Genomic and genetic analysis of oral stem cells

口腔干细胞的基因组和遗传分析

• 批准号: 9770830
• 负责人: Rose-Anne Romano
• 金额: $ 15.89万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9770830
• 关键词: ATAC-seq; Address; Affect; Animals; Architecture; Area; Automobile Driving; Biological Assay; Biological Models; Cell Cycle Arrest; Cell Lineage; Cell physiology; Cells; Cellular biology; Chromatin; Critical Pathways; Cues; Development; Disease; Embryo; Environment; Epigenetic Process; Epithelial; Epithelial Cells; Equilibrium; Expression Profiling; Foundations; Future; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Genome; Genomics; Goals; Homeostasis; Injury; Knowledge; Light; Mediating; Molecular; Molecular Conformation; Morphogenesis; Mus; Natural regeneration; Normal tissue morphology; Oral; Oral cavity; Organ; Pathway interactions; Play; Population; Process; Property; Protein Isoforms; Regulation; Regulator Genes; Regulatory Element; Role; Shapes; Signal Pathway; Signal Transduction; Small Interfering RNA; Stem cells; Stratification; Techniques; Technology; Time; Tissue Engineering; Tissue-Specific Gene Expression; Tissues; Transcriptional Regulation; Transgenic Mice; Work; base; cell type; epigenomics; experimental study; genetic analysis; genome-wide; improved; in vivo; in vivo Model; innovation; insight; interest; keratinocyte; knock-down; mouse model; next generation sequencing; novel; novel therapeutic intervention; oral cavity epithelium; oral tissue; progenitor; prospective; regenerative; repaired; self-renewal; stem; stem cell division; tissue regeneration; tool; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

PROJECT SUMMARY The tightly regulated balance between proliferation and differentiation of basal stem/progenitor cells of the oral epithelium is critical for proper tissue development, repair, renewal, and to maintain homeostasis. Therefore, the development of new tools and strategies directed at identifying transcriptional and signaling networks underlying stem/progenitor cell function of the oral epithelium are critical. Hence, our goal is to examine the molecular mechanisms of the transcriptional and gene-regulatory mechanisms that control stem/progenitor cell function of the oral epithelium with the ultimate goal for using the knowledge gained from such studies towards stem cell regenerative-based therapies and tissue engineering approaches. It is well established that ΔNp63 plays a critical role in epithelial regenerative function as ΔNp63-null animals fail to develop several epithelial-rich organs including those of the oral cavity. However, our current knowledge of how Np63 interacts with and shapes the chromatin and transcriptional regulatory environment of the stem/progenitor cells of the oral epithelium, is lacking. To address these knowledge gaps, we will utilize an enriched population of oral epithelial stem/progenitor cells obtained from novel ΔNp63-GFP transgenic mice to study two major areas of interest. First, we will perform both clonogenic and functional assays to compare the abilities of ΔNp63-GFPhi, ΔNp63-GFPlow, ΔNp63neg and ΔNp63-GFPhi-KD (ΔNp63 specific inducible knockdown in ΔNp63-GFPhi cells using siRNA mediated strategies) oral epithelial cells to retain their progenitor capabilities in organospheres (Aim1A). Furthermore, we will perform transcriptomic profiling (RNA-seq) to generate global gene expression profiles of ΔNp63-GFPhi, ΔNp63-GFPlow, ΔNp63neg and ΔNp63-GFPhi-KD to better understand the Np63-dependent gene regulatory mechanisms that are important for oral epithelial stem/progenitor cell biology (Aim1B). Such studies are important, since they will identify for the first time the gene expression profile of oral epithelial stem/progenitor cells on a broad and dynamic scale. Second, to examine the global status of the chromatin architecture of oral epithelia cells, we will perform ATAC-seq experiments with ΔNp63- GFPhi, ΔNp63-GFPlow, ΔNp63neg and ΔNp63-GFPhi-KD cells to identify the ΔNp63 dependent and independent regulatory chromatin environment that are important for stem/progenitor cell function (Aim 2). Collectively, our approach using a genetically-defined model system and cutting-edge next generation sequencing technology will better elucidate the transcriptomic and epigenomic landscape of oral stem/progenitor cells and shed light on the ΔNp63-governed transcriptional regulatory network and signaling pathways. This work is highly innovative and significant because our proposed use of sophisticated genetic tools, in vivo models and genome-wide profiling assays to examine fundamental transcriptional control mechanisms will lead to new discoveries important for oral epithelial stem cell based regenerative strategies used to treat and regenerate oral tissues following injury, damage or in diseased states.

项目总结 骨髓瘤基础干/祖细胞增殖与分化之间的严格调控平衡 口腔上皮对于正常的组织发育、修复、更新和维持动态平衡是至关重要的。 因此，针对识别转录和信号转导的新工具和策略的开发 口腔上皮干细胞/祖细胞功能的基础网络是至关重要的。因此，我们的目标是 研究控制转录和基因调控机制的分子机制 口腔上皮的干/祖细胞功能，最终目的是利用从 这类研究以干细胞再生疗法和组织工程方法为基础。这很好 证实ΔNp63在上皮再生功能中起关键作用，而ΔNp63缺失的动物无法 发育几个富含上皮组织的器官，包括口腔的器官。然而，我们目前对 Np63如何与染色质和转录调控环境相互作用并塑造 缺乏口腔上皮的干/祖细胞。为了解决这些知识差距，我们将利用 新型ΔNp63-绿色荧光蛋白转基因小鼠口腔上皮干/祖细胞的富集群 研究两个主要感兴趣的领域。首先，我们将执行克隆和功能分析，以比较 ΔNp63-GFPhi、ΔNp63-GFP low、ΔNp63neg和ΔNp63-GFPhi-KD(ΔNp63特异性可诱导基因敲除)的能力 在Δnp63-GFPhi细胞中使用siRNA介导的策略)口腔上皮细胞保留其祖细胞能力 在有机球中(Aim1a)。此外，我们将执行转录特征分析(RNA-seq)来生成全局 ΔNp63-GFPhi、ΔNp63-Gfplow、ΔNp63neg和ΔNp63-GFPhi-KD的基因表达谱 口腔上皮干/祖细胞依赖Np63的基因调控机制 生物学(Aim1B)。这样的研究很重要，因为他们将第一次确定基因的表达 口腔上皮干/祖细胞在广泛和动态范围内的分布。第二，审视全球 口腔上皮细胞染色质构筑的现状，我们将用Δnp63- GFPhi、ΔNp63-GFP low、ΔNp63neg和ΔNp63-GFPhi-KD细胞鉴定ΔNp63依赖和非依赖 对干细胞/祖细胞功能很重要的染色质调节环境(目标2)。总的来说，我们的 使用基因定义的模型系统和尖端的下一代测序技术的方法 将更好地阐明口腔干细胞/祖细胞的转录和表观基因组图景，并揭示 在ΔNp63控制的转录调控网络和信号通路上。这项工作具有很高的 创新性和重大意义，因为我们建议使用复杂的遗传工具，体内模型和 全基因组图谱分析检查基本转录控制机制将导致新的 口腔上皮干细胞再生策略用于治疗和再生的重要发现 受伤、损坏或患病状态后的口腔组织。