4.4 Freshwater transfer affects DNA methylation in crucial genes
involved in maintaining hydromineral balance
We showed that a freshwater transfer affects expression changes in genes
that were differentially methylated, which suggests that a change in
salinity could induce an altered pattern of DNA methylation, which in
turn could have functional consequences and allow sea bass to display
phenotypic variation through gene expression changes linked to
hydromineral balance.
Gene expression changes in fish gills following a salinity change have
been shown in numerous species (Leguen et al., 2015; Qin et al., 2022)
whereas studies on DNA methylation changes in different salinity
conditions are scarce. We focused in this study on genes that are
involved in osmoregulation, cell volume regulation and acid base
regulation and that display gene expression as well as DNA methylation
changes (Table 1). Among them, the gene encoding for water channel
Aquaporin 3 (aqp3) is highly induced in FW vs SW
(log2FC=3.3), and hypomethylated at promoter and gene body levels. This
gene is expressed in ionocytes and is known to be overexpressed in gills
of numerous species in FW, notably for the basolateral release of water
from ionocytes to the serosal fluid to prevent cell swelling (Cutler and
Cramb., 2002; Giffard-Mena et al., 2007). In studies on mammal gastric
carcinoma, aqp3 was shown to be hypermethylated at its promoter
and first exon, which limited its expression (Wang et al., 2019). In
fish, aqp3 gene expression is controlled by cortisol and
prolactin, where prolactin induces its expression and cortisol decreases
its expression in gills of Mozambique tilapia Oreochromis
mossambicus (Breves et al., 2016). Interestingly, in D. labrax,the gene nr3c1 encoding for the glucocorticoid receptor was
repressed and hypomethylated at its gene body (GB). Moreover, theprlra gene encoding for one of the two prolactin receptor
paralogs was upregulated in FW vs SW (log2FC=1.93) as shown
previously (L’Honoré et al. 2020) and hypomethylated at promoter and GB
levels. This is consistent with the observed upregulation and
hypomethylation (at promoters, first exons or introns) of the ‘prolactin
signaling pathway’. DNA methylation changes of genes encoding for
hormone receptors could be key regulators of osmoregulatory processes.
To our knowledge, no data are available on the effect of methylation
changes on prlr expression in fish. However, studies on KO mice
brains with selective disruption of the dopamine D2 receptor in neurons,
have shown an upregulation of prlr correlated with decreased
methylation of their promoters (Brie et al., 2020).
Regarding ion channels and transporters that are involved in salt
uptake, we identified chloride channels clcn2 and clcn3that were upregulated in FW vs SW (with log2FC of 2.63 and 0.71
respectively). clcn2 was hypermethylated at GB level.
Interestingly we found another paralog of clcn2 , that was
downregulated in FW (log2FC=-2.13) and hypomethylated in GB. Both Clcn2
and Clcn3 channels have been localized in basolateral membranes of
ionocytes and are suspected to transport Cl- to the
blood for its uptake (Tang et al., 2010; Bossus et al., 2013).clcn2 expression and protein changes according to salinity have
been shown in several studies, although with sometimes contrasting data
(Root et al., 2021; Bossus et al., 2013) that might be linked to the
presence of two clcn2 paralogs that were not differentiated.
Several genes encode for
Na+/K+-ATPase, which is a key active
ion transporting pump expressed in basolateral membranes of gill
ionocytes. As expected, the major paralog (atp1a1a ) encoding for
subunit NKAα1 was upregulated in FW vs SW as shown previously by
Blondeau-Bidet et al. (2019) in D. labrax gills. We also observed
a hypomethylation at promoter and GB levels. atp1a3 (encoding for
the NKAα3 subunit), which has not been investigated so far in D.
labrax gills, was also upregulated and hypomethylated in promoters. InD. labrax SW-type ionocytes, the apical chloride channel CFTR and
basolateral cotransporter NKCC1 are crucial proteins involved in salt
secretion (Lorin-Nebel et al. 2006; Bodinier et al., 2009). Both genes
encoding for these proteins were downregulated in FW and hypermethylated
at GB level. Fougere et al. (2020) showed that focal adhesion kinases
are, according to their phosphorylation state, colocalized with apical
tight junctions and CFTR in apical membranes of ionocytes ofFundulus heteroclitus, and are involved in ion secretion by these
cells. We identified two ptk2 genes encoding for focal adhesion
kinase 1 which were downregulated (log2FC=-0.4113 and -0.7749) and
hypomethylated (GB and promoters) but the functional link between Ptk2
with apically localized proteins in ionocytes remains to be shown inD. labrax . V-type H+ ATPase (VHA) is another
important pump expressed in fish gill ionocytes. It is involved in
H+ secretion (for acid-base balance) coupled to
transepithelial Na+ uptake (for osmoregulation). We
identified three genes encoding for VHA that showed expression and
methylation changes upon FW transfer (Table 1). Together, all these data
point to significant methylation changes in key genes involved in hyper-
and hypo-osmoregulation as well as acid-base regulation. This is
consistent with the statement that salinity affects the plastic
responses through DNA methylation changes, to maintain hydromineral
balance, as already mentioned in other species (Heckwolf et al. 2020).