BR-C Z4 and FoxJ interact to regulate expression of a chitin synthase gene CHSA-2b in the pupal wing discs of the silkworm, Bombyx mori

doi:10.1016/j.ibmb.2019.103264

Insect Biochemistry and Molecular Biology

Volume 116, January 2020, 103264

https://doi.org/10.1016/j.ibmb.2019.103264 Get rights and content

Highlights

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20E stimulates pupa-specific transcription of CHSA-2b in Bombyx wing discs.
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FoxJ enhances CHSA-2b transcription by directly binding to a Fox site in its promoter.
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BR-C Z4 regulates CHSA-2b by inhibiting the expression of FoxJ.
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FoxJ up-regulates multiple silkworm pupal genes.

Abstract

Elaborate regulation of tissue- and stage-specific expression of genes is prerequisite for insect development. The hormone 20-hydroxyecdysone (20E) initiates metamorphosis by regulating the expression of a series of genes. However, how 20E orderly regulates the pupa-specific expression of genes remains unclear. In this study, we report a regulatory mechanism for the pupa-specific expression of chitin synthase A 2b (CHSA-2b) in Bombyx mori. We found that Broad-Complex Z4 (BR-C Z4) was up-regulated by 20E just before pupation, while transcription factor FoxJ and CHSA-2b were up-regulated during the pupal stage. There is a Fox cis-regulatory element in the CHSA-2b promoter region, and FoxJ protein bound to this element, enhancing the CHSA-2b transcription during the pupal stage. In addition to CHSA-2b, FoxJ also up-regulated the expression of 16 out of 19 pupa-specific genes tested. However, at the prepupal stage, 20E-induced BR-C Z4 inhibited the FoxJ transcription, indirectly inhibiting the CHSA-2b transcription. These data suggest that at the pre-pupation stage, 20E-induced BR-C Z4 inhibited the expression of pupa-stage genes like CHSA-2b by inhibiting the expression of FoxJ; by the pupal stage, the expression of BR-C Z4 decreased, releasing its inhibition on FoxJ, which then up-regulated the expression of the pupa-specific genes. This study explains the elaborate regulation of the pupa-specific gene expression during metamorphosis in B. mori.

Graphical abstract

Introduction

The stage- and tissue-specific expression of genes is important for insect morphogenesis. Ecdysteroids can regulate the stage- and cell-specific nuclear receptors and transcription factors to mediate insect development, such as midgut remodeling (Harker et al., 2013; Parthasarathy and Palli, 2007). In the Bombyx mori wing disc, there are 58 cuticle proteins specifically expressed during the prepupal stage (Ou et al., 2014). Many genes that are involved in chitin degradation and synthesis pathways are significantly up-regulated during the larva-pupa transition (Ou et al., 2014). Chitin is a polymer of N-acetylglucosamine and a component of insect epidermis, wings, trachea and the midgut peritrophic membrane (Merzendorfer, 2006; Moussian, 2010; Rezende et al., 2008; Tellam and Eisemann, 2000). Chitin synthase (CHS), including epidermal and midgut chitin synthase (CHSA and CHSB), catalyzes the synthesis of chitin. The RNA interference (RNAi) of CHSA in Drosophila melanogaster, Tribolium castaneum and Ostrinia furnacalis results in abnormal embryonic development (Ostrowski et al., 2002) and molting difficulty (Arakane et al., 2005; Qu and Yang, 2012). In some Lepidoptera, an extra alternative splicing in CHSA occurs in the second exon (Qu and Yang, 2012). CHSA-2a is expressed throughout growth and development, while CHSA-2b is only expressed in the wing disc at the mid-pupal stage (Xu et al., 2017). B. mori CHSA-2b RNAi resulted in the malfunction of wing development (Xu et al., 2017). These results indicate that stage- and tissue-specific genes are involved in successful development of insect tissues and organs.

