Molecular functional characterization of the setdb1 and its potential target gene sox5 illuminate the histone modification-mediated orchestration of gonadal development in Chinese tongue sole (Cynoglossus semilaevis)

SET (SuVar3-9, Enhancer of Zeste, Trithorax) domain bifurcated histone lysine methyltransferase 1, setdb1, is the predominant histone lysine methyltransferase catalyzing H3K9me3. Prior studies have illustrated that setdb1 and H3K9me3 critically regulate sex differentiation and gametogenesis. However, the molecular details by which setdb1 is involved in these processes in fish have been poorly reported. Here, we cloned and characterized the setdb1 ORF (open reading frame) sequence from Chinese tongue sole (Cynoglossus semilaevis). The setdb1 ORF sequence was 3,669 bp, encoding a 1,222-amino-acid protein. Phylogenetic analysis showed that setdb1 was structurally conserved. qRT-PCR revealed that setdb1 had a high expression level in the testes at 12 mpf (months post fertilization). Single -cell RNA-seq data at 24 mpf indicated that setdb1 was generally expressed in spermatogenic cells at each stage except for sperm and was centrally expressed in oogonia. H3K9me3 modification was observed in gonads with the immunofluorescence technique. Furthermore, the overexpression experiment suggested that sox5 was a candidate target of setdb1. sox5 was abundantly expressed in male and pseudomale gonads at 24 mpf. Single -cell RNA-seq data showed that sox5 was mainly expressed in spermatogonia and its expression gradually declined with differentiation. Taken together, our findings imply that setdb1 regulates sox5 transcription in gonads, which provides molecular clues into histone modification-mediated orchestration of sex differentiation and gametogenesis.