Epigenomic Analysis of Temperature-dependent Sex Determination

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Temperature-dependent gender determination was first reported nearly 50 years ago in an African lizard. It has since been shown that temperature determines gender in some fish and amphibians, many lizards, numerous turtles, and all crocodilians. Yet, the mechanism that converts temperature into a biological signal for male versus female development is not known. The Rhen lab has identified novel temperature sensitive genes that respond quickly to temperature change in snapping turtle embryos. These particular genes are known to turn on or turn off gene expression during embryonic development in other species by modifying chromosomal structure. Such factors are also known as epigenetic regulators and may play a role in gender determination. The proposed studies will test the hypothesis that epigenetic mechanisms regulate expression of genes required for testis or ovary development. Ultimately, this research will advance our understanding of temperature-dependent gender determination and provide insight into the role epigenetic mechanisms play in testis and ovary development in all vertebrates. Educational activities will enhance learning through semester long projects in the classroom, improve undergraduate research experiences through extended internships of 2-3 years, and increase the participation of women and underrepresented minorities in biology. Outreach activities will train Rhen lab members to communicate science more effectively to the public, disseminate findings to a wider audience through production of a mini-documentary about temperature-dependent gender determination, and target a demographic that is neglected in STEM outreach through an informal class for older Americans.The Rhen lab has developed the common snapping turtle as a model to study unique and conserved mechanisms of gender determination in vertebrates. The overarching hypothesis is that temperature-dependent changes in histone methylation play a key role in regulating transcriptional programs for testis versus ovary development. Experiments test the specific hypothesis that Polycomb Repressive Complex 2 and lysine-specific demethylase 6B regulate methylation of histone H3 on lysine 27 (H3K27) in gonads from embryos incubated under male and female thermal regimes. Polycomb Repressive Complex 2 methylates histone H3 at lysine 27. Trimethylated histone H3 (H3K27me3) is a stable epigenetic mark that represses transcription. Conversely, lysine-specific demethylase 6B allows gene expression by demethylating H3K27me3 to H3K27me2 and H3K27me1. In Aim 1, chromatin immunoprecipitation (ChIP) and quantitative PCR will be used to analyze temperature effects on H3K27 methylation at conserved gender-determining loci. In Aim 2, ChIP-Sequencing will be used to analyze temperature effects on genome-wide patterns of H3K27 methylation in embryonic gonads. In Aim 3, experiments will test whether Polycomb Repressive Complex 2 and lysine-specific demethylase 6B are causally involved in gender determination by perturbing gene expression and/or using pharmacological inhibitors of these proteins. This research will clarify the molecular mechanisms underlying temperature-dependent expression of gender-determining genes. Findings will be presented in public databases, in peer-reviewed publications, and at national/international meetings.