DNA Methylation of the Gonadal Aromatase (cyp19a) Promoter Is Involved in Temperature-Dependent Sex Ratio Shifts in the European Sea Bass

et al. (2011) DNA Methylation of the Gonadal Aromatase (cyp19a) Promoter Is Involved in Temperature-Dependent Sex Ratio Shifts in the European Sea Bass. PLoS Genet 7(12): e1002447. doi:10.1371/journal.pgen.1002447 DNA Methylation of the Gonadal Aromatase (cyp19a ) Promoter Is Involved in Temperature-Dependent Sex Ratio Shifts in the European Sea Bass Laia Navarro-Martn 0 1 Jordi Vin as 0 1 Laia Ribas 0 1 Noelia Daz 0 1 Arantxa Gutie rrez 0 1 Luciano Di Croce 0 1 Francesc Piferrer 0 1 Emma Whitelaw, Queensland Institute of Medical Research, Australia 0 a Current address: Centre for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa , Ottawa , Canada b Current address: Laboratori d'Ictiologia Gene`tica, Departament de Biologia, Universitat de Girona , Girona , Spain 1 1 Institut de Cie`ncies del Mar , Consejo Superior de Investigaciones Cient ficas (CSIC), Barcelona , Spain , 2 Centre de Regulacio Gen o`mica (CRG)/ICREA and Univeristat Pompeu Fabra (UPF) , Barcelona , Spain Sex ratio shifts in response to temperature are common in fish and reptiles. However, the mechanism linking temperature during early development and sex ratios has remained elusive. We show in the European sea bass (sb), a fish in which temperature effects on sex ratios are maximal before the gonads form, that juvenile males have double the DNA methylation levels of females in the promoter of gonadal aromatase (cyp19a), the enzyme that converts androgens into estrogens. Exposure to high temperature increased the cyp19a promoter methylation levels of females, indicating that induced-masculinization involves DNA methylation-mediated control of aromatase gene expression, with an observed inverse relationship between methylation levels and expression. Although different CpGs within the sb cyp19a promoter exhibited different sensitivity to temperature, we show that the increased methylation of the sb cyp19a promoter, which occurs in the gonads but not in the brain, is not a generalized effect of temperature. Importantly, these effects were also observed in sexually undifferentiated fish and were not altered by estrogen treatment. Thus, methylation of the sb cyp19a promoter is the cause of the lower expression of cyp19a in temperature-masculinized fish. In vitro, induced methylation of the sb cyp19a promoter suppressed the ability of SF-1 and Foxl2 to stimulate transcription. Finally, a CpG differentially methylated by temperature and adjacent to a Sox transcription factor binding site is conserved across species. Thus, DNA methylation of the aromatase promoter may be an essential component of the long-sought-after mechanism connecting environmental temperature and sex ratios in vertebrate species with temperature-dependent sex determination. - Funding: LN-M and ND were supported by predoctoral scholarships and JV and LR by postdoctoral grants from the Spanish Ministry of Science and Innovation (MCINN). Research was funded by MCINN projects Sexgene (AGL2006-01359), Aquagenomics (CDS2007-0002), and Epigen-Aqua (AGL2010-15939) to FP and by MICINN project BFU2010-18692 and AGAUR grant to LDC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The sex ratio is a crucial demographic parameter important for population viability that is established by the processes of sex determination and differentiation. The sex determination mechanisms in vertebrates include genotypic sex determination (GSD), temperature-dependent sex determination (TSD) or a combination of both. In TSD, the temperature experienced during a particular time during early development, referred to as the thermosensitive period (TSP), irreversibly determines gonadal sex. TSD is well established in reptiles and fish [1]. Regardless of the sex determining system, in non-mammalian vertebrates the androgen-to-estrogen ratio determines whether a sexually undifferentiated gonad sexually differentiates into a testis or ovary. This sex steroid ratio depends of the activity of the enzyme aromatase, Cyp19a, the product of the cyp19a gene, which irreversibly converts androgens into estrogens. Further, in ectothermic vertebrates, the effects of environmental temperature on sex ratios are mediated by changes in cyp19a expression. Thus, in reptiles with TSD, exposure to female-promoting temperatures is invariably associated with gonadal cyp19a upregulation, whereas exposure to male-producing temperatures is associated with cyp19a suppression [2,3]. In all fish species analyzed so far, more males are produced with increasing temperatures [4]. The masculinizing effects of high temperature are also invariably caused by an inhibition of cyp19a expression and enzymatic activity [57]. Thus, regardless of the animal group and the sex determining mechanism considered, cyp19a regulation is a key player in the sex ratio response to temperature in vertebrates. Unfortunately, the molecular mechanism by which temperature affects cyp19a has remained elusive [1,8], and this is most important since identifying environmental cues and their perception and transduction mechanisms is a central focus of eco-devo research [9]. Gorelick [10] hypothesized that different methylation patterns of virtually identical sex chromosomes in species with TSD could be altered by small environmental changes, hence determining the Temperature changes during early embryonic and/or larval stages are able to modify sex ratios in fish and reptiles. However, the underlying mechanism by which temperature is able to modify the molecular pathways that developing gonads follow to become ovaries or testes is still unknown. One of the most interesting questions raised from previous studies with our model species, the European sea bass, was how temperature could affect the developmental fate of the gonads at a time when they were not even formed in the most rudimentary manner. This was the telltale sign of an epigenetic mechanism. In this study, DNA methylation levels of the aromatase promoter were analyzed in European sea bass exposed to different temperatures during early developmental stages. Aromatase is the enzyme that converts androgens (male hormones) into estrogens (female hormones), which are essential for ovarian development in all non-mammalian vertebrates. We show that increased temperature during a critical period in early development is able to increase DNA methylation of the aromatase promoter, preventing aromatase gene expression. We conclude that gonadal aromatase promoter methylation is most likely part of the long-sought-after mechanism connecting temperature and environmental sex determination in vertebrates. sex of individuals. He also proposed that sex differences are initially determined by different patterns of methylation on nuclear DNA of females and males. Recently, review (...truncated)