4.5 Paracellular permeability-related genes are hypomethylated and upregulated
In clinical studies on mammal cancer where disruption of cell-cell junctions is frequently found, investigations have shown a link between claudin overexpression and DNA hypomethylation for several claudins (Li et al., 2018), including cldn4 (Kwon et al., 2011). In fish gills, the tightening of the gill epithelium is a natural and essential process in fresh water in order to avoid excess water entry and limit passive ion loss. Euryhaline species have to constantly adjust gill permeability through the expression of tight or leaky junctions. A striking result of this study is the consistent promoter hypomethylation and upregulation of genes involved in paracellular permeability and the formation of tight junctions. In both analyses (KEGG pathway and GO-term enrichment analyses), tight junctions appeared among the most significant pathways. In sticklebacks, Metzger and Schulte (2018) also showed that the category cell-cell-junction appeared among the top ten enriched cellular component GO categories comparing differentially methylated cytosines in different salinity conditions, which highly suggests that methylation changes control the expression of these genes in fish. In another study comparing slow-twitch and fast-twitch muscles in the teleost Pseudocaranx dentex, Li et al. (2022) also identified a correlation between DNA methylation and gene expression levels in genes involved in cell junctions (tight and adherens junctions). Tight junctions are composed of multiple membrane-spanning proteins such as occludins, claudins and several junctional adhesion molecules (Chasiotis et al., 2012). Claudins are very diverse in fish and mammals (Engelund et al., 2012) with a total of 61 cldn genes in European sea bass including all paralogs. Among the different genes that show differential expression and methylation levels, cldn4and cldn8 are supposed to be involved in increasing the epithelial tightness (Bagherie-Lachidan et al., 2009) which is essential in freshwater environments. We found two genes encoding for Claudin 8 with a significant upregulation (log2FC: 1.44 and 0.68) and hypomethylation. In gills of puffer fish (Tetraodon nigroviridis ), one claudin 8 paralog (Tncldn8d ) also showed an increased expression in FW, but not the 3 others (Bagherie-Lachidan et al., 2009). The overexpression of cldn 4 in FW was shown in other teleost species, like tilapia (Oreochromis mossambicus ) (Tipsmark et al., 2008), killifish (Whitehead et al., 2012), and rainbow trout (Oncorhynchus mykiss ) (Leguen et al., 2015). We identified two genes encoding for D. labrax Claudin 4 paralogs that were both upregulated in FW (log2FC: 0.87 and 1.47) and hypomethylated in promoters as well as other key genes involved in tight junction assembling (cgn , f11r, marveld3 ). Given the enrichment of the tight junction pathway for upregulation and hypomethylation, there is some evidence that claudin promoters, first exons or introns are a preferential target for differential methylation in changing salinity environments in euryhaline D. labrax . Studies in other teleost species on the effect of methylation on the tight junction pathway are required to confirm this trend in fish.