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).