Temperature Shift Alters DNA Methylation and Histone Modification Patterns in Gonadal Aromatase (cyp19a1) Gene in Species with Temperature-Dependent Sex Determination.

Yuiko Matsumoto,Brette Hannigan,David Crews

doi:10.1371/journal.pone.0167362

Abstract

The environment surrounding the embryos has a profound impact on the developmental process and phenotypic outcomes of the organism. In species with temperature-dependent sex determination, gonadal sex is determined by the incubation temperature of the eggs. A mechanistic link between temperature and transcriptional regulation of developmental genes, however, remains elusive. In this study, we examine the changes in DNA methylation and histone modification patterns of the aromatase (cyp19a1) gene in embryonic gonads of red-eared slider turtles (Trachemys scripta) subjected to a temperature shift during development. Shifting embryos from a male-producing temperature (MPT) to a female-producing temperature (FPT) at the beginning of the temperature-sensitive period (TSP) resulted in an increase in aromatase mRNA expression while a shift from FPT to MPT resulted in decreased expression. DNA methylation levels at CpG sites in the promoter of the aromatase gene were high (70–90%) at the beginning of TSP, but decreased in embryos that were incubated at constant FPT and those shifted from MPT to the FPT. This decrease in methylation in the promoter inversely correlated with the expected increase in aromatase expression at the FPT. The active demethylation under the FPT was especially prominent at the CpG site upstream of the gonad-specific TATA box at the beginning of TSP and spread downstream of the gene including exon1 as the gonad development progressed. In embryos incubated at FPT, the promoter region was also labeled by canonical transcriptional activation markers, H3K4me3 and RNA polymerase II. A transcriptional repression marker, H3K27me3, was observed in temperature-shifted gonads of both temperature groups, but was not maintained throughout the development in either group. Our findings suggest that DNA hypomethylation and H3K4me3 modification at the aromatase promoter may be a primary mechanism that releases a transcriptional block of aromatase to initiate a cascade of ovarian differentiation.

Highlights

Primordial gonads are initially bipotential; namely, having the capacity to develop into either testes or ovaries when they are generated from the coelomic surface of mesonephrous tissue during embryonic development
When mRNA levels were compared between two control temperatures, male-producing temperature (MPT) and femaleproducing temperature (FPT), the gonadal expression at stage 16 tended to be higher in the FPT group relative to those in the MPT group (P = 0.05, Wilcoxon rank sum; S2 Fig)
The expression of aromatase in differentiating gonads have been examined in various species, the actual role of aromatase during gonad sex determination is often regarded as a secondary effect; that is to facilitate the differentiation process that follows once the gonadal trajectory is fixed by the environmental cue

Summary

Introduction

Primordial gonads are initially bipotential; namely, having the capacity to develop into either testes or ovaries when they are generated from the coelomic surface of mesonephrous tissue during embryonic development. Differentiation of the embryonic gonads into one of the developmental trajectories (i.e., testes or ovaries) depends on either heritable genetic factors (i.e., genotypic sex determination, GSD) or the physical and biotic environment (i.e., environmental sex determination, ESD). The gonadal sex of several fish species with sex chromosomes is influenced by exposure to extreme temperatures, which produces a skewed sex ratio [3,7]. This form of temperature-driven ESD system is known as temperature-dependent sex determination or TSD. In a TSD system, the initial mechanism linking an upstream trigger, i.e., ambient temperatures, and the downstream modifiers involved in the regulation of relevant gene expression remain largely unknown

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Conclusion