiúros/genética , Braquiúros/crescimento & desenvolvimento , Sistema Enzimático do Citocromo P-450/genética , Proteína DEAD-box 20/biossíntese , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA Mensageiro/genética , Fator Esteroidogênico 1/biossíntese , Vitelogeninas/genéticaRESUMO
During the final instar larvae of holometabolous insects, a pulse of 20-hydroxyecdysone (20E) and a drop in juvenile hormone (JH) trigger larval-pupal metamorphosis. In this study, two LdFTZ-F1 cDNAs (LdFTZ-F1-1 and LdFTZ-F1-2) were cloned in Leptinotarsa decemlineata. Both LdFTZ-F1-1 and LdFTZ-F1-2 were highly expressed just before or right after each molt, similar to the expression pattern of an ecdysteroidogenesis gene LdSHD. Ingestion of an ecdysteroid agonist halofenozide (Hal) enhanced LdFTZ-F1-1 and LdFTZ-F1-2 expression in the final larval instar. Conversely, a decrease in 20E by feeding a double-stranded RNA (dsRNA) against LdSHD repressed the expression. Moreover, Hal rescued the expression levels in LdSHD-silenced larvae. Thus, 20E peaks seem to induce the transcription of LdFTZ-F1s. Furthermore, ingesting dsLdFTZ-F1 from a common fragment of LdFTZ-F1-1 and LdFTZ-F1-2 successfully knocked down both LdFTZ-F1s, and impaired pupation. Finally, knocking down LdFTZ-F1s significantly repressed the transcription of three ecdysteroidogenesis genes, lowered 20E titer, and reduced the expression of two 20E receptor genes. Silencing LdFTZ-F1s also induced the expression of a JH biosynthesis gene, increased JH titer, but decreased the mRNA level of a JH early-inducible gene. Thus, LdFTZ-F1s are involved in the regulation of pupation by modulating 20E and JH titers and mediating their signaling pathways.
Assuntos
Besouros/fisiologia , Ecdisterona/metabolismo , Proteínas de Insetos/fisiologia , Metamorfose Biológica , Fatores de Transcrição/fisiologia , Animais , Proteínas de Ligação a DNA , Proteínas de Drosophila , Interferência de RNARESUMO
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.