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

研究不对称分裂细胞中组蛋白遗传的新方法的开发和应用

• 批准号: 10709475
• 负责人: Jason Thomas Palladino
• 金额: $ 7.18万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10709475
• 关键词: Aging; Biological; Biological Models; Cancerous; Cell Cycle Progression; Cell Differentiation process; Cell Lineage; Cell Nucleus; Cell division; Cell physiology; Cells; Chromatin; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Cues; DNA; DNA Methylation; DNA Sequence; Daughter; Detection; Deterioration; Development; Developmental Biology; Disease; Dissection; Drosophila genus; Epigenetic Process; Female; Fluorescent in Situ Hybridization; Ganglia; Gene Expression; Genetic Variation; Genome; Guide RNA; Health; Histones; Homeostasis; Human; Infertility; Inheritance Patterns; Inherited; Intestines; Intrinsic factor; Knowledge; Label; Light; Malignant Neoplasms; Methodology; Methods; Mitosis; Mitotic; Molecular; Mothers; Mus; Muscular Dystrophies; Normal Cell; Onset of illness; Organelles; Organism; Pattern; Phenotype; Play; Post-Translational Protein Processing; Process; Proliferating; Proteins; RNA; RNA library; Research; Resolution; Role; S phase; Spermatocytes; Structure; Technology; Testing; Time; Tissues; Transgenic Organisms; Variant; Work; adult stem cell; age related; cancer cell; cancer type; cell age; cell behavior; cell type; design; embryonic stem cell; epigenetic memory; epigenetic regulation; epigenomics; experimental study; genetic information; genomic locus; germline stem cells; in vivo; male; nerve stem cell; neuroblast; novel; segregation; self-renewal; stem cell differentiation; stem cell homeostasis; stem cell model; stem cells; tissue degeneration; tool

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.

正确的发育取决于不对称细胞分裂（ACD），该过程将干细胞分开 产生新的干细胞和一个分化细胞。许多指导ACD的固有和外在因素具有 被发现和表征。然而，染色质对细胞效果测定的贡献很差 理解。以前，我们的实验室发现了组蛋白和组蛋白后翻译后修饰的不对称性 （HPTM）果蝇雄性生殖细胞的遗传。该过程的进一步解剖表明它的功能和 以三个步骤进行调节：1）在S期间建立组蛋白不对称； 2）组蛋白不对称是 在M期间区分； 3）遗传的不对称组蛋白的读数指导细胞周期 有丝分裂出口后的进展。有趣的是，这些不对称的破坏导致两种干细胞损失 和过度增殖表型，表明不对称组蛋白遗传是一个必不可少的过程 组织健康。此外，此过程的恶化可能很常见，包括与年龄有关的疾病 组织变性和癌症。我假设不对称组蛋白遗传是一种通用机制 这在发育过程中的干细胞稳态和细胞污染测定中起着至关重要的作用。但是，我们的 目前，单个细胞内特定基因座的组蛋白和HPTM的能力在此时受到严重限制。因此，我们 迫切需要研究新工具来研究不对称组蛋白遗传的作用和后果 发育，干细胞稳态和细胞命运测定。在此提案中，我将1）扩大我们的 了解在不对称分裂细胞中的整个组蛋白遗传的普遍性，作用和模式 2）开发一种新颖的方法，用于标记非重复基因座以跟踪表观基因组特征。 首先，我将表达用果蝇神经元干细胞中的光旋转dendra2标记的组蛋白 血统。神经细胞（NBS），其后代神经节母细胞（GMC）和传输的中间体 神经元祖细胞（INP）是一个良好的干细胞模型系统。诱导405nm的光线 光转换，我将观察旧（红色）与I型和II中的新（绿色）组蛋白的遗传模式 NBS。这些研究将1）扩大我们对ACD中组蛋白遗传的了解，以及2）揭示组蛋白是否是否 遗传模式随着不对称分裂的细胞的年龄而变化并失去效力（例如INP）。 传统上，在特定基因组位置跟踪表观基因组信息是使用的 鱼。但是，鱼所需的热变性与 检测与DNA相关蛋白的检测。为了克服这一限制，我将使寡头技术适应 设计指南RNA库将DCAS9绑定到特定的非重复基因座。这将使我能够跟踪 具有单细胞分辨率的特定基因组基因座的组蛋白和HPTM遗传模式。 这些作品都将增强我们对ACD潜在机制的理解，并提供新的 工具促进了研究和临床环境中表观基因组改变的检测。