Transcriptional Regulation of Cuticular Protein Glycine-Rich13 Gene Expression in Wing Disc of Bombyx mori, Lepidoptera

Abstract

Cuticular protein genes are good models to study the molecular mechanisms of signaling by ecdysteroids, which regulate molting and metamorphosis in insects. The present research demonstrates on hormonal regulation and analysis of the regulatory sequences and transcription factors important for Bombyx mori cuticular protein glycine-rich13 (CPG13) gene expression. Expression of CPG13 was strong at prepupal stage in wing tissues of B. mori. CPG13 expression was induced by the addition of 20E, which was inhibited by cycloheximide in the wing disc. The upstream region of the CPG13 gene was analyzed using a transient reporter assay with a gene gun system and identified two BR-Z2 binding sites to be important cis-acting elements for the transcription activation of the luciferase reporter gene by an ecdysone pulse. Site-directed mutagenesis of these sites, followed by introduction into wing discs, significantly decreased the reporter activity. It was found that the regions carrying the binding sites for the ecdysone-responsive transcription factor BR-Z2 were responsible for the hormonal enhancement of the reporter gene activity in wing discs. Mutation of the BR-Z2 binding sites decreased the reporter activity suggesting that the BR-Z2 isoform can bind to the upstream region of the cuticle protein gene, CPG13 and activates its expression.

Transcriptional Profiling Shows that BHR4 and E74A Are the Regulators of CPH33 and CPH34 Gene Expression

“…Current studies have showed that BR-C, E74A and βFTZ-F1 regulate different cuticular protein genes [9,10,11,16,17,20]. Thus, these ecdysone-responsive transcription factors are suggested to regulate their target genes, and the series of their expression would bring about insect metamorphosis.…”

“…Thus, cuticular protein genes, which have different developmental profiles, were induced by different types of ecdysone signal, and these things were described in a recent review [7].The determination of insect genomic sequences also brought about the comprehensive analysis of cuticular-protein gene expression and the analysis of its regulation by transcription factors [8][9][10][11][12][13][14][15][16][17]. Cuticular protein genes have different expression profiles [8][9][10][11][12][13][14][15][16][17] and regulatory systems by ecdysone-responsive transcription factors [9][10][11][12][13][14][15][16][17][18][19]. From this, insect cuticular protein genes are suggested to be a suitable material for clarifying the regulatory mechanism of ecdysone-responsive transcription factors.…”

“…Expression of CPR55, BMWCP2 and BMWCP5 was induced by an ecdysone pulse through a transcription factor, βFTZ-F1, which bound to their upstream binding sites and increased their promoter activity [10,16,17,20]. Recent experimental results proved that CPR95 was regulated by E74A and BmorCPG13 by BR-C Z2 through a reporter assay by using wing discs [9,11]. Thus, it was proved that the expression of the cuticular protein genes of B. mori was regulated by ecdysone-responsive transcription factors.…”

Genes encoding cuticular proteins are components of the Nimrod gene cluster in Drosophila

“…By using NCBI and UniProt BLAST the Vajk proteins were found to be 49 to 80% identical to several cuticular proteins identified in other insect species, such as Bombyx mori (Ali et al, 2015;Suetsugu et al, 2013), Danaus plexippus (Zhan et al, 2011), Operophtera brumata (Derks et al, 2015), Anopheles gambiae (He et al, 2007), Papilio polytes (Futahashi et al, 2012) and Papilio xuthus Futahashi et al, 2012).…”

Transcriptional regulation of cuticular genes during insect metamorphosis

“…Developmental profiles and reporter assays also suggest that the expression of Group 3 CP genes BmorCPH33 and BmorCPR34 is regulated by BHR4 (44,59). Group 4 expression is believed to be regulated by BR-C, as the expression profiles of BmorCPG11, BmorCPG13, and BmorCPH30 are similar to the expression profile of BR-C, and a BR-C binding site is located upstream of BmorCPG11 and BmorCPG13 (47,48,60) (48 Ref3). The expression of the Group 5 genes can be observed after the wandering stage.…”