DNA methylation-mediated feminization in male Cynoglossus semilaevis with homozygous dmrt1 knockout

Dmrt1 is a conserved master male-sex-determining gene in vertebrates. In the tongue sole ( Cynoglossus semilaevis ), an economically important fish with a ZZ/ZW sex determination system and remarkable female growth superiority, loss of dmrt1 leads to a feminized phenotype in ZZ males. However, the underlying epigenetic regulatory mechanism remains unclear. To investigate this, we performed whole-genome bisulfite sequencing (WGBS) on the brain and gonads of wild-type and dmrt1 ⁻/⁻ males, integrated with RNA-seq data. Compared to wild-type males, we identified 2,993 and 15,173 differentially methylated regions (DMRs) in the brain, and 261,627 and 13,401 DMRs in the gonads of wild-type females and dmrt1 ⁻/⁻ males, respectively. This indicates that dmrt1 deficiency is associated with extensive alterations in DNA methylation. Further analysis revealed 49 genes whose promoter methylation levels were significantly negatively correlated with their expression. Functional enrichment analysis associated these genes with diverse biological processes including signal transduction and cellular communication, protein modification, metabolic processes, and cell adhesion. Crucially, several are potential key factors in sex regulation, influencing: sex determination and early gonadal development ( wt1 , nanos1 , tmem88 ); gametogenesis and germ cell function ( chd1l , pdia4 , serpine2 , ak1 , hgsnat , mmp14 , map7d2b , exoc3l4 , slc6a15 , slc27a4 , stc2 ); and cellular architecture and hormonal signaling ( dag1 , adamts17 , kalrn , pde8a , lats2 , flna , tas1r3 , krt13 , hebp2 , igfbp3 , ugt3 ). Overall, these findings provide novel molecular evidence and a set of candidate pathways for understanding the epigenetic mechanisms of sex determination in teleost fish.