The methylome of Biomphalaria glabrata and other mollusks: enduring modification of epigenetic landscape and phenotypic traits by a new DNA methylation inhibitor

Nelia Luviano,Slavica Ivanovic,Fleur Gawehns,Cristian Chaparro,Patrice David,Céline Cosseau,Christoph Grunau,Paola B Arimondo,Marie Lopez,Koen Verhoeven

doi:10.1186/s13072-021-00422-7

Abstract

Background5-Methylcytosine (5mC) is an important epigenetic mark in eukaryotes. Little information about its role exists for invertebrates. To investigate the contribution of 5mC to phenotypic variation in invertebrates, alteration of methylation patterns needs to be produced. Here, we apply new non-nucleoside DNA methyltransferase inhibitors (DNMTi) to introduce aleatory changes into the methylome of mollusk species.ResultsFlavanone inhibitor Flv1 was efficient in reducing 5mC in the freshwater snails Biomphalaria glabrata and Physa acuta, and to a lesser degree, probably due to lower stability in sea water, in the oyster Crassostrea gigas. Flv1 has no toxic effects and significantly decreased the 5mC level in the treated B. glabrata and in its offspring. Drug treatment triggers significant variation in the shell height in both generations. A reduced representation bisulfite-sequencing method called epiGBS corroborates hypomethylation effect of Flv1 in both B. glabrata generations and identifies seven Differential Methylated Regions (DMR) out of 32 found both in Flv1-exposed snails and its progeny, from which 5 were hypomethylated, demonstrating a multigenerational effect. By targeted bisulfite sequencing, we confirmed hypomethylation in a locus and show that it is associated with reduced gene expression.ConclusionsFlv1 is a new and efficient DNMTi that can be used to induce transient and heritable modifications of the epigenetic landscape and phenotypic traits in mollusks, a phylum of the invertebrates in which epigenetics is understudied.

Highlights

DNA methylation is an epigenetic mark that can be associated with changes in gene function without changes in the underlying DNA sequence [29]
DNA methyltransferase inhibitors (DNMTi) Flv1 and Flv2 are suitable for pulse treatment in freshwater To induce changes in DNA methylation that were sufficiently strong to lead to phenotypic changes but not strong enough to have toxic effects, we intended to apply a brief DNMTi pulse of only a couple of hours
In F1 generation, we found significant differences in shell height (p = 1.37e-13) and mollusk’s weight (p = 0.00277) between treatments, and multiple pairwise-comparisons, indicated that the Reduced representation of bisulfite‐sequencing epigenotyping-by-sequencing method (epiGBS) reduces sequencing effort roughly 10 × and allows for reliable evaluation of global 5mC level and identification of differentially methylated sites and regions To obtain a clearer picture of where hypomethylation occurred in the epigenomes of the DNMTi exposed populations and their offspring, we adapted a reduced representation technique that was originally developed for mosaic methylation of plants: epigenotyping by sequencing

Summary

Introduction

DNA methylation is an epigenetic mark that can be associated with changes in gene function without changes in the underlying DNA sequence [29]. DNA methylation is assumed to be evolutionary ancient, but its functions and patterns are very diversified. In many invertebrates, a common type of DNA methylation is the “mosaic” pattern consisting in large domains of methylated DNA separated by large domains of unmethylated DNA [40]. Another pattern is a very low level [36] or a total absence of DNA methylation [3, 11]. Higher GBM is believed to be associated with active transcription in vertebrates and invertebrates, while promoter methylation in vertebrates is associated with repression of gene expression [71]

Objectives

Methods

Results

Conclusion