Heavy metal induced epigenetic modifications

The present project deals with the influence of heavy metal pollution on epigenetic responses in the common earthworm Lumbricus terrestris, and with the persistence and heritability of metal induced epigenetic alterations. The main questions this project will answer are: Are metal detoxification processes and earthworm tolerance to metals governed through epigenetic mechanisms? and Can heavy metal pollution induce long lasting and even heritable epigenetic alterations in earthworms? To assess the detoxification processes in earthworms exposed to a low dose of cadmium (Cd), this proposal will assess the expression of main metal detoxifying gene (metallothionein (MT)) and its potential epigenetic regulation which will be monitored by methylation sensitive high resolution melting (MS-HRM). Genome wide DNA methylation status will be assessed using the cutting edge next generation sequencing tool, bisulfite-converted restriction site associated DNA sequencing (bsRADseq). Additionally, genes involved in DNA methylation and demethylation (DNMT1, DNMT2, DNMT3, TET) will be defined and quantified to further explain DNA methylation alterations. The above mentioned analyses will be performed in earthworms after Cd exposure and after a period of recovery, as well as in their F1 and F2 progeny. Furthermore, development of tolerance mechanisms in earthworms reared in low Cd dose polluted soil as well as in their progeny will be tested upon high Cd dose exposure and assessed by the above mentioned methods. The project novelty lies in the unique synergy of experts from different disciplines as well as in novel methodology which will allow us to examine the issue of metal induced epigenetic perturbations from different angles. The results of this study will generate novel information on epigenetic alterations, and their persistence and heritability in metal polluted environments. Furthermore, the project will, for the first time, define the epigenetic regulation of metal detoxification genes and fill the existing knowledge gaps on the regulatory mechanisms of metal detoxification in invertebrates.

The main result of this project is a detailed view of DNA methylation changes and their association with gene expression in the earthworm Lumbricus terrestris exposed to the heavy metal cadmium (Cd). DNA methylation was assessed at global, genome-wide and gene-specific levels. Cd-induced dose-dependent global DNA hypermethylation was detected at the level of cytosine methylation and, for the first time, at the level of adenine methylation in the genome. Genes involved in DNA methylation/demethylation, including DNMT1, DNMT3 and TET genes, could not explain the observed methylation effects, and neither gene expression nor protein activity showed significant changes between treatment groups. At the level of gene methylation, we observed slight, yet insignificant, changes in intron and exon methylation of the main Cd detoxifying gene, metallothionein (MT2). However, this change in methylation could not be linked to MT2 gene expression, linking to the methylation independent gene regulation. Furthermore, we were able to obtain a high-resolution picture of genome-wide methylation changes at the nucleotide level using next-generation sequencing tools. Global hypermethylation was not reflected across the genome, and high-throughput sequencing rather revealed a dose-response Cd induced DNA hypomethylation at the genome-wide level. Most of the methylation changes occurred within the gene bodies. Interestingly, at higher Cd treatment, differentially methylated regions were associated with a group of "metal ion stress response" genes, suggesting an epigenetic background governing the organism's response to Cd treatment. Transcriptome analysis revealed differential expression of genes involved in DNA damage and integrity, oxidative stress and metal stress response. In addition, genes involved in metabolism, binding and catalytic activity were among the groups of genes most affected by cadmium treatment. The multi-OMIC approach used in this study, combining epigenetic and transcriptomic endpoints, provided valuable and high-resolution data on organismal responses to heavy metal stress. This approach is highly valuable but rarely done in environmental epigenetics and is of great importance for both basic epigenomic research and the applied field of ecotoxicology in terms of epigenetic biomarker development.