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

描述(申请人提供)：睾丸畸胎瘤发生在人类良性的，非精原细胞瘤的生殖细胞肿瘤，其特征是分化成不同的特定细胞和组织类型。这些罕见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，并掌握了有关这些细胞命运的编程和调节它们之间过渡的机制的重要线索。虽然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，导致了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上却没有。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，这是一种与斑马鱼生殖细胞调节有关的基因的小鼠同源基因。DND1是一种RNA结合蛋白，针对几个参与生殖细胞分化和细胞周期控制的已知转录本，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在PGC发育的过渡阶段调控许多控制生殖细胞编程的靶点。我们建议使用无偏Y3H筛选来确定DND1的mRNA客户，并使用一种计算方法来确定相互作用的miRNAs和可能的菌株差异，这些差异可能解释了对畸胎瘤形成的不同敏感性。我们还计划创建DND1的条件标记等位基因，并将其删除(1)在生殖细胞指定/迁移阶段，(2)在生殖细胞向精原细胞原转化的胎儿阶段，以及(3)在出生后的动物中。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，并免疫沉淀DND1与其体内mRNA靶标结合。我们还将在E14.5从敏感(129)和不敏感(B6)菌株中分离野生型和突变生殖细胞，以获得全球表达谱。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菌株对肿瘤形成的敏感性的转录和转录后机制。对肿瘤形成敏感的菌株的翻译机制与转录网络的整合与人类生殖细胞肿瘤有直接的临床相关性。 公共卫生相关性：生殖细胞对生殖健康至关重要。胚胎发育受阻会导致不育或生殖细胞肿瘤的形成。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变的机制。虽然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是共同的。

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.