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

项目摘要 干细胞依靠不对称的细胞分裂来维持其干细胞种群并产生 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特性， 它们依赖于表观遗传信息。表观遗传信息可以被定义为调节基因的因素 表达发生变化，而不改变主要的DNA序列。表观遗传调节因子Polycomb基团 (PCG)，是一个保守的转录抑制复合体，调节许多干细胞谱系。减少了…… PCG功能会导致干细胞的丧失，而PcG活性的增加会导致 干细胞谱系中的祖细胞，因此肿瘤生长。因此，PcG活动的准确控制是 是正常发育、组织动态平衡和再生所必需的。通过使用雄性果蝇 作为一个模型系统，我们的实验室研究了表观遗传学对干细胞维持、增殖、 和差异化。在果蝇的睾丸中，有两类干细胞，即生殖系干细胞 (GSCs)和囊干细胞(CySCs)。陈实验室之前的研究报告说，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- 由表观遗传调节器自主调节。最近，PcG的Polycomb(Pc)子组件已经 在果蝇生殖系的早期生殖细胞中，被发现定位于单个主要的小点。在……里面 此外，Pc点还富含DNA复制起始和组蛋白部位的身体成分。至 确定这个丰富的Pc点的功能，首先，我想描述一下主要的结构 通过一系列基因突变分析确定关键的Pc点 成核组分及其相互作用。然后，我将研究Pc标点是如何通过 纯化Pc蛋白，进行液滴形成、单分子FRET等生物物理检测 研究不同小点成分的分子相互作用来创建结构。最后，我会 通过检测Pc的突变如何影响它的能力，在体外和体内研究这个小点的功能。 体外相分离，然后将该Pc变异体加入体内突变背景中，以检测其表型 对生殖系的影响。这些研究的结果应该有助于我对筹委会的形成有更深入的了解 小点及其在生殖细胞DNA复制启动中的潜在作用。通过这项工作，我希望提升 我们在干细胞和染色质生物学领域的基本知识将在未来应用于治疗 癌症的治疗。由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，以及我将从同事导师那里得到的支持，将使我能够完成我的论文工作 成为一名全面发展的科学家，准备在一家专注于研究的机构从事博士后工作。

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.