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

基本信息

批准号：
10388699
负责人：
Jason Thomas Palladino
金额：
$ 6.76万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
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/10388699
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 Detection Deterioration Development Developmental Biology Disease Dissection Drosophila genus Epigenetic Process Female Fluorescent in Situ Hybridization Ganglia Gene Expression Genetic Variation Genome Genomics Guide RNA Health Histones Homeostasis Human Infertility Inheritance Patterns Inherited Intestines Intrinsic factor Knowledge Label Light Location 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 Proteins RNA RNA library Regulation Research Resolution Role S phase Spermatocytes Structure Technology Testing Time Tissues Transgenic Organisms Variant Work adult stem cell age related base 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 progenitor self-renewal stem cell differentiation stem cell homeostasis stem cell model stem cells tissue degeneration tool

项目摘要

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），这是干细胞分裂的过程产生更新的干细胞和分化细胞。许多引导 ACD 的内在和外在因素被发现并表征。然而，染色质对细胞命运决定的贡献却很薄弱。明白了。此前，我们实验室发现了组蛋白和组蛋白翻译后修饰的不对称性 (hPTM) 果蝇雄性生殖细胞的遗传。对这个过程的进一步剖析揭示了它的功能和分三步调节：1) 在 S 期建立组蛋白不对称性； 2) 组蛋白不对称性是在M期期间表现出色； 3）遗传性不对称组蛋白的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重要的过程组织健康。此外，这一过程的恶化在包括与年龄相关的疾病中可能很常见组织退化和癌症。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命运决定中起着至关重要的作用。然而，我们的目前，追踪单细胞内特定位点的组蛋白和 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、干细胞稳态和细胞命运决定。在本提案中，我将 1) 扩大我们的范围了解不对称分裂细胞中组蛋白遗传的普遍性、作用和模式 2）开发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，我将在果蝇神经元干细胞中表达带有光转换 Dendra2 标记的组蛋白血统。神经母细胞 (NB)、其后代神经节母细胞 (GMC) 和转运放大中间体神经祖细胞（INP）是一个经过充分研究的干细胞模型系统。 405nm光诱导后光转换，我将观察 I 型和 II 型中旧（红色）组蛋白与新（绿色）组蛋白的遗传模式注意。这些研究将 1) 扩展我们对 ACD 中组蛋白遗传的认识，2) 揭示组蛋白是否遗传模式随着不对称分裂细胞的衰老和丧失效力（例如 INP）而改变。传统上，跟踪特定基因组位置的表观基因组信息是使用鱼。然而，FISH 所需的热变性部分或完全不兼容 DNA相关蛋白的检测。为了克服这个限制，我将采用 Oligopaint 技术设计指南 RNA 文库，用于将 dCas9 束缚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位点的组蛋白和 hPTM 遗传模式。这些工作将增强我们对 ACD 基础机制的理解，并提供新的促进研究和临床环境中表观基因组改变检测的工具。