Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University, Taian 271018, Shandong, China; Key Laboratory of Efficient Utilization of Non-grain Feed Resources (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Shandong Agricultural University, Taian 271018, Shandong, China.
Department of Biochemistry and Molecular Biology, Institute of Marine and Environmental Technology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Curr Biol. 2023 Dec 4;33(23):5057-5070.e5. doi: 10.1016/j.cub.2023.10.044. Epub 2023 Nov 22.
Sex determination in many fish species is remarkably plastic and temperature sensitive. Nile tilapia display a genetic sex-determination system (XX/XY). However, high-temperature treatment during critical thermosensitive periods can induce XX females into XXm pseudo-males, and this phenomenon is termed temperature-induced sex reversal (TISR). To investigate the molecular mechanism of TISR in Nile tilapia, we performed Iso-seq analysis and found a dramatic effect of high temperature on gene alternative splicing (AS). Kdm6bb histone demethylase showed a novel AS at intron 5 that generates Kdm6bb_tv1 transcripts without intron 5 and Kdm6bb_tv2 with intron 5. Kdm6bb_tv1 encodes a full-length protein while Kdm6bb_tv2 encodes a truncated protein. Expression analysis revealed that intron 5 splicing of Kdm6bb is male and gonad biased at larval stage, and only gonad biased at adult stage. High-temperature treatment induced intron 5 splicing in the gonads of XX and XY fish, resulting in increased Kdm6bb_tv1 expression. To directly test the role of Kdm6bb_tv1 in Nile tilapia TISR, we knocked out expression of Kdm6bb_tv1. However, Kdm6bb_tv1 homozygous mutants showed embryonic lethality. Overexpression of Kdm6bb_tv1, but not Kdm6bb_tv2, induced sex reversal of XX females into pseudo-males. Overexpression of Kdm6bb_tv1, as with high-temperature treatment, modified the promotor region of Gsdf and Dmrt1 by demethylating the trimethylated lysine 27 of histone 3 (H3K27me3), thereby increasing expression. Collectively, these studies demonstrate that AS of Kdm6bb intron 5 increases the expression of Kdm6bb_tv1, which acts as a direct link between high temperature and activation of Gsdf and Dmrt1 expression, leading to male sex determination.
在许多鱼类物种中，性别决定具有显著的可塑性和温度敏感性。尼罗罗非鱼表现出一种遗传性别决定系统（XX/XY）。然而，在关键热敏期高温处理会诱导 XX 雌性变成 XXm 假雄性，这种现象被称为温度诱导性别反转（TISR）。为了研究尼罗罗非鱼 TISR 的分子机制，我们进行了 Iso-seq 分析，发现高温对基因选择性剪接（AS）有显著影响。Kdm6bb 组蛋白去甲基酶在 5 号内含子处表现出一种新的 AS，产生没有 5 号内含子的 Kdm6bb_tv1 转录本和具有 5 号内含子的 Kdm6bb_tv2 转录本。Kdm6bb_tv1 编码全长蛋白，而 Kdm6bb_tv2 编码截短蛋白。表达分析显示，Kdm6bb 的 5 号内含子剪接在幼虫期具有雄性和性腺偏倚性，而在成年期仅具有性腺偏倚性。高温处理诱导 XX 和 XY 鱼性腺中的 5 号内含子剪接，导致 Kdm6bb_tv1 表达增加。为了直接测试 Kdm6bb_tv1 在尼罗罗非鱼 TISR 中的作用，我们敲除了 Kdm6bb_tv1 的表达。然而，Kdm6bb_tv1 纯合突变体表现出胚胎致死性。Kdm6bb_tv1 的过表达，但不是 Kdm6bb_tv2 的过表达，将 XX 雌性诱导成假雄性。Kdm6bb_tv1 的过表达，与高温处理一样，通过去甲基化组蛋白 3（H3K27me3）上的三甲基赖氨酸 27 修饰 Gsdf 和 Dmrt1 的启动子区域，从而增加表达。总之，这些研究表明 Kdm6bb 内含子 5 的 AS 增加了 Kdm6bb_tv1 的表达，Kdm6bb_tv1 作为高温和激活 Gsdf 和 Dmrt1 表达之间的直接联系，导致雄性性别决定。
