Methyl-CpG binding domain (MBD)2/3 specifically recognizes and binds to the genomic mCpG site with a ß-sheet in the MBD to affect embryonic development in Bombyx mori.

Methyl-CpG (mCpG) binding domain (MBD) proteins especially bind with methylated DNA, and are involved in many important biological processes; however, the binding mechanism between insect MBD2/3 and mCpG remains unclear. In this study, we identified 2 isoforms of the MBD2/3 gene in Bombyx mori, MBD2/3-S and MBD2/3-L. Binding analysis of MBD2/3-L, MBD2/3-S, and 7 mutant MBD2/3-L proteins deficient in ß1-ß6 or α1 in the MBD showed that ß2-ß3-turns in the ß-sheet of the MBD are necessary for the formation of the MBD2/3-mCpG complex; furthermore, other secondary structures, namely, ß4-ß6 and an α-helix, play a role in stabilizing the ß-sheet structure to ensure that the MBD is able to bind mCpG. In addition, sequence alignment and binding analyses of different insect MBD2/3s indicated that insect MBD2/3s have an intact and conserved MBD that binds to the mCpG of target genes. Furthermore, MBD2/3 RNA interference results showed that MBD2/3-L plays a role in regulating B. mori embryonic development, similar to that of DNA methylation; however, MBD2/3-S without ß4-ß6 and α-helix does not alter embryonic development. These results suggest that MBD2/3-L recognizes and binds to mCpG through the intact ß-sheet structure in its MBD, thus ensuring silkworm embryonic development.

Homeobox protein A1-like and DNA methylation regulate embryo-specific Zinc finger protein 615 gene expression and embryonic development in the silkworm Bombyx mori.

DNA methylation and transcription factors play roles in gene expression and animal development. In insects, DNA methylation modifies gene bodies, but how DNA methylation and transcription factors regulate gene expression is unclear. In this study, we investigated the mechanism that regulates the expression of Bombyx mori Zinc finger protein 615 (ZnF 615), which is a downstream gene of DNA methyltransferase 1 (Dnmt1), and its effects on the regulation of embryonic development. By progressively truncating the ZnF 615 promoter, it was found that the -223 and -190 nt region, which contains homeobox (Hox) protein cis-regulatory elements (CREs), had the greatest impact on the transcription of ZnF 615. RNA interference (RNAi)-mediated knockdown and overexpression of Hox family genes showed that Hox A1-like can enhance the messenger RNA level of ZnF 615. Further studies showed that Hox A1-like regulates ZnF 615 expression by directly binding to the -223 and -190 nt region of its promoter. Simultaneous RNAi-mediated knockdown or overexpression of Hox A1-like and Dnmt1 significantly inhibited or enhanced the regulatory effect of either gene alone on ZnF 615 expression, suggesting that both DNA methylation of gene bodies and binding of transcription factors to promoters are essential for gene expression. RNAi-mediated knockdown of Hox A1-like and Dnmt1 showed that the embryonic development was retarded and the hatching rate was decreased. Taken together, these data suggest that Hox A1-like and DNA methylation enhance the expression of ZnF 615, thereby affecting the development of B. mori embryos.

DNA methylation-mediated expression of zinc finger protein 615 affects embryonic development in Bombyx mori.

Cell division and differentiation after egg fertilization are critical steps in the development of embryos from single cells to multicellular individuals and are regulated by DNA methylation via its effects on gene expression. However, the mechanisms by which DNA methylation regulates these processes in insects remain unclear. Here, we studied the impacts of DNA methylation on early embryonic development in Bombyx mori. Genome methylation and transcriptome analysis of early embryos showed that DNA methylation events mainly occurred in the 5' region of protein metabolism-related genes. The transcription factor gene zinc finger protein 615 ( ZnF615) was methylated by DNA methyltransferase 1 (Dnmt1) to be up-regulated and bind to protein metabolism-related genes. Dnmt1 RNA interference (RNAi) revealed that DNA methylation mainly regulated the expression of nonmethylated nutrient metabolism-related genes through ZnF615. The same sites in the ZnF615 gene were methylated in ovaries and embryos. Knockout of ZnF615 using CRISPR/Cas9 gene editing decreased the hatching rate and egg number to levels similar to that of Dnmt1 knockout. Analysis of the ZnF615 methylation rate revealed that the DNA methylation pattern in the parent ovary was maintained and doubled in the offspring embryo. Thus, Dnmt1-mediated intragenic DNA methylation of the transcription factor ZnF615 enhances its expression to ensure ovarian and embryonic development.

Dynamic transcriptome analysis of Bombyx mori embryonic development.

Bombyx mori has been extensively studied but the gene expression control of its embryonic development is unclear. In this study, we performed transcriptome profiling of six stages of B. mori embryonic development using RNA sequencing (RNA-seq). A total of 12 894 transcripts were obtained from the embryos. Of these, 12 456 transcripts were shared among the six stages, namely, fertilized egg, blastoderm, germ-band, organogenesis, reversal period, and youth period stages. There were 111, 48, 41, 54, 77, and 107 transcripts specifically expressed during the six stages, respectively. By analyzing weighted gene correlation networks and differently expressed genes, we found that during embryonic development, many genes related to DNA replication, transcription, protein synthesis, and epigenetic modifications were upregulated in the early embryos. Genes of cuticle proteins, chitin synthesis-related proteins, and neuropeptides were more abundant in the late embryos. Although pathways of juvenile hormone and the ecdysteroid 20-hydroxyecdysone, and transcription factors were expressed throughout the embryonic development stages, more regulatory pathways were highly expressed around the organogenesis stage, suggesting more gene expression for organogenesis. The results of RNA-seq were confirmed by quantitative real-time polymerase chain reaction of 16 genes of different pathways. Nucleic acid methylation and seven sites in histone H3 modifications were confirmed by dot blot and western blot. This study increases the understanding of the molecular mechanisms of the embryonic developmental process and information on the regulation of B. mori development.

Signal transducer and activator of transcription is involved in the expression regulation of ecdysteroid-induced insulin-like growth factor-like peptide in the pupal wing disc of silkworm, Bombyx mori.

In insects, 20-hydroxyecdysone (20E) and insulin-like growth factor-like peptides (IGFLPs) regulate the development of imaginal discs. However, how IGFLPs are up-regulated to impact the development of the pupal wing disc is still unclear. In this study, we investigated the expression regulation of IGFLP in the pupal wing disc of silkworm, Bombyx mori. We confirmed that B. mori IGFLP (BmIGFLP) was mainly expressed in the pupal wing disc and the expression of BmIGFLP could be significantly induced by 20E. Bioinformatics analysis of BmIGFLP promoter sequence revealed three cis-regulation elements (CREs) of signal transducer and activator of transcription (STAT), which is a key component in the Janus-activated kinase / STAT pathway. Luciferase activity assays showed that two CREs enhanced the transcriptional activity of BmIGFLP. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that BmSTAT proteins in the nuclear extracts of B. mori pupal wing discs and BmN cells could only bind to the STAT CRE3, indicating that STAT CRE3 activated by BmSTAT enhances BmIGFLP expression at pupal stages. Although 20E could not enhance the expression of BmSTAT, 20E enhanced the nucleus translocation of BmSTAT to bind with the STAT CRE3 in the BmIGFLP promoter. The increase of transcriptional activity of the STAT CRE3 by overexpression of BmSTAT and addition of 20E in BmN cells confirmed this result. Taken together, all data indicate that BmSTAT is one of the transcription factors activating 20E-induced BmIGFLP expression in the pupal wing disc.