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

项目概要 干细胞依靠不对称细胞分裂来维持其干细胞群并产生 分化的细胞。为了使干细胞在不对称分裂后继续保持其细胞身份， 他们依赖于他们的表观遗传信息。表观遗传信息可以定义为调节基因的因素 表达发生变化而不改变原始 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，可调节许多干细胞谱系。减少 PcG 功能可导致干细胞损失，而 PcG 活性增加可导致干细胞过度增殖 干细胞谱系中的祖细胞，从而促进肿瘤生长。因此，精确控制 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，我们的实验室研究干细胞维持、增殖、 和差异化。果蝇睾丸内有两种干细胞群，即生殖干细胞 （GSC）和囊肿干细胞（CySC）。陈实验室之前的研究报告称，PcG 基因 在 CySCs 谱系中发挥作用，调节 GSC 的身份和活性，提供对非细胞- 表观遗传调节剂的自主调节。最近，PcG 的 Polycomb (Pc) 子组件已 被发现定位于果蝇生殖系内早期生殖细胞的单个主要泪点中。在 此外，Pc 泪点还富含 DNA 复制起始和组蛋白基因座体成分。至 确定这个富集Pc punctum的功能，首先，我想表征一下主要结构 通过一系列基因破坏分析确定 Pc 泪点的关键组成部分 成核成分及其相互作用。然后，我将研究 Pc 泪点是如何形成的 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，我会 通过检查 PC 突变如何影响其能力，研究该泪点的体外和体内功能 在体外进行相分离，然后将该 PC 变体添加到体内突变体背景中，以检查其表型 对种系的影响。这些研究的结果应该可以帮助我深入了解个人计算机的形成 泪点及其在生殖细胞 DNA 复制启动中的潜在作用。我希望通过这项工作提高 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 癌症的治疗。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共同导师的支持将使我能够完成我的论文工作 并成为一名全面发展的科学家，准备在以研究为重点的机构攻读博士后职位。

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.