Regulation of Germ Cell Pluripotency Through The RNA-Binding Protein, DND1

描述（由申请人提供）：睾丸畸胎瘤作为良性、非精原细胞生殖细胞肿瘤出现在人类中，其特征在于多种特定细胞和组织类型的分化。这些不寻常的肿瘤反映了生殖细胞、胚胎干细胞和肿瘤细胞之间的密切关系，并掌握了有关编程这些细胞命运和调节它们之间转变的机制的重要线索。尽管畸胎瘤在啮齿动物模型中几乎是未知的，但在 20 世纪 70 年代发现了一种自发的小鼠突变 Ter，它导致睾丸畸胎瘤的高发病率，特别是在 129/SvJ 遗传背景上，但在其他品系（如 C57BL/6J）上则不然。在大多数遗传背景下，Ter-/- XY 生殖细胞在出生时完全消失。最近，Ter 突变被定位到 Dnd1，Dnd1 是参与斑马鱼生殖细胞调节的基因的小鼠直系同源物。 DND1 是一种 RNA 结合蛋白，针对参与生殖细胞分化和细胞周期控制的几种已知转录本，并保护它们免受 miRNA 介导的降解。根据我们的初步数据，我们假设 DND1 调节许多控制 PGC 发育过渡阶段生殖细胞编程的靶标。我们建议使用无偏 Y3H 筛选来识别 DND1 的 mRNA 客户端，并使用计算方法来识别相互作用的 miRNA 和可能解释对畸胎瘤形成的不同敏感性的可能的菌株差异。我们还计划创建 Dnd1 的条件标记等位基因，并在 (1) 生殖细胞特化/迁移阶段、(2) 在生殖细胞向睾丸中的原精原细胞转变的胎儿阶段以及 (3) 在出生后动物中删除它。我们将使用这个标记的等位基因来确定 DND1 在发育过程中的细胞定位，并对与其体内 mRNA 靶标结合的 DND1 进行免疫沉淀。我们还将从易感菌株 (129) 和非易感菌株 (B6) 中分离出 E14.5 处的野生型和突变生殖细胞，以获得全局表达谱。这些数据将与 DND1 靶标数据相关联，以阐明控制生殖细胞周期进展和对肿瘤形成的菌株敏感性的转录和转录后机制。翻译机制与肿瘤形成应变敏感性的转录网络的整合与人类生殖细胞肿瘤具有直接的临床相关性。 公共卫生相关性：生殖细胞对于生殖健康至关重要。它们的发育受到干扰会导致不育或形成生殖细胞肿瘤。通过整合转录和转录后信息的水平，我们将确定 DND1（一种 RNA 结合蛋白）控制生殖细胞的细胞周期和编程转换的机制。尽管 DND1 是生殖细胞特有的，但我们预计所涉及的调节机制可能对许多多能干细胞群体来说是常见的。

DESCRIPTION (provided by applicant): Testicular teratomas arise in humans as benign, non-seminomatous germ cell tumors characterized by the differentiation of a diverse array of specific cell and tissue types. These unusual tumors reflect the close relationship between germ cells, embryonic stem cells, and tumor cells, and hold important clues about the mechanisms that program these cell fates and regulate the transitions between them. Although teratomas were virtually unknown in rodent models, a spontaneous mouse mutation, Ter, was discovered in the 1970s that led to a high incidence of testicular teratomas, specifically on the 129/SvJ genetic background but not on other strains such as C57BL/6J. On most genetic backgrounds Ter-/- XY germ cells disappear completely by the time of birth. Recently, the Ter mutation was mapped to Dnd1, the mouse ortholog of a gene involved in germ cell regulation in zebrafish. DND1 is an RNA-binding protein that targets several known transcripts involved in germ cell differentiation and cell cycle control and protects them from miRNA-mediated degradation. Based on our preliminary data, we hypothesize that DND1 regulates many targets that control germ cell programming at transitional stages of PGC development. We propose to use an unbiased Y3H screen to identify the mRNA clients of DND1, and a computational approach to identify interacting miRNAs and possible strain differences that may account for the different sensitivity to teratoma formation. We also plan to create a conditional tagged allele of Dnd1 and delete it (1) at germ cell specification/migration stages, (2) during the fetal stage of germ cell transition to pro- spermatogonia in the testis and (3) in postnatal animals. We will use this tagged allele to determine the cellular localization of DND1 during development, and to immunoprecipitate DND1 bound to its in vivo mRNA targets. We will also isolate wild type and mutant germ cells at E14.5 from both the susceptible (129) and non-susceptible (B6) strains to obtain global expression profiles. These data will be correlated with DND1 target data to elucidate the transcriptional and post-transcriptional mechanisms governing germ cell cycle progression and strain sensitivity to tumor formation. The integration of translational mechanisms with transcriptional networks underlying strain sensitivity to tumor formation has immediate clinical relevance to human germ cell tumors. PUBLIC HEALTH RELEVANCE: Germ cells are crucial to reproductive health. Disruptions of their development lead to sterility or the formation of germ cell tumors. By integrating levels of transcriptional and post-transcriptional information, we will identify the mechanisms through which DND1, an RNA-binding protein, controls the cell cycle and programming transitions of germ cells. Although DND1 is specific to germ cells, we anticipate that the regulatory mechanisms involved may be common to many pluripotent stem cell populations.