3.6 Freshwater-triggered gene expression changes associated with hyper- and hypomethylation
The distribution of differentially methylated and expressed genes was examined in different genomic contexts (Fig. 8). There was a balanced distribution between upregulated and downregulated genes in FW, ranging from 46-58 % for upregulation and 42-54 % for downregulation when considering hypomethylated genes (exons and introns) and promoters (Fig. 8A). In contrast, in the context of hypermethylation, 62-81 % of the genes and promoters were downregulated and only 19 to 38 % were upregulated (Fig. 8B). It is worth noting that in the first introns, we found the highest proportion (81 %) of downregulated genes in the context of hypermethylation.
KEGG pathway analysis was performed to show the functional enrichment of DEGs and DMGs (Fig. 9 and Table 1, Table 7S). We focused on differentially methylated genes at promoters, first exons or first introns and their expression changes upon salinity transfer. Some pathways involved in ‘regulation of actin cytoskeleton’, ‘focal adhesion’, and ‘calcium signaling pathway’ were statistically enriched for both DEGs and DMGs. Genes encoding for functions related to these pathways had their expression repressed and displayed either a hypermethylation or a hypomethylation in FW (Fig. 9). Regarding the ‘calcium signaling pathway’ category, we identified genes encoding for the SERCA (Sarco Endoplasmic Reticulum Calcium ATPase) pump (atp2a2 ) and several voltage-dependent calcium channels. In several KEGG categories, we identified tropomyosin (tpm1 ) as being hypermethylated and downregulated, as well as genes involved in and in ‘integrin signaling’ (itga 1 , 4 , 5 ,9 ).
The ‘tight junction’ category was enriched, as in the previous analysis, and showed genes that were mainly hypomethylated and upregulated in FWvs SW or hypomethylated and downregulated in FW vs SW (Fig. 9). Ten genes encoding for Claudins were differently expressed and were enriched in DMRs (Table 1). Among them, eight were upregulated in FW and hypomethylated, mainly at promoter level. In this category, we also identified one paralog of tight-junction protein 2 (tjp2 ), the cytoskeleton-associated cingulin-like protein (cgn ), MarvelD3, a transmembrane component of tight junctions, and the junctional adhesion molecule a-like (f11r ). These genes were all hypomethylated and upregulated.
The ‘focal adhesion pathway’ was enriched in downregulated genes that were either hyper- or hypo-methylated. As indicated previously, we identified several genes involved in integrin signaling with some of them being also hypomethylated (itga 3 , 6 , 7 ) and a transcription factor, focal adhesion kinase (ptk2 ), that was downregulated and hypomethylated. The pathway ‘regulation of actin cytoskeleton’ was significantly enriched with mainly downregulated genes that were either hyper- or hypomethylated in FW, as indicated previously. Regarding metabolism, the KEGG pathways ‘glycosphingolipid biosynthesis’ and ‘glycerophospholipid metabolism’ and ‘sphingolipid signaling pathway’ were also enriched for DEGs and DMRs (Table 7S).
In the ‘mineral absorption’ pathway, only two genes were highlighted, including one gene encoding for chloride channel 2 (clcn2, one of the two paralogs) and one encoding for the copper transporterctr1 (slc31a1 ). Both genes appeared hypermethylated and upregulated in FW. We also noticed the upregulation of the prolactin signaling pathway with genes enriched for hypomethylation, as the prolactin receptor (prlr ) (Table 7S).