3.4 The effect of freshwater transfer on DNA methylation and gene expression dynamics
We identified 17 265 differentially methylated regions (DMRs) inD. labrax exposed to FW vs SW conditions (Table 3S) (p-value <0.05) and these DMRs were uniformly spread within the 24 chromosomes (Fig. 5A). 66% of the DMRs were hypomethylated (n = 11 375) whereas 34% of the DMRs were hypermethylated (n = 5890) in FW. The majority of the DMRs were found in genes (48 %), among them 7 % and 41 % were present in exons and introns, respectively (Fig. 5B). In genes, we identified 2367 hypermethylated regions and 4652 hypomethylated regions in FW vs SW conditions. Promoter and intergenic regions contained 9 % and 43 % of DMRs, respectively. In promoters, 582 DMRs were hypermethylated and 1481 were hypomethylated in FW vs SW (Table 3S).
Considering exons and introns (Fig. 5C, D), DMRs showed an average percentage of 73 % of hypomethylation and 27 % of hypermethylation. DMRs were less frequent in the first (0.01 %) and last (0.005%) exons (Fig. 5C). Higher percentages of DMRs were observed in the internal exons (i. e. exons located in non-flanking regions of genes), with an average DMR percentage by bp of 0.0173 %. We observed the same trend in the introns, with lowest percentages of DMRs observed in the first (0.0044 %) and last (0.0048 %) introns, whereas DMRs in internal introns were 1.2-1.4 times more frequent (0.0062 %).
Among the differentially methylated gene bodies and promoters, we performed an enrichment analysis of gene ontology terms (GO-terms) (Fig. 6A, Table 4S and 6S). We identified several GO-terms that were associated to actin cytoskeleton organization and regulation, as well as GO-terms associated to bicellular tight junctions. We also identified GO categories linked to mitochondrial metabolism as H+-transporting ATP synthase activity and oxidoreductase activity as well as proton channel activity (black stars, Fig. 6A). The most significant GO-terms of the DNA methylome analysis are listed in Table 4S.
A differential analysis was performed to identify gene expression patterns between FW and SW exposed fish. We found 5144 differentially expressed genes (DEGs), among which 2316 were upregulated and 2828 were downregulated (Table 5S). The enrichment analysis of GO-terms (Fig. 6B, Table 4S and 6S) identified functional categories involving genes that were upregulated in fresh water linked to mitochondrial metabolism. These include genes involved in the tricarboxylic acid (TCA) cycle (as dihydrolipoamide S-succinyltransferase dlst , succinate dehydrogenases sdha, sdhd, sdhb and mitochondrial citrate synthase precursor cs ), genes linked to the ‘electron transport chain’, ‘electron transfer activity’ and ‘ATP biosynthetic process’. Genes linked to methionine metabolism as s-methyl-5-thioadenosine phosphorylase (mtap ) were upregulated. We also identified upregulated genes involved in other metabolic processes (peptide metabolic process, cellular amino acid metabolic process) as well as ATPase and proton transporter activity (see next paragraph on ATPases). In contrast, genes with molecular functions linked to membrane rafts and membrane microdomains were downregulated in fresh water. The most significant GO-terms of the transcriptome analysis are listed in Table 4S.