During the pupal stage, insect wings composed of chitin and cuticle proteins develop along with extracellular matrix remodeling, cellular morphological changes and cuticle deposition (Zhang et al., 2013). Pupal development is regulated by the molting hormone 20-hydroxyecdysone (20E), which binds to a heterodimeric receptor complex consisting of Ecdysone Receptor (EcR) and Ultraspiracle (USP). The 20E/EcR/USP complex up-regulates ecdysis-associated primary response genes, such as Broad-Complex (BR-C), E75 and E74 (Burtis et al., 1990; DiBello et al., 1991; Segraves and Hogness, 1990). These early expressed transcription factors then activate the expression of secondary response genes, such as Ftz-F1, DHR3, DHR39 and E78B (Thummel, 2001), and enhance the expression of later expressed genes that are directly associated with molting and metamorphosis events. BR-C is a key member of the 20E regulatory pathway (Bayer et al., 1996). It encodes a Broad-Complex-Tramtrack-Bric-a-brac (BTB) domain protein with one of four alternatively spliced C2H2 zinc-finger motifs Z1-Z4 (DiBello et al., 1991; Konopova and Jindra, 2008). BR-C proteins have important regulatory roles during the larva-pupa transition and in sex determination in insects (Emery et al., 1994; Huet et al., 1993; Ito et al., 2013). In B. mori, many pupa-specific Wing Cuticle Proteins (WCPs) have been identified (Takeda et al., 2001), and 20E up-regulates the expression of WCP10 (Noji et al., 2003), WCP2 (Nita et al., 2009), cuticle protein GlyGlyTyr-repeat 1 (CPG1) (Suzuki et al., 2002) and WCP4 (Deng et al., 2012). The 20E-EcR/USP-BRC Z4-POUM2 is the pathway that regulates WCP4 expression during the prepupal stage (Deng et al., 2012, 2015). This indicates that pupal genes are up-regulated by 20E cascade signals. In addition to pupa-specific genes, non-stage-specific genes contribute to wing development, but their regulatory mechanism may be different. For example, in the B. mori pupa, two alternative splicing variants of epidermal chitin synthase, CHSA-2a and CHSA-2b, are expressed at different stages in response to a 20E pulse or 20E to ensure wing development (Xu et al., 2017). CHSA-2a is expressed in the epidermis and wing of larvae, pupae and adults, while CHSA-2b is expressed only in the wing during the mid-pupal stage.

How are these genes expressed at the pupal stage? In B. mori, the mechanism of stage- and tissue-specific expression of CHSA-2b shows that different rates of DNA methylation can result in different expression levels of CHSA-2b in the epidermis and wing disc. The down-regulated DNA methyltransferase (Dnmt1) allows the pupa-specific transcription factor Deaf1 to bind the same Dnmt1-bound position in the CHSA-2b promoter (Xu et al., 2018). In addition, the transcription factor forkhead box A (Fox A) inhibits pupa-specific WCP4 expression before the pupal stage by binding to the WCP4 promoter. Its down-regulation at the pupal stage indirectly enhances pupa-specific expression of WCP4 (Hu et al., 2019). The down-regulation of Dnmt1 and Fox A at pupal stage explains why CHSA-2b and WCP4 are expressed at the pupal stage of B. mori but not at the larval stage. However, it is unclear why these two 20E-induced genes are not expressed earlier when the 20E titer is high.

We studied the regulation of 20E-induced expression of CHSA-2b as an example of a pupal gene. We found that 20E-responsive FoxJ was inhibited by BR-C Z4 before the early pupal stage, but it was then up-regulated at the mid-pupal stage, thereby ensuring CHSA-2b expression. Our results show how a combination of 20E-pathway factors regulates pupa-specific gene expression.

Section snippets

Insects and cell line

B. mori strain Dazao was obtained from the Research and Development Center of the Sericultural Research Institute of the Academy of Agricultural Sciences of Guangdong Province, China. Larvae were reared on fresh mulberry leaves at 25 °C and a 12:12 h (L:D) photoperiod in an incubator located in a clean and temperature-controlled chamber.

The B. mori cell line (Bm12), originally developed from ovarian tissues (Khurad et al., 2009) was maintained at 28 °C in Grace medium (Invitrogen, Carlsbad, CA,

BR-C Z or Fox cis-regulatory element in the −734 to −716 nt region of the promoter enhancs CHSA-2b gene transcription

To investigate the 20E-related pathways regulating CHSA-2b expression in B. mori pupae, we analyzed the 20E-related cis-regulatory elements in the CHSA-2b promoter. Three known 20E-related cis-regulatory elements were predicated, including BR-C Z1 at −732 to −714 nt, BR-C Z4 or Fox at −734 to −716 nt, and E74A at −1658 to −1644 nt of the promoter. The sequence of −734 to −716 nt includes potential BR-C Z4 (taaaTAAAcaaa) and Fox (aaaTAAAcaaaca) cis-regulatory elements, where both cis-regulatory

Discussion

Wing disc development is a significant process in the holometabolous insect pupa. The wing disc consists of undifferentiated and proliferating cells. The disc undergoes dramatic morphological changes and eventually evaginates from the body to form the pupal wing disc (Klein, 2001). Pupal development is regulated by an ecdysteroid cascade, including 20E signaling pathway genes such as E74, E75, BR-C, Ftz and HR3 and other transcription factors (Burtis et al., 1990; DiBello et al., 1991; Segraves

Acknowledgments

This work was supported by the National High Technology Research and Development Program of China (863 Program) (No. 2011AA10A204-3).

