Dnmt1 has an essential function despite the absence of CpG DNA methylation in the red flour beetle Tribolium castaneum

Nora K E Schulz,Maike F Diddens-De Buhr,Frank Lyko,C Isabel Wagner,Joachim Kurtz,Julia Ebeling,Günter Raddatz

doi:10.1038/s41598-018-34701-3

Abstract

Epigenetic mechanisms, such as CpG DNA methylation enable phenotypic plasticity and rapid adaptation to changing environments. CpG DNA methylation is established by DNA methyltransferases (DNMTs), which are well conserved across vertebrates and invertebrates. There are insects with functional DNA methylation despite lacking a complete set of Dnmts. But at least one of the enzymes, DNMT1, appears to be required to maintain an active DNA methylation system. The red flour beetle, Tribolium castaneum, lacks Dnmt3 but possesses Dnmt1 and it has been controversial whether it has a functional DNA methylation system. Using whole genome bisulfite sequencing, we did not find any defined patterns of CpG DNA methylation in embryos. Nevertheless, we found Dnmt1 expressed throughout the entire life cycle of the beetle, with mRNA transcripts significantly more abundant in eggs and ovaries. A maternal knockdown of Dnmt1 caused a developmental arrest in offspring embryos. We show that Dnmt1 plays an essential role in T. castaneum embryos and that its downregulation leads to an early developmental arrest. This function appears to be unrelated to DNA methylation, since we did not find any evidence for this modification. This strongly suggests an alternative role of this protein.

Highlights

Insect methylation patterns can be highly diverse[15,16]
This implies that the cytosines that remain after the bisulfite treatment can be either attributed to incomplete conversion or sequencing errors and no CpG DNA methylation is present in the embryos
Using whole-genome bisulfite sequencing (WGBS), we did not observe any recognizable evidence for CpG DNA methylation in T. castaneum embryos

Summary

Introduction

Insect methylation patterns can be highly diverse[15,16]. In cockroaches and some lepidopterans, DNA methylation can reach levels similar to those observed in vertebrates and plants[15,17]. On the other hand, using an approach based on methylation sensitive restriction endonucleases, which lacks the sensitivity and specificity of WGBS, Feliciello et al.[34] found evidence for DNA methylation in early embryos, whereas larvae, pupae and adults were found to be mostly devoid of methylation While this raised the possibility of an active demethylation process in later developmental stages, resembling the epigenetic reprogramming in mammalian embryos[35,36], additional bisulfite sequencing of satellite DNA indicated that these fragments remain heavily methylated throughout the entire lifetime of the individual, even in non-CpG contexts[34]. The controversial evidence of CpG DNA methylation in T. castaneum demands for additional, functional studies in this important insect model organism. We here investigate the existence of CpG DNA methylation and the role of Dnmt[1] in T. castaneum using WGBS and knockdown of Dnmt[1] using maternal RNAi

Methods

Results

Conclusion