Study mechanisms and biological significance underlying enrichment of Polycomb protein with DNA replication machinery components in Drosophila male germline

研究果蝇雄性种系中多梳蛋白与 DNA 复制机制成分富集的机制和生物学意义

• 批准号: 9976339
• 负责人: Velinda Vidaurre
• 金额: $ 4.55万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9976339
• 关键词: Affect; Affinity; Automobile Driving; Bacteria; Binding; Biological; Biological Assay; Biological Models; Biology; Biophysics; Cell Cycle; Cell Differentiation process; Cell Lineage; Cell Maintenance; Cell division; Cells; Characteristics; Chimeric Proteins; Chromatin; Codon Nucleotides; Communities; Complex; Cyst; DNA; DNA Sequence; DNA biosynthesis; Data; Defect; Development; Disease; Drosophila genus; Drosophila melanogaster; Epigenetic Process; Excision; Fluorescence Recovery After Photobleaching; Future; Gene Expression; Genes; Genetic; Genetic Transcription; Germ Cells; Germ Lines; Hematopoietic stem cells; Histones; Homeostasis; In Vitro; Institution; Kinetics; Knowledge; Label; Laboratories; Laboratory Study; Lead; Ligands; Light; Liquid substance; Maintenance; Measures; Meiosis; Mentors; Messenger RNA; Methyltransferase; Mitotic; Modification; Molecular; Molecular Conformation; Mutate; Mutation; Natural regeneration; Nomarski Interference Contrast Microscopy; Nuclear; PRC1 Protein; Phase; Phenotype; Physical condensation; Plasmids; Polycomb; Positioning Attribute; Postdoctoral Fellow; Proteins; RNA; Replication Initiation; Reporting; Research; Resources; Role; S Phase; Scientist; Series; Site; Sodium Chloride; Solubility; Structure; Surface; TATA-Binding Protein Associated Factors; Testing; Testis; Therapeutic; Tissues; Transgenes; Universities; Variant; Work; adult stem cell; base; biophysical properties; cancer therapy; cell type; epigenetic regulation; experimental study; fluidity; germline stem cells; histone modification; in vivo; insight; internal control; knock-down; leukemia; male; mutant; single-molecule FRET; stem cell population; stem cells; tumor; tumor growth

Project Summary Stem cells rely on asymmetric cell division to maintain their stem cell population and give rise to differentiated cells. In order for stem cells to continue to maintain their cell identity post asymmetric division, they rely on their epigenetic information. Epigenetic information can be defined as factors that regulate gene expression changes without altering the primary DNA sequences. The epigenetic regulator, Polycomb group (PcG), is a conserved transcription repressive complex that regulates many stem cell lineages. A decrease in PcG function can lead to a loss of stem cells, while an increase of PcG activity can cause overproliferation of progenitor cells in stem cell lineages and thus tumor growth. As a result, accurate control of PcG activity is necessary for normal development, tissue homeostasis and regeneration. By using the Drosophila male germline as a model system, our lab investigates epigenetic regulation on stem cell maintenance, proliferation, and differentiation. Within the Drosophila testis, there are two populations of stem cells, the germline stem cells (GSCs) and the cyst stem cells (CySCs). Previous studies from the Chen Laboratory reported that PcG genes act in the CySCs lineage to regulate GSC identity and activity, providing a new understanding of non-cell- autonomous regulation by an epigenetic regulator. More recently, the Polycomb (Pc) subcomponent of PcG has been found to localize into a single major punctum in early stage germ cells within the Drosophila germ line. In addition, the Pc punctum is also enriched for DNA replication initiation and histone locus body components. To determine the function of this enriched Pc punctum, first, I would like to characterize the main structural component of the Pc punctum by a series of genetic disruption assays to determine the key Pc punctum components for nucleation and their interactions. Then, I will study how the Pc punctum is able to form by purifying Pc protein to perform biophysical assays such as droplet formation and single molecule FRET to investigate the molecular interactions of different punctum components to create the structure. Finally, I will investigate the function of this punctum in vitro and in vivo, by examining how mutations in Pc affect its ability to phase separate in vitro and then adding this Pc variant to a mutant background in vivo, to examine its phenotypic effects on the germline. The results of these studies should help me to gain insight as to the formation of the Pc punctum and its potential role in DNA replication initiation in germ cells. Through this work I hope to enhance our basic knowledge in the stem cell and chromatin biology fields to be applied in the future to therapeutic treatment of cancers. The ample scientific resources and supportive scientific community provided by Johns Hopkins University, and the support I will receive from my co-mentors, will allow me to complete my thesis work and become a well-rounded scientist ready to pursue a post-doctoral position at a research focused institution.

项目摘要 干细胞依靠不对称细胞分裂来维持其干细胞群体，并产生 分化细胞为了使干细胞在不对称分裂后继续保持其细胞身份， 他们依赖于表观遗传信息表观遗传信息可以定义为调控基因表达的因子 表达改变而不改变一级DNA序列。表观遗传调节因子Polycomb组 (PcG)是一种保守的转录抑制复合物，调节许多干细胞谱系。减少 PcG功能可导致干细胞的损失，而PcG活性的增加可导致干细胞的过度增殖。 干细胞谱系中的祖细胞，从而导致肿瘤生长。因此，准确控制PcG活性是 正常发育、组织稳态和再生所必需的。通过使用雄性果蝇 生殖系作为模型系统，我们的实验室研究了干细胞维持，增殖， 和差异化。在果蝇的睾丸中，有两类干细胞， （GSC）和囊肿干细胞（CySC）。Chen实验室以前的研究报告说，PcG基因 在CySC谱系中起作用，调节GSC的身份和活性，为非细胞- 由表观遗传调节器进行自主调节。最近，PcG的Polycomb（Pc）子组件已经 在果蝇生殖系内的早期生殖细胞中发现定位于单个大泪点。在 此外，Pc点还富含DNA复制起始和组蛋白基因座体成分。到 为了确定这种富集的Pc点的功能，首先，我想表征主要结构 通过一系列的遗传破坏试验来确定Pc punctum的关键组分 成核组分及其相互作用。然后，我将研究Pc点是如何形成的， 纯化Pc蛋白以进行生物物理测定，例如液滴形成和单分子FRET， 研究不同泪点成分的分子相互作用以创建结构。最后要 通过研究Pc突变如何影响其在体外和体内的功能， 体外相分离，然后将该Pc变体加入到体内突变背景中，以检查其表型 对生殖细胞的影响。这些研究的结果应该可以帮助我更深入地了解个人电脑的形成 生殖点及其在生殖细胞DNA复制起始中的潜在作用。通过这项工作，我希望提高 我们在干细胞和染色质生物学领域的基础知识将在未来应用于治疗 治疗癌症。约翰斯提供的充足的科学资源和支持性的科学界 霍普金斯大学，以及我的合作导师的支持，将使我能够完成我的论文工作 并成为一个全面的科学家准备追求在研究为重点的机构博士后职位。