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Posttranscriptional regulation of Dmrt1 during sexual development in Medaka

青鳉性发育过程中 Dmrt1 的转录后调控

基本信息

批准号：
264892131
负责人：
Dr. Amaury Herpin
金额：
--
依托单位：
Lehrstuhl für Entwicklungsbiochemie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2014
资助国家：
德国
起止时间：
2013-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/264892131?language=en
关键词：
Posttranscriptional regulation Dmrt1 during sexual

项目摘要

The development of males and females and bisexual reproduction is the most prevalent mode in metazoans. Despite the generality of the phenomenon itself the mechanisms how sex is determined are very different among various groups and have evolved repeatedly and independently. The underlying molecular pathways can change quickly during evolution, even within closely related groups of organisms. The factors involved in sex determination and gonad differentiation can also be substantially different. A single exception is the transcription factor Dmrt1, which is highly conserved in all metazoans and has been shown to be involved in sexual development from flies and worms up to humans. In vertebrates a precise regulation of gene expression with respect to cell type specificity, time of development and transcript level is requested to ensure a proper function in sex determination. We are using the small aquarium fish model medaka (Oryzias latipes) to study the role of Dmrt1 in vertebrate sexual development and on a more general level to contribute to an understanding of the evolution of the high plasticity of sex determining mechanisms. We have shown earlier that in medaka a duplicated copy of dmrt1 (dmrt1bY) is the master male sex-determining gene. We found that regulation of dmrt1bY expression most critically occurs on the posttranscriptional level through a cell type specific and developmental time dependent differential mRNA stability. We identified a protein-binding motif in the 3' UTR of dmrt1bY to be responsible for this regulation. Interestingly, this motif is highly conserved in dmrt1 homologues of invertebrates and vertebrates, including human. In this project we want to identify the proteins which interact with the dmrt1 3' UTR and regulate its expression, as well as their interaction with other germline and gonad determining genes. On the one hand, we propose to study two candidate proteins that have been reported to bind to two different sequences, which both together make up the dmrt1 3' UTR consensus binding motif. This will be done after recombinant expression of both proteins and assaying their binding activities in vitro. In parallel, we will take an unbiased approach and screen for binding factors by affinity column purification. For functional characterization, we will study the expression pattern of the 3' UTR box binding proteins and further on use factor overexpression, gene knockdown and gene knockout approaches to validate their role in dmrt1 posttranscriptional expression regulation. Finally, the factors characterized in the fish model will be studied in mouse for their role in sexual development. This should contribute to a better understanding how the highly conserved sex determining factor Dmrt1 exerts its function in vertebrates and increase the knowledge about the complex regulatory interactions that lead to determination of the gonad anlage towards testis or ovary development in vertebrates.

雌雄同体的发育和双性生殖是后生动物中最普遍的模式。尽管这一现象本身具有普遍性，但在不同的群体中，决定性别的机制却非常不同，而且是反复而独立地进化的。在进化过程中，潜在的分子途径可以迅速改变，甚至在密切相关的生物群体中也是如此。涉及性别决定和性腺分化的因素也可能有本质上的不同。唯一的例外是转录因子Dmrt1，它在所有后生动物中都是高度保守的，并被证明与从苍蝇和蠕虫到人类的性发育有关。在脊椎动物中，基因表达需要根据细胞类型特异性、发育时间和转录水平进行精确调控，以确保其在性别决定中发挥适当的作用。我们正在使用小型观赏鱼模型medaka （Oryzias latipes）来研究Dmrt1在脊椎动物性发育中的作用，并在更一般的水平上有助于理解性别决定机制的高度可塑性的进化。我们之前已经表明，在medaka中，dmrt1 （dmrt1bY）的复制拷贝是主要的雄性性别决定基因。我们发现dmrt1bY表达的调控最关键地发生在转录后水平，通过细胞类型特异性和发育时间依赖性的差异mRNA稳定性。我们在dmrt1bY的3' UTR中发现了一个蛋白质结合基序，负责这种调节。有趣的是，该基序在无脊椎动物和脊椎动物（包括人类）的dmrt1同源物中高度保守。在这个项目中，我们想要鉴定与dmrt1 3' UTR相互作用并调节其表达的蛋白，以及它们与其他生殖系和性腺决定基因的相互作用。一方面，我们建议研究两个候选蛋白，这两个候选蛋白已被报道与两个不同的序列结合，这两个序列共同构成dmrt1 3' UTR共识结合基序。这将在这两种蛋白的重组表达和测定它们在体外的结合活性之后进行。同时，我们将采取无偏的方法，通过亲和柱纯化筛选结合因子。在功能表征方面，我们将研究3' UTR盒子结合蛋白的表达模式，并进一步利用因子过表达、基因敲低和基因敲除的方法来验证它们在dmrt1转录后表达调控中的作用。最后，将在小鼠中研究鱼类模型中特征的因素在性发育中的作用。这将有助于更好地理解高度保守的性别决定因子Dmrt1如何在脊椎动物中发挥其功能，并增加对复杂的调节相互作用的了解，这些相互作用导致了脊椎动物睾丸或卵巢发育性腺的决定。