No abstract

Background: DNA methylation, as an essential epigenetic modification found in mammals and plants, has been implicated to play an important role in insect reproduction. However, the functional role and the regulatory mechanism of DNA methylation during insect organ or tissue development are far from being clear. Results: Here, we found that DNA methylation inhibitor (5-aza-dC) treatment in newly molted pupae decreased the chitin content of pupal wing discs and adult wings and resulted in wing deformity of Bombyx mori. Transcriptome analysis revealed that the up-regulation of chitinase 10 (BmCHT10) gene might be related to the decrease of chitin content induced by 5-aza-dC treatment. Further, the luciferase activity assays demonstrated that DNA methylation suppressed the promoter activity of BmCHT10 by down-regulating the transcription factor, homeobox protein araucan (Bmara). Electrophoretic mobility shift assay, DNA pull-down and chromatin immunoprecipitation demonstrated that Bmara directly bound to the BmCHT10 promoter. Therefore, DNA methylation is involved in keeping the structural integrity of the silkworm wings from unwanted chitin degradation, as a consequence, it promotes the wing development of B. mori. Conclusions: This study reveals that DNA methylation plays an important role in the wing development of B. mori. Our results support that the indirect transcriptional repression of a chitin degradation-related gene BmCHT10 by DNA methylation is necessary to keep the proper wing development in B. mori.

Cyclic adenosine monophosphate (cAMP) response element binding protein (CREB)‐binding protein (CBP or CREBBP) plays important roles in regulating gene transcription and animal development. However, the process by which CBP is up‐regulated to impact insect development is unknown. In this study, the regulatory mechanism of Bombyx mori CBP (BmCBP) expression induced by 20‐hydroxyecdysone (20E) was investigated. In the Bo. mori cell line, DZNU‐Bm‐12, 20E enhanced BmCBP transcription and histone H3K27 acetylation. BmCBP RNA interference (RNAi) resulted in decreased histone H3K27 acetylation. Additionally, the luciferase activity analysis revealed that the transcription factor, Bo. mori CCAAT/enhancer‐binding protein gamma (BmC/EBPg), activated BmCBP transcription, which was suppressed by BmC/EBPg RNAi and promoted by BmC/EBPg overexpression. Electrophoretic mobility shift assay and chromatin immunoprecipitation results demonstrated that BmC/EBPg could bind to the C/EBP cis‐regulatory elements in two positions of the BmCBP promoter. Moreover, BmC/EBPg transcription was enhanced by the 20E receptor (BmEcR), which bound to the BmC/EBPg promoter. BmEcR RNAi significantly inhibited the transcriptional levels of BmC/EBPg and BmCBP in the presence of 20E. Furthermore, the BmEcR‐BmC/EBPg pathway regulated the acetylation levels of histone H3K27. Altogether, these results indicate that BmEcR enhances the expression of BmC/EBPg, which binds to the BmCBP promoter, activates BmCBP expression and leads to histone H3K27 acetylation.