Epigenetic regulation of sex: the role of DNA methylation and zbtb38 in zebrafish sex differentiation and heat-induced masculinization

There is increasing evidence that global change can threaten biodiversity by inducing skewed sex ratios. Accumulating evidences support a role of epigenetics, mainly DNA methylation, in sex differentiation. The aim of the present work was to investigate the potential role of zbtb38, a transcriptional factor that binds to methylated promoters, in sex differentiation and/or maintenance in zebrafish. We analyzed the methylation and transcription level of zbtb38 in males, females and undifferentiated individuals raised at standard or high temperature, a masculinizing factor. Results were compared to those obtained for genes already known to be involved in sex differentiation/maintenance (cyp19a1a, foxl2a, dmrt1). All genes presented a sex-specific pattern of DNA methylation and transcription but the most significant differences between sexes were observed for zbtb38. Moreover, a highly significant positive correlation was observed between the methylation level of zbtb38 and cyp19a1a, which encodes an enzyme that converts androgens into estradiol. However, while the hypermethylation of cyp19a1a was associated with its down-regulation, an inverse relationship was observed for zbtb38, providing a basis for mutual antagonism. Furthermore, zbtb38 was the only gene for which its transcription level was affected by temperature, being up-regulated in females that escaped to masculinization. Finally, despite embryos presented a paternal methylome, zbtb38 was the only gene for which its methylation level rapidly changed during early development to reach intermediate values between males and females at the larval stage, ie a bi-potential state. Our results strongly support a strategic role of DNA methylation and zbtb38 in sex differentiation and maintenance.