DNA methylation on N6-adenine in lepidopteran Bombyx mori

Increasing knowledge of DNA methylation that occurs on the sixth position of adenine (N6-methyladenine, 6 mA) has emerged as a novel epigenetic mark in eukaryotes and plays an important role in regulating gene transcription, DNA replication and repair, transposable activities, and others. Here, we show DNA 6 mA methylation is present in Bombyx mori, a lepidopteran model insect, and identify the 6 mA methyltransferase, METTL4, and 6 mA demethylase, NMAD, which regulate the levels of 6 mA in embryogenesis and cultured cells of B. mori. Importantly, RNAi knockdown of METTL4 and NMAD not only induce cell cycle arrest at G1 phase but also result in defects of chromosome alignments at metaphase. We further demonstrate that 6 mA methylation is widely distributed across the genome of B. mori by 6 mA-Seq and primarily enriched in the regulatory regions as well as gene bodies. Integrated analysis of 6 mA-Seq and RNA-Seq reveals that 6 mA methylation in B. mori is preferentially related with lowly expressed genes and negatively correlated with active gene transcription, which provides a novel regulatory mechanism of DNA 6 mA methylation on target genes. Altogether, these data identify 6 mA methylation in B. mori and demonstrate a crucial role of 6 mA signaling in controlling cell cycle progression.