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 RNAimediated 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.
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 β1−β6 or α1 in the MBD showed that β2−β3‐turns in the β‐sheet of the MBD are necessary for the formation of the MBD2/3–mCpG complex; furthermore, other secondary structures, namely, β4−β6 and an α‐helix, play a role in stabilizing the β‐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 β4−β6 and α‐helix does not alter embryonic development. These results suggest that MBD2/3‐L recognizes and binds to mCpG through the intact β‐sheet structure in its MBD, thus ensuring silkworm embryonic development.
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