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

通过 RNA 结合蛋白 DND1 调节生殖细胞多能性

基本信息

批准号：
8513346
负责人：
Blanche Capel
金额：
$ 28.07万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8513346
关键词：
Accounting Affect Alleles Animals Benign Binding Birth Caenorhabditis elegans Cell Cycle Progression Cell Cycle Regulation Cell Differentiation process Cell Lineage Cell Survival Cells Client Data Development Drosophila genus Embryo Female Fertilization Gatekeeping Genes Genetic Genome Germ Cells Germ cell tumor Goals Histocompatibility Testing Human Human Cell Line Incidence Lead Mammals Maps Mediating Meiosis Messenger RNA MicroRNAs Mitosis Molecular Molecular Profiling Mus Mutant Strains Mice Mutation Nonsense Mutation Orthologous Gene Outcome Pathway interactions Play Pluripotent Stem Cells Proteins RNA-Binding Proteins Regulation Reproductive Health Rodent Model Role Spermatogonia Staging Sterility Structure of primordial sex cell Teratoma Testicular Teratoma Testis Time Transcript Zebrafish base cell motility cell type clinically relevant embryonic stem cell fetal in vivo male migration mutant neoplastic cell nonseminomatous germ cell tumor pluripotency postnatal programs public health relevance stem cell population tumor tumor initiation

项目摘要

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.

描述（由申请人提供）：睾丸畸胎瘤在人类中以良性，非鼻膜细胞肿瘤的形式出现，其特征是分化了多种特定细胞和组织类型的阵列。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，并掌握了对这些细胞命运进行编程并调节其之间的过渡的重要线索。尽管在啮齿动物模型中实际上未知畸胎瘤，但在1970年代发现了自发的小鼠突变TER，导致睾丸畸胎瘤的高发生率，特别是在129/SVJ遗传背景上，但没有在其他菌株上，例如C57BL/6J。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，将TER突变映射到DND1，这是斑马鱼中涉及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，它靶向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调节了许多在PGC开发过渡阶段控制生殖细胞编程的目标。我们建议使用公正的Y3H屏幕来识别DND1的mRNA客户端，并采用计算方法来识别相互作用的miRNA和可能的菌株差异，这可能解释了对畸胎瘤形成的不同敏感性。我们还计划在生殖细胞规格/迁移阶段中创建有条件的DND1等位基因，并在生殖细胞规格/迁移阶段中删除（1），在生殖细胞的胎儿阶段过渡到睾丸中的细胞类药物，在产后动物中（3）。我们将使用此标记的等位基因来确定发育过程中DND1的细胞定位，并免疫沉淀DND1与其体内mRNA靶标结合。我们还将与E14.5处的野生型和突变细胞分离，从易感（129）和不敏感（B6）菌株中分离出来，以获得全局表达谱。这些数据将与DND1目标数据相关，以阐明管理生殖细胞周期进程以及对肿瘤形成的应变敏感性的转录和转录机制。对肿瘤形成的应变敏感性的转录机制与转录网络的整合具有与人类生殖细胞肿瘤的临床相关性。公共卫生相关性：生殖细胞对于生殖健康至关重要。它们发育的破坏会导致无菌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过渡的机制。尽管DND1是针对生殖细胞的，但我们预计所涉及的调节机制可能是许多多能干细胞群体共有的。