MeDIP-seq and nCpG analyses illuminate sexually dimorphic methylation of gonadal development genes with high historic methylation in turtle hatchlings with temperature-dependent sex determination

Abstract

BackgroundDNA methylation alters gene expression but not DNA sequence and mediates some cases of phenotypic plasticity. Temperature-dependent sex determination (TSD) epitomizes phenotypic plasticity where environmental temperature drives embryonic sexual fate, as occurs commonly in turtles. Importantly, the temperature-specific transcription of two genes underlying gonadal differentiation is known to be induced by differential methylation in TSD fish, turtle and alligator. Yet, how extensive is the link between DNA methylation and TSD remains unclear. Here we test for broad differences in genome-wide DNA methylation between male and female hatchling gonads of the TSD painted turtle Chrysemys picta using methyl DNA immunoprecipitation sequencing, to identify differentially methylated candidates for future study. We also examine the genome-wide nCpG distribution (which affects DNA methylation) in painted turtles and test for historic methylation in genes regulating vertebrate gonadogenesis.ResultsTurtle global methylation was consistent with other vertebrates (57% of the genome, 78% of all CpG dinucleotides). Numerous genes predicted to regulate turtle gonadogenesis exhibited sex-specific methylation and were proximal to methylated repeats. nCpG distribution predicted actual turtle DNA methylation and was bimodal in gene promoters (as other vertebrates) and introns (unlike other vertebrates). Differentially methylated genes, including regulators of sexual development, had lower nCpG content indicative of higher historic methylation.ConclusionsOurs is the first evidence suggesting that sexually dimorphic DNA methylation is pervasive in turtle gonads (perhaps mediated by repeat methylation) and that it targets numerous regulators of gonadal development, consistent with the hypothesis that it may regulate thermosensitive transcription in TSD vertebrates. However, further research during embryogenesis will help test this hypothesis and the alternative that instead, most differential methylation observed in hatchlings is the by-product of sexual differentiation and not its cause.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-017-0136-2) contains supplementary material, which is available to authorized users.

Thermal Response of Epigenetic Genes Informs Turtle Sex Determination with and without Sex Chromosomes

“…Differential transcription has been documented for many TSD and GSD turtle homologs of the genes in the regulatory network of mammalian gonadal development [Lovell-Badge et al, 2002;Eggers et al, 2014], and some evidence exists that epigenetic modifications such as DNA and histone methylation mediate some of the observed differential tran-scription in TSD taxa [Matsumoto et al, 2013;Radhakrishnan et al, 2017a;Ge et al, 2018]. A recent study demonstrated that differential transcription of epigenetic genes can be a powerful tool to identify candidate epigenetic processes involved in sexual development in TSD turtles that help guide subsequent functional assays [Ge et al, 2018].…”

“…Epigenetic mechanisms (methylation and piRNA [Piwi protein-associated RNA]) have been proposed to underlie sex determination in a number of species [Okamoto et al, 2011;Kuroki et al, 2013;Kiuchi et al, 2014;Venegas et al, 2016;Radhakrishnan et al, 2017a], and few genes of the epigenetic machinery have been reported to respond differentially to temperature in TSD taxa such as Kdm6b (also called JMJD3) in alligator [Yatsu et al, 2016] and slider turtles [Czerwinski et al, 2016], and JARID2 and JMJD3 in Jacky dragons that have a mixed GSD/TSD system [Deveson et al, 2017], both related to changes in histone methylation. Following the lead from such differential transcription evidence, it was recently demonstrated that the higher gene expression of Kdm6b at male-producing temperatures (MPT) in slider turtles yields higher KDM6D protein levels which act to remove the triple methylation of the histone H3K27 bound at the promoter of Dmrt1 , thus activating this testis development gene as part of the male differentiation cascade [Ge et al, 2018].…”

Embryonic Temperature Programs Phenotype in Reptiles

“…Epigenetic mechanisms have been shown to play a role in environmental regulation of several traits in animals. In recent years, attempts have been made to survey epigenetic mechanisms to understand how temperature programs phenotype including studies of DNA methylation and histone modifications in several reptiles with TSD (Navarro-Martin et al, 2011;Matsumoto et al, 2013Matsumoto et al, , 2016Czerwinski et al, 2016;Yatsu et al, 2016;Fan et al, 2017;Radhakrishnan et al, 2017Radhakrishnan et al, , 2018Ge et al, 2018;Martín-del-Campo et al, 2019).…”

<b><i>Hox</i></b> Genes in Reptile Development, Epigenetic Regulation, and Teratogenesis

“…Interestingly, sex determination in turtles seems to be epigenetically regulated since some studies have shown differential temperature-dependent DNA methylation levels between ovaries and testis in turtles presenting TSD [Matsumoto et al, 2013;2016;Venegas et al, 2016;Radhakrishnan et al, 2017]. Likewise, epigenetic mechanisms may be involved in the regulation of gene expression networks defining shape in the early embryo.…”