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.

项目摘要 基本茎/祖细胞的增殖和分化之间的严格调节平衡 口腔上皮对于适当的组织发展，修复，更新和维持稳态至关重要。 因此，开发旨在识别转录和信号传导的新工具和策略 口腔上皮的茎/祖细胞功能的基础网络至关重要。因此，我们的目标是 检查控制转录和基因调节机制的分子机制 口服上皮的茎/祖细胞功能，其最终目标是使用从中获得的知识 这种研究基于干细胞再生的疗法和组织工程方法的研究。很好 确定ΔNP63在上皮再生功能中起着至关重要的作用，因为Δnp63-null动物无法 开发几个包括口腔的上皮器官，包括口腔的器官。但是，我们目前对 NP63如何与染色质和转录调节环境相互作用并塑造 缺乏口腔上皮的茎/祖细胞。为了解决这些知识差距，我们将利用 从新型ΔNP63-GFP转基因小鼠获得的口服上皮干/祖细胞的富集群体 研究两个主要感兴趣的领域。首先，我们将同时执行clogenic和功能分析以比较 ΔNP63-GFPHI，ΔNP63-GFPLOW，ΔNP63NEG和ΔNP63-GFPHI-KD的能力（ΔNP63特定诱导型敲低 在ΔNP63-GFPHI细胞中使用siRNA介导的策略）口服上皮细胞以保持其祖细胞能力 在组织球（AIM1A）中。此外，我们将执行转录组分析（RNA-SEQ）以生成全局 ΔNP63-GFPHI，ΔNP63-GFPLOW，ΔNP63NEG和ΔNP63-GFPHI-KD的基因表达谱图更好地理解 NP63依赖性基因调节机制，对口腔上皮茎/祖细胞很重要 生物学（AIM1B）。这样的研究很重要，因为它们将首次识别基因表达 口服上皮茎/祖细胞在广泛而动态的尺度上的特征。第二，检查全球 口服上皮细胞染色质结构的状态，我们将用ΔNP63-进行ATAC-SEQ实验 GFPHI，ΔNP63-GFPLOW，ΔNP63NEG和ΔNP63-GFPHI-KD细胞以识别ΔNP63依赖性和独立 对茎/祖细胞功能很重要的调节染色质环境（AIM 2）。总体而言，我们的 使用一般定义的模型系统和最先进的下一代测序技术的方法 将更好地阐明口腔茎/祖细胞的转录组和表观基因组景观 在ΔNP63的转录调节网络和信号通路上。这项工作很高 创新和意义 全基因组分析测定法检查基本的转录控制机制将导致新的 对于用于治疗和再生的口服上皮干细胞再生策略至关重要的发现 受伤，损害或患病状态后的口腔组织。