MBD2/3 specifically recognizes and binds to the genomic mCpG site with a β-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 two 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 seven 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, the MBD2/3 RNAi 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. This article is protected by copyright. All rights reserved.