Abstract
Stage-specific gene expression governs metamorphosis of the silkworm, Bombyx mori. B. mori wing cuticle protein gene 4 (BmWCP4) is an essential gene for wing disc development expressed specifically during pupation. BmWCP4 transcription is suppressed at the larval stage by unknown mechanisms, which we sought to elucidate here. Bioinformatics analysis predicted seven potential Forkhead box (Fox) cis-regulatory elements (CREs) in the BmWCP4 promoter region, and we found that Fox CRE6 contributes to suppression of BmWCP4 expression. Electrophoretic mobility shift (EMSA) and DNA pull-down assays revealed that BmFoxA suppressed activity at the BmWCP4 promoter by specifically binding to the Fox CRE6. The expression level of BmFoxA in the wing discs was higher during the larval stage than at the pupal stage. In contrast, expression of another transcription factor, BmSAGE, increased over the course of development. Of note, the hormone 20-hydroxyecdysone (20E), which governs molting in insects, suppressed BmFoxA expression in the wing discs and up-regulated that of BmSage EMSA and cell co-transfection assays indicated that BmSAGE interacted with BmFoxA and suppressed its binding to the Fox CRE6, thereby releasing BmFoxA-mediated suppression of BmWCP4 In summary, higher BmFoxA expression during the larval stage suppresses BmWCP4 expression by binding to the Fox CRE6 on the BmWCP4 promoter. During metamorphosis, BmSAGE forms a complex with BmFoxA to relieve this repression, initiating BmWCP4 expression. Taken together, this study reveals a switchlike role for BmFoxA in regulating BmWCP4 expression and provides new insights into the regulatory regulation of wing disc development in insects.
Highlights
Stage-specific gene expression governs metamorphosis of the silkworm, Bombyx mori
The results showed that the Forkhead box (Fox) CRE7 could increase the activity of BmWCP4 promoter by 3.0-fold over the core promoter that did not contain any Fox cis-regulatory elements (CREs)
These results suggested that the Fox CRE1, Fox CRE4, Fox CRE5, Fox CRE6, and Fox CRE7 might be involved in the regulation of the BmWCP4 expression
Summary
Stage-specific gene expression governs metamorphosis of the silkworm, Bombyx mori. B. mori wing cuticle protein gene 4 (BmWCP4) is an essential gene for wing disc development expressed during pupation. Electrophoretic mobility shift (EMSA) and DNA pull-down assays revealed that BmFoxA suppressed activity at the BmWCP4 promoter by binding to the Fox CRE6. Fkh is the first FoxA protein identified in Drosophila, participating in the differentiation of embryo cells, autophagy, phospholipid metabolism, and hormone signal pathway [13, 14]. It is required for the salivary gland development in Drosophila melanogaster and inhibits apoptosis in salivary glands or activates the expression of some important genes, such as Senseless (Sens) [15,16,17]. In Spodoptera litura, SlFoxA regulates the expression of ABCC2 enhanced GFP; aa, amino acids; qRT-PCR, quantitative real-time PCR; ANOVA, analysis of variance; EMSA, electrophoretic mobility shift assay; NP-40, Nonidet P-40
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have