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.

正常的发育依赖于不对称细胞分裂（ACD），这是一个分裂干细胞的过程。 产生更新的干细胞和分化细胞。许多内在和外在因素引导ACD， 被发现和鉴定。然而，染色质对细胞命运决定的贡献很小， 明白此前，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 （hPTM）遗传在果蝇雄性生殖细胞。对这一过程的进一步剖析揭示了它的功能， 在三个步骤中调节：1）组蛋白不对称性在S期建立; 2）组蛋白不对称性在S期建立。 在M期区分; 3）遗传的不对称组蛋白的读出引导细胞周期 有丝分裂退出后的进展。有趣的是，这些不对称性的破坏会导致干细胞丢失， 和过度增殖表型，这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有关的疾病中可能是常见的。 组织变性和癌症。我假设不对称组蛋白遗传是一种普遍机制 它在发育过程中的干细胞稳态和细胞命运决定中起着关键作用。但我们的 目前，在单细胞内的特定位点追踪组蛋白和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。在这个建议中，我将1）扩大我们的 理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，我将在果蝇神经干细胞中表达标记有光转换蛋白Dendra 2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 神经元祖细胞（INPs）是一种被充分研究的干细胞模型系统。405 nm光诱导后 通过光转换，我将观察I型和II型中旧（红色）与新（绿色）组蛋白的遗传模式 NB。这些研究将1）扩大我们对ACD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的细胞老化和失去效力而改变（例如INPs）。 在特定基因组位置处跟踪表观基因组信息传统上使用 鱼.然而，FISH所需的热变性与其部分或完全不相容。 DNA相关蛋白的检测。为了克服这一限制，我将采用Oligopaint技术， 设计引导RNA文库，用于将dCas 9连接到特定的非重复基因座。这能让我追踪 组蛋白和hPTM遗传模式在特定的基因组位点与单细胞分辨率。 这些工作将加深我们对ACD机制的理解，并提供新的 在研究和临床环境中促进检测表观基因组改变的工具。