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.