Epigenomic Analysis of Temperature-dependent Sex Determination

大约50年前，在一种非洲蜥蜴身上首次报道了温度依赖性的性别决定。从那以后，温度决定了一些鱼类和两栖动物、许多蜥蜴、许多海龟和所有鳄鱼的性别。然而，将温度转换为雄性与雌性发育的生物信号的机制尚不清楚。Rhen实验室已经发现了新的温度敏感基因，这些基因对鳄龟胚胎中的温度变化迅速做出反应。已知这些特定基因在其他物种的胚胎发育期间通过修改染色体结构来开启或关闭基因表达。这些因素也被称为表观遗传调节因子，可能在性别决定中发挥作用。拟议的研究将测试假设，即表观遗传机制调节睾丸或卵巢发育所需的基因表达。最终，这项研究将促进我们对温度依赖性性别决定的理解，并深入了解表观遗传机制在所有脊椎动物睾丸和卵巢发育中的作用。教育活动将通过课堂上的学期项目加强学习，通过2-3年的延长实习改善本科生的研究经验，并增加妇女和代表性不足的少数民族在生物学中的参与。外联活动将培训Rhen实验室成员更有效地向公众传播科学，通过制作关于温度依赖性性别决定的迷你纪录片向更广泛的受众传播研究结果，通过为美国老年人开设的非正式课程，针对STEM推广中被忽视的人口群体。在脊椎动物中的决定。最重要的假设是，温度依赖性的组蛋白甲基化的变化在调节睾丸与卵巢发育的转录程序中起着关键作用。实验测试的具体假设，即Polycomb抑制复合物2和赖氨酸特异性脱甲基酶6 B调节组蛋白H3的甲基化赖氨酸27（H3 K27）在性腺从男性和女性的热制度下孵育的胚胎。多梳抑制复合物2在赖氨酸27处甲基化组蛋白H3。三甲基化组蛋白H3（H3 K27 me 3）是一种稳定的表观遗传标记，抑制转录。相反，赖氨酸特异性脱甲基酶6 B通过将H3 K27 me 3脱甲基为H3 K27 me 2和H3 K27 me 1来允许基因表达。在目标1中，染色质免疫沉淀（ChIP）和定量PCR将用于分析温度对H3 K27甲基化在保守的性别决定基因座的影响。在目标2中，ChIP测序将用于分析温度对胚胎性腺中H3 K27甲基化全基因组模式的影响。在目标3中，实验将通过干扰基因表达和/或使用这些蛋白质的药理学抑制剂来测试多梳抑制复合物2和赖氨酸特异性脱甲基酶6 B是否因果地参与性别决定。这项研究将阐明性别决定基因表达温度依赖性的分子机制。调查结果将在公共数据库、同行评审出版物和国家/国际会议上公布。

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.