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

Abstract 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 β 1− β 6 or α 1 in the MBD showed that β 2− β 3‐turns in the β ‐sheet of the MBD are necessary for the formation of the MBD2/3–mCpG complex; furthermore, other secondary structures, namely, β 4− β 6 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 β 4− β 6 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.