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

DND1 在雄性生殖细胞细胞周期停滞期间介导表观遗传重编程

• 批准号: 10490349
• 负责人: Blanche Capel
• 金额: $ 39.05万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-17 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10490349
• 关键词: ATAC-seq; Actins; Address; Adult; Affect; Antibodies; Binding; Biological Assay; Cell Cycle Arrest; Cell Cycle Regulation; Cells; Chimeric Proteins; Chromatin; Chromatin Structure; Clone Cells; DNA Methylation; DNA Modification Methylases; Data; Development; Disease; Enzymes; Epigenetic Process; Gametogenesis; Gelatin; Genus Mentha; Germ Cells; Gonadal structure; Heterogeneity; Histones; Hour; Image; Immunoprecipitation; In Situ; Individual; Knowledge; Label; Lamins; Lead; Maintenance; Maps; Mediating; Messenger RNA; Methylation; Mitotic; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Lamin; Nuclear Lamina; Pathway interactions; Pattern; Physiological; Population; Post-Transcriptional Regulation; Process; Proteins; RNA; RNA Stability; RNA-Binding Proteins; Reporter; Role; Slide; Spermatocytes; Teratoma; Tertiary Protein Structure; Testing; Time; Transcript; Transgenic Mice; Translating; Translations; Western Blotting; Work; base; epigenetic regulation; epigenome; histone modification; in vivo; insight; lens; loss of function; male; male fertility; novel; pluripotency; scaffold; stem cell fate; stem cell population; stem cells; subfertility; transcriptome; transcriptome sequencing; transdifferentiation

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.

抽象的 众所周知，在建立成年精子干细胞（SSC）之前，染色质 雄性生殖细胞（MGC）在长时间的有丝分裂停滞期间经过广泛的表观遗传重编程 （G0）。但是，有关此过程的许多关键问题仍未得到解决。做所有的MGC进行 类似的重新编程，还是对于会导致SSC的特定子群体选择的过程？ 甲基化，组蛋白修饰和染色质结构的变化如何变化 途径并稳定SSC命运？这些变化取决于细胞周期停滞吗？在这里，我们建议 通过RNA结合蛋白（RBP）死端1（DND1）的镜头解决这些重要问题。 DND1对于维持MGC身份至关重要，因为DND1功能的丧失导致细菌的跨差异 细胞会导致体细胞命运或破坏G0和形成畸胎瘤。我们最近开发了一个转基因 小鼠系中DND1和GFP之间的功能融合蛋白来自内源性 基因座（DND1GFP）。该记者在G0：DND1-LO细胞和DND1-HI期间区分了两个MGC种群 细胞，仅占人群的5-12％。 RNA-Seq表明DND1转录水平以及 在DND1-GFP-HI细胞中，多个表观遗传调节剂的转录水平高出5-10倍。此外， 使用针对DND1-GFP的抗体确定了多个表观遗传调节剂的RNA免疫沉淀（RIP） 这是G0期间DND1的靶标。这些靶标包括DNA甲基转移酶（DNMT），酶 SETDB1，施加核层固定相关的抑制性组蛋白标记（H3K9me3），五个都铎王朝域 蛋白质（TDRDS）和四个与矩阵相关的依赖性调节剂（SMARC） 脚手架。我们假设（1）在DND1-HI细胞中DND1控制转录后调节 表观遗传修饰符通过稳定其转录物和门控翻译和（2） 这些表观遗传调节剂与内部核膜（INM）相关 维护途中。该项目将测试MGC之间异质性的重要性，即关键 RBP的功能，DND1在表观遗传重编程中和细胞周期停滞的作用。在AIM 1中，我们将 确定DND1-HI细胞是代表克隆人还是瞬态群体，以及转录本是否代表 表观遗传调节剂受到保护，并在DND1-HI或DND1-LO细胞中差异化。在AIM 2中，我们将 MAP表观遗传学随时间变化，并确定它们在DND1-HI和DND1-LO细胞中是否有所不同。目标 3，我们将确定发生变化的染色质结构域是否定位于核层粘蛋白，并且 是否通过阻断与层粘连蛋白的关联或破坏细胞周期停滞而破坏变化。这个项目 将解决MGC之间异质性的重要性以及细胞周期停滞和核的作用 薄荷。我们预计这些结果将发现生理，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能力的情况。