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).