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Metformin-induced AMPK activation suppresses larval growth and molting probably by disrupting 20E synthesis and glycometabolism in fall webworm, Hyphantria cunea Drury
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Citation Excerpt :
Our results were consistent with the results in T. castaneum after treated with AICAR (Jiang et al., 2020). In addition, metformin also upregulated the expression of HcBr-C, a positive transcriptional regulator of chitin synthetase involved in the regulation of HcCHS (Karim et al., 1993; Zhang et al., 2020). Based on the results of this study, we speculated that metformin restrained the growth and molting of H. cunea larvae probably through three aspects (as shown in Fig. 10).
Metformin, considered to be a potent AMPK activator, is widely used for clinical therapy of cancer and diabetes due to its distinct function in regulating cell energy balance and body metabolism. However, the effect of metformin-induced AMPK activation on the growth and development of insects remains largely unknown. In the present study, we focused on the role of metformin in regulating the growth and development of Hyphantria cunea, a notorious defoliator in the forestry. Firstly, we obtained the complete coding sequences of HcAMPKα2, HcAMPKβ1, HcAMPKγ2 from H. cunea, which encoded a protein of 512, 281, and 680 amino acids respectively. Furthermore, the phylogenetic analysis revealed that these three subunits were highly homologous with the AMPK subunits from other lepidopteran species. According to the bioassay, we found metformin remarkably restrained the growth and development of H. cunea larvae, and caused molting delayed and body weight reduced. In addition, expressions of HcAMPKα2, HcAMPKβ1, and HcAMPKγ2 were upregulated 3.30-, 5.93- and 5.92-folds at 24 h after treatment, confirming that metformin activated AMPK signaling at the transcriptional level in H. cunea larvae. Conversely, the expressions of two vital Halloween genes (HcCYP306A1 and HcCYP314A1) in the 20E synthesis pathway were remarkably suppressed by metformin. Thus, we presumed that metformin delayed larval molting probably by impeding 20E synthesis in the H. cunea larvae. Finally, we found that metformin accelerated glycogen breakdown, elevated in vivo trehalose level, promoted chitin synthesis, and upregulated transcriptions of the genes in chitin synthesis pathway. Taken together, the findings provide a new insight into the molecular mechanisms by which AMPK regulates carbohydrate metabolism and chitin synthesis in insects.
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Analysing the wing-specific CHS1 splice variant 2b from B. mori, however, revealed a more complex picture as CHS1-2b expression was, unlike CHS1-2a, upregulated in pupal wings in response to 20E injection (Xu et al., 2017). The 20E-triggered gene regulatory network that activated CHS1-2b expression in the pupal stage appeared to involve the FoxJ transcription factor, which is repressed during the prepupal stage by BR-C Z4 (Zhang et al., 2020a). Somewhat different observations were made in L. migratoria (Liu et al., 2018c).
Chitin, a linear structural polysaccharide is a major constituent of insect procuticle. Chitin is embedded in a complex matrix of proteins, which contributes significantly to the physicochemical and physiological properties of the cuticle. The wide range of properties of individual cuticles at specific anatomical locations and developmental stages is attributable to difference in chitin/protein ratio, protein composition of the matrix, cross-linking, presence of minerals and the degree of dehydration. The epidermal cells that underlie the cuticle orchestrate the timing of appearance of individual layers of the cuticle and its composition and organization. This process is particularly important during molting, when the old cuticle is replaced by a new one. Molting fluid contains an assortment of enzymes that deproteinize the cuticular matrix to expose the chitin crystallites to chitinolytic enzymes. A variety of enzymes of chitin metabolism participate in the synthesis, modification, and turnover of cuticular components and in cross-linking and tanning of proteins. Chemical inhibitors and RNA interference strategies targeted against the chitin metabolic enzymes and cuticular proteins represent specific and effective strategies for insect control.
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¹: These authors equally contributed to the study.

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ReviewBR-C Z4 and FoxJ interact to regulate expression of a chitin synthase gene CHSA-2b in the pupal wing discs of the silkworm, Bombyx mori

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Insects and cell line

BR-C Z or Fox cis-regulatory element in the −734 to −716 nt region of the promoter enhancs CHSA-2b gene transcription

Discussion

Acknowledgments

Insect Biochem. Mol. Biol.

Dev. Biol.

Cell

Insect Biochem. Mol. Biol.

Dev. Biol.

Insect Biochem. Mol. Biol.

J. Biol. Chem.

Biochim. Biophys. Acta

Curr. Opin. Genet. Dev.

Methods

Insect Biochem. Mol. Biol.

Comp. Biochem. Physiol. B Biochem. Mol. Biol.

Insect Biochem. Mol. Biol.

J. Insect Physiol.

Insect Biochem. Mol. Biol.

Insect Biochem. Mol. Biol.

Insect Biochem. Mol. Biol.

Dev. Cell

Insect Biochem. Mol. Biol.

Insect Biochem. Mol. Biol.

Cell

Insect Biochem. Mol. Biol.

The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix

Insect Mol. Biol.

20-Hydroxyecdysone activates Forkhead box O to promote proteolysis during Helicoverpa armigera molting

Development

Homeodomain POU and Abd-A proteins regulate the transcription of pupal genes during metamorphosis of the silkworm, Bombyx mori

Proc. Natl. Acad. Sci. U. S. A

Review
BR-C Z4 and FoxJ interact to regulate expression of a chitin synthase gene CHSA-2b in the pupal wing discs of the silkworm, Bombyx mori

BR-C Z or Fox cis-regulatory element in the −734 to −716 nt region of the promoter enhancs CHSA-2b gene transcription