Development and Application of a Novel Method to Study Histone Inheritance in Asymmetrically Dividing Cells

正常发育依赖于不对称细胞分裂(ACD)，这是一种分裂干细胞的过程 产生更新的干细胞和分化细胞。指导ACD的许多内在和外在因素 已被发现并确定了其特征。然而，染色质对细胞命运决定的贡献很小。 明白了。在此之前，我们的实验室发现组蛋白和组蛋白翻译后修饰的不对称性。 果蝇雄性生殖细胞的(HPTM)遗传。对这一过程的进一步剖析揭示了它的功能和 组蛋白的调节分三步进行：1)组蛋白不对称是在S期建立的；2)组蛋白不对称是在 在M期区分；3)遗传的不对称组蛋白读出指导细胞周期 有丝分裂退出后的进展。有趣的是，破坏这些不对称性会导致两种干细胞的丢失 和过度增殖的表型，表明不对称的组蛋白遗传是 组织健康。此外，这一过程的恶化在包括年龄相关的疾病中可能是常见的 组织退化和癌症。我假设不对称的组蛋白遗传是一种普遍的机制 这在干细胞动态平衡和发育过程中决定细胞命运方面起着至关重要的作用。然而，我们的 目前，追踪单个细胞内特定位点上的组蛋白和hPTM的能力受到严重限制。因此，我们 迫切需要新的工具来研究不对称组蛋白遗传的作用和后果 发育、干细胞动态平衡和细胞命运决定。在这项提议中，我将扩大我们的 了解组蛋白遗传在不对称分裂的细胞和细胞中的渗透性、作用和模式 2)发展了一种标记非重复基因座以追踪表观基因组特征的新方法。 首先，我将在果蝇神经干细胞中表达标记了光转化Dendra2的组蛋白 血统。神经母细胞(NBS)及其后代神经节母细胞(GMC)和转运放大中间体 神经前体细胞(INPs)是一种研究较多的干细胞模型系统。跟随405 nm光诱导 光转化，我将观察I型和II型中旧(红色)和新(绿色)组蛋白的遗传模式 国家广播公司。这些研究将1)扩大我们对ACD中组蛋白遗传的认识，2)揭示组蛋白是否 随着细胞不对称分裂、衰老和失去活力，遗传模式也会发生变化(例如INP)。 跟踪特定基因组位置的表观基因组信息传统上是使用 鱼。然而，鱼所需的热变性部分或完全不符合 DNA相关蛋白的检测。为了克服这一限制，我将使寡色画技术 设计指南RNA文库，将dCas9与特定的、非重复的基因座连接起来。这将使我能够跟踪 组蛋白和hPTM在特定基因组座位上的遗传模式与单细胞分辨率。 这些工作将加深我们对ACD潜在机制的理解，并为我们提供新的 便于检测研究和临床环境中的表观基因组变化的工具。

Proper development depends on asymmetric cell division (ACD), a process by which dividing stem cells produce a renewed stem cell and a differentiating cell. Many intrinsic and extrinsic factors guiding ACD have been found and characterized. Yet, the contribution of chromatin to cell-fate determination is poorly understood. Previously, our lab discovered asymmetries in histone and histone post-translational modification (hPTM) inheritance in Drosophila male germ cells. Further dissection of this process revealed it functions and is regulated in three-steps: 1) histone asymmetry is established during S-phase; 2) histone asymmetry is distinguished during M-phase; 3) the readout of the inherited asymmetric histones guides cell cycle progression following mitotic exit. Interestingly, disruption of these asymmetries results in both stem cell loss and overproliferation phenotypes, suggesting that asymmetric histone inheritance is an essential process in tissue health. Further, deterioration of this process may be common among diseases including age-related tissue degeneration and cancer. I hypothesize that asymmetric histone inheritance is a general mechanism that plays a crucial role in stem cell homeostasis and cell-fate determination during development. However, our ability to track histones and hPTMs at specific loci within single cells is severely limited at this time. Thus, we are in urgent need of new tools to study the roles and consequences of asymmetric histone inheritance in development, stem cell homeostasis, and cell-fate determination. In this proposal, I will 1) broaden our understanding of pervasiveness, roles, and patterns of histone inheritance in asymmetrically dividing cells and 2) develop a novel method for labeling non-repetitive loci to track epigenomic features. First, I will express histones tagged with the photoconvertible Dendra2 in Drosophila neuronal stem cell lineages. Neuroblasts (NBs), their progenies ganglion mother cells (GMCs), and transit-amplifying intermediate neuronal progenitors (INPs) are a well-studied stem cell model system. Following 405nm light induced photoconversion, I will observe the inheritance patterns of old (red) versus new (green) histones in type I and II NBs. These studies will 1) expand our knowledge on histone inheritance in ACD, and 2) reveal whether histone inheritance patterns change as asymmetrically dividing cells age and lose potency (e.g. INPs). Tracking epigenomic information at specific genomic locations has traditionally been performed using FISH. However, the heat denaturation required for FISH is either partially or completely incompatible with detection of DNA-associated proteins. To overcome this limitation, I will adapt Oligopaint technologies to design guide RNA libraries for tethering dCas9 to specific, non-repetitive loci. This will allow me to track histone and hPTM inheritance patterns at specific genomic loci with single-cell resolution. These works will both enhance our understanding of the mechanisms underlying ACD and provide new tools facilitating the detection of epigenomic alterations in research and clinical settings.