表观遗传因子Kdm6b在红耳龟温度依赖型性别决定中的作用及其机制研究

Temperature-dependent sex determination represents an extraordinary model of phenotypic plasticity, however its underlying molecular mechanism has long been a mystery. The key step to decipher the TSD mechanism is to answer how the ambient temperature regulates the expression of genes responsible for initiation of gonadal differentiation. Epigenetic mechanism, capable of integrating environmental factors and regulation of gene expression, is hypothesized to serve as a potential regulatory mechanism of TSD, however, the direct evidence is lacking. Based on the previous RNA-seq data, histone demethylase Kdm6b is selected as the candidate epigenetic regulator. First, the expression profile and temperature-sensitivity of Kdm6b in early embryonic gonads of the red-eared slider turtle (TSD species) will be examined. Most importantly, the loss- and gain-of-function analysis of Kdm6b will be subsequently performedin turtle embryos, through the previously developed lentivirus mediated gene knockdown and knock-in system. Meanwhile, The screen and identification of target genes of Kdm6b will be performed through ChIP-seq and rescue experiments. Finally, the way how Kdm6b functions on the target genes will be analyzed. This study aims to elucidate the functional role of Kdm6b in TSD and its regulatory mechanism, and to provide the direct genetic evidence of contribution of epigenetic mechanism to TSD, which lays the foundation for further deciphering of TSD mechanism.

温度依赖型性别决定（TSD）是一种非常奇特的表型可塑性现象，其分子机理长期是个谜。破译TSD机制的关键在于解析温度是通过何种机制调控性腺分化启动基因的表达？作为整合环境因素和基因表达的媒介，表观遗传被推测是一种潜在的TSD调控机制，但一直缺乏直接的遗传证据。基于前期RNA-seq数据，本项目拟以红耳龟为TSD动物模型，分析组蛋白去甲基化酶基因Kdm6b在早期胚胎性腺中的表达规律和温度敏感性；采用慢病毒介导的基因敲低和过表达技术，重点进行Kdm6b的功能缺失和获得（loss- and gain-of-function）研究；通过ChIP-seq和功能回复实验（rescue）对Kdm6b下游靶基因进行筛选和功能鉴定；最后分析Kdm6b对靶基因的调控方式。本课题旨在阐明Kdm6b在红耳龟TSD中的生物学功能及其调控机制，提供表观遗传调控TSD的直接证据，为进一步破译TSD机制奠定基础。

作为整合环境因素和基因表达的媒介，表观遗传被认为是一种潜在的TSD（温度依赖型性别决定）调控机制，但一直缺乏直接的遗传证据。鉴于此，本项目以红耳龟为TSD动物模型，以表观遗传因子Kdm6b为研究对象，通过表达分析，功能缺失和获得研究，功能回复以及ChIP实验等，开展了Kdm6b的表达及其上下游调控机制研究，发现了（1）Kdm6b的表达方式属于温度依赖型，且能够快速响应温度变化；（2）Kdm6b缺失和过表达均会导致性别逆转；（3）KDM6B通过消除启动子区H3K27三甲基化标记，直接促进雄性性别决定基因Dmrt1转录，从而启动雄性发育。并且Dmrt1的过表达（或敲低）能够恢复由Kdm6b敲低（或过表达）引起的性逆转；（4）高温（产雌温度）下，降低pSTAT3水平会导致性腺由雄转雌，而同时敲低Kdm6b能够恢复此性逆转；（5）高温（产雌温度）下，降低pCaMKII水平会导致雌向雄性逆转，而同时激活pSTAT3能够使性腺重新回到雌性分化方向；（6）高温（产雌温度）促进性腺体细胞Ca2+内流，通过pCaMKII激活pSTAT3，pSTAT3直接绑定在Kdm6b的启动子区，抑制其转录，继而抑制雄性性别决定基因Dmrt1的表达，最终促进红耳龟雌性发育。基于以上结果，本项目揭示了Kdm6b在红耳龟TSD中必需的关键作用，并明确了Kdm6b的上下游调控机制及通路（温度-Ca2+-pCaMKII-pSTAT3-Kdm6b-Dmrt1），首次直接证明了表观遗传对TSD的调控作用，率先揭开了长期困扰学界的TSD机制的神秘面纱，提出了“温度-表观遗传-性别决定基因”假说和“pSTAT3-Kdm6b保守盒”的TSD调控模型，为“表观遗传调控TSD”提供了直接的遗传证据，为彻底破译TSD机制奠定了重要基础。