DND1 Mediates Epigenetic Reprogramming During Cell Cycle Arrest In Male Germ Cells

摘要 众所周知，在成熟精原干细胞（SSCs）建立之前， 雄性生殖细胞（MGCs）在长时间的有丝分裂停滞期间经历广泛的表观遗传重编程 （G0）。然而，关于这一进程的一些关键问题仍然没有答案。是否所有MGCs都要接受 类似的重编程，或者该过程对于将产生SSC的特定亚群是选择性的？ 甲基化、组蛋白修饰和染色质结构沉默的变化如何影响发育 途径和稳定SSC的命运？这些变化依赖于细胞周期停滞吗？在此，我们建议 通过RNA结合蛋白（RBP）死端1（DND 1）的透镜来解决这些重要问题。 DND 1是维持MGC身份所必需的，因为DND 1功能的丧失导致生殖细胞的转分化。 细胞向体细胞命运或G 0破坏和畸胎瘤形成。我们最近开发了一种转基因的 小鼠系，其中DND 1和GFP之间的功能性融合蛋白从内源性 基因座（Dnd 1GFP）。该报告者区分了G 0期的两种MGC群体：DND 1-lo细胞和DND 1-hi细胞 细胞，仅占人口的5-12%。RNA-seq显示，Dnd 1转录水平以及 在DND 1-GFP-hi细胞中，多种表观遗传调节因子的转录水平高5-10倍。此外，委员会认为， 使用抗DND 1-GFP抗体的RNA免疫沉淀（RIP）鉴定了多种表观遗传调节因子 在G 0期是DND 1的目标。在这些靶标中有DNA甲基转移酶（Dnmts）， Setdb 1，即施加核纤层相关的抑制性组蛋白标记（H3 K9 me 3），5个Tudor结构域 蛋白（Tdrds）和四种与基质相关的肌动蛋白依赖性调节因子（Smarcs） 脚手架我们推测（1）在DND 1-hi细胞中，DND 1控制着转录后调控， 表观遗传修饰剂通过稳定其转录和门控翻译和（2），许多 这些表观遗传调节因子与内核膜（INM）依赖性 维护GO。该项目将测试MGCs之间异质性的重要性，即关键因素 RBP，DND 1在表观遗传重编程中的功能，以及细胞周期阻滞的作用。在目标1中，我们 确定DND 1-hi细胞是否代表克隆或瞬时群体，以及 表观遗传调节因子在DND 1-hi或DND 1-lo细胞中受到保护并差异翻译。在目标2中，我们 绘制随时间推移的表观遗传变化，并确定它们在DND 1-hi和DND 1-lo细胞中是否不同。在Aim中 3，我们将确定发生变化的染色质结构域是否定位于核纤层蛋白， 是否通过阻断与核纤层蛋白的结合或破坏细胞周期停滞来破坏变化。这个项目 将解决MGCs之间的异质性的意义和细胞周期阻滞的作用和核 核纤层我们希望这些结果将揭示生理，疾病相关或环境的脆弱性 改变表观遗传机制并影响配子发生和男性生育力的条件。

ABSTRACT It is well known that prior to the establishment of adult spermatogonial stem cells (SSCs), the chromatin in male germ cells (MGCs) undergoes extensive epigenetic reprogramming during a long period of mitotic arrest (G0). However, a number of critical questions about this process remain unanswered. Do all MGCs undergo similar reprogramming or is the process selective for a specific sub-population that will give rise to the SSCs? How do changes in methylation, histone modifications, and chromatin structure silence developmental pathways and stabilize SSC fate? Do these changes depend on cell cycle arrest? Here, we propose to address these important questions through the lens of the RNA-binding protein (RBP) dead end 1 (DND1). DND1 is essential to maintain MGC identity since loss of Dnd1 function results in trans-differentiation of germ cells to somatic fates or disruption of G0 and formation of teratomas. We recently developed a transgenic mouse line in which a functional fusion protein between DND1 and GFP is expressed from the endogenous locus (Dnd1GFP). This reporter distinguishes two MGC populations during G0: DND1-lo cells and DND1-hi cells, which represent only 5-12% of the population. RNA-seq revealed that Dnd1 transcript levels as well as transcript levels for multiple epigenetic regulators are 5-10 fold higher in DND1-GFP-hi cells. Furthermore, RNA immunoprecipitation (RIP) using antibodies against DND1-GFP identified multiple epigenetic regulators that are targets of DND1 during G0. Among these targets are DNA methyltransferases (Dnmts), the enzyme Setdb1, that imposes the nuclear lamina associated repressive histone mark (H3K9me3), five Tudor domain proteins (Tdrds), and four actin dependent regulators (Smarcs) that function in association with a matrix scaffold. We hypothesize (1) that in DND1-hi cells, DND1 controls the post-transcriptional regulation of epigenetic modifiers by stabilizing their transcripts and gating translation and (2) that many of these epigenetic regulators function in association with the inner nuclear membrane (INM) dependent upon maintenance of GO. This project will test the significance of heterogeneity among MGCs, the critical function of the RBP, DND1 in epigenetic reprogramming, and the role of cell cycle arrest. In Aim 1, we will determine whether DND1-hi cells represent a clonal or transient population, and whether transcripts of epigenetic regulators are protected and differentially translated in DND1-hi or DND1-lo cells. In Aim 2, we will map epigenetic changes over time, and determine whether they differ in DND1-hi and DND1-lo cells. In Aim 3, we will determine whether chromatin domains undergoing change are localized to the nuclear lamins, and whether changes are disrupted by blocking association with lamins or disrupting cell cycle arrest. This project will address the significance of heterogeneity among MGCs and the role of cell cycle arrest and the nuclear lamins. We expect these results will uncover vulnerabilities to physiological, disease-related, or environmental conditions that alter epigenetic mechanisms and affect gametogenesis and male fertility.