Genomic and genetic analysis of oral stem cells

项目摘要 基底干/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育、修复、更新和维持体内平衡是至关重要的。 因此，开发针对识别转录和信号传导的新工具和策略， 构成口腔上皮干/祖细胞功能基础的网络是至关重要的。因此，我们的目标是 研究控制基因表达的转录和基因调控机制的分子机制， 口腔上皮干/祖细胞功能的研究，最终目标是利用从 这些研究将有助于干细胞再生疗法和组织工程方法的发展。公 确定Δ Np 63在上皮再生功能中起关键作用，因为Δ Np 63缺失动物不能 发育出几个富含上皮的器官，包括口腔的那些。然而，我们目前对 Np 63如何与细胞的染色质和转录调控环境相互作用并塑造其形状， 口腔上皮干/祖细胞缺乏。为了弥补这些知识差距，我们将利用 从新的Δ Np 63-GFP转基因小鼠获得的口腔上皮干/祖细胞富集群体， 研究两个主要的兴趣领域。首先，我们将进行克隆形成和功能测定，以比较 Δ Np 63-GFPhi、Δ Np 63-GFPlow、Δ Np 63 neg和Δ Np 63-GFPhi-KD（Δ Np 63特异性诱导敲低）的能力 在Δ Np 63-GFPhi细胞中使用siRNA介导的策略）口腔上皮细胞以保留其祖细胞能力 在有机微球中（Aim 1A）。此外，我们还将进行转录组学分析（RNA-seq），以生成全局 Δ Np 63-GFPhi、Δ Np 63-GFPlow、Δ Np 63 neg和Δ Np 63-GFPhi-KD的基因表达谱，以更好地理解 对口腔上皮干/祖细胞重要JNP 63依赖性基因调控机制 生物学（Aim 1B）。这些研究很重要，因为它们将首次确定基因表达， 口腔上皮干/祖细胞的广泛和动态规模的概况。第二，考察全球 为了了解口腔上皮细胞染色质结构的状态，我们将用Δ Np 63- GFPhi、Δ Np 63-GFP low、Δ Np 63 neg和Δ Np 63-GFP hi-KD细胞，以鉴定Δ Np 63依赖性和非依赖性 调节染色质环境对干/祖细胞功能很重要（Aim 2）。总体而言，我们 使用基因定义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/祖细胞的转录组学和表观基因组学景观， 关于Δ Np 63控制的转录调节网络和信号传导途径。这项工作是高度 创新性和重要性，因为我们建议使用复杂的遗传工具，体内模型， 全基因组分析检测基本的转录控制机制将导致新的 发现重要的口腔上皮干细胞为基础的再生策略用于治疗和再生 口腔组织损伤后，损害或在患病状态。

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.