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: 10642896
• 负责人: Blanche Capel
• 金额: $ 38.66万
• 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/10642896
• 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; Dissociation; Enzymes; Epigenetic Process; Gametogenesis; Gelatin; Genetic Transcription; Genus Mentha; Germ Cells; Gonadal structure; Heterogeneity; Histones; Hour; Image; Immunoprecipitation; In Situ; Individual; Knowledge; Label; Lamins; 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; 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.

摘要 众所周知，在成体精原干细胞(SSCs)建立之前，体内的染色质 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得到回答。是否所有的MGC都要经过 类似的重新编程，还是该过程对特定的子群体有选择性，从而产生SSC？ 甲基化、组蛋白修饰和染色质结构的改变如何抑制发育 路径和稳定的SSC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(DND1)的镜头解决这些重要问题。 DND1是维持MGC特性所必需的，因为DND1功能的丧失会导致生殖细胞的转分化 细胞对体细胞命运或G0的破坏和畸胎瘤的形成。我们最近开发出了一种转基因 DND1和GFP之间的功能性融合蛋白从内源性表达的小鼠系 基因座(Dnd1GFP)记者在G0期区分了两个MGC群体：DND1-lo细胞和DND1-Hi细胞 细胞，只占人口的5%-12%。RNA-seq显示DND1转录水平以及 在DND1-GFP-HI细胞中，多种表观遗传调控因子的转录水平高出5-10倍。此外， 利用抗DND1-GFP抗体的RNA免疫沉淀(RIP)鉴定多种表观遗传调控因子 这些都是G0期间DND1的目标。这些靶标包括DNA甲基转移酶(DNMT)，一种酶 SetDB1，它强加了核层相关的抑制性组蛋白标记(H3K9me3)，5个Tudor结构域 蛋白质(Tdrd)和四个肌动蛋白依赖调节因子(Smarcs)与基质结合发挥作用 脚手架。我们假设(1)在DND1-HI细胞中，DND1控制转录后调控 通过稳定表观遗传修饰物的转录和门控翻译，以及(2)许多 这些表观遗传调节因子的作用与依赖内核膜(INM)有关 在维护围棋时。该项目将测试MGC之间的异构性的重要性，关键是 RBP、DND1在表观遗传重编程中的作用，以及细胞周期停滞的作用。在目标1中，我们将 确定DND1-HI细胞代表的是克隆性还是暂时性群体，以及 表观遗传调节因子在DND1-hi或DND1-lo细胞中受到保护和差异翻译。在目标2中，我们将 绘制表观遗传学随时间的变化图，并确定它们在DND1-HI和DND1-LO细胞中是否不同。在AIM 3，我们将确定正在变化的染色质结构域是否定位于核层，以及 是否通过阻断与Lamins的关联或扰乱细胞周期停滞来扰乱变化。这个项目 将讨论mGC之间异质性的重要性，以及细胞周期停滞和核 小羊羔。我们预计这些结果将揭示生理、疾病相关或环境方面的脆弱性 改变表观遗传机制并影响配子发生和雄性生育的条件。