Elucidating the role of p63 and transcriptional control mechanisms in progenitor cells of the salivary gland

项目摘要 唾液腺干/祖细胞增殖和分化之间的微妙平衡 前列腺（SG）必须严格调节，以确保适当的形态发生，稳态和再生。 正常SG功能的改变，临床上通常表现为唾液分泌减少，与疾病相关 如干燥综合征和头颈部鳞状细胞癌患者的γ-照射治疗。 目前，唾液过少的治疗选择和恢复正常SG功能的机制仍然存在 有限公司因此，开发新的工具和策略，以确定转录和 SG的干/祖细胞功能的信号传导网络，最终目标是用于干细胞 基于再生的疗法和组织工程方法是至关重要的。因此，我们的目标是检查 SG的干/祖细胞的转录组景观，并确定和表征调控基因。 这个细胞群体的网络。已经确定Δ Np63在上皮再生中起关键作用， 由于Δ Np63缺失动物不能发育包括SG在内的几个富含上皮的器官，但我们的 目前缺乏关于Δ Np63如何调节SG干/祖细胞中基因表达的知识。 因此，研究p63驱动的调控网络是更好地理解生物学的关键一步。 的SG祖细胞和指导治疗SG功能障碍的新策略。解决这些 由于知识上的差距，我们将利用从新型Δ Np63-GFP转基因小鼠中获得的SG干/祖细胞， 研究两个主要的兴趣领域。首先，我们将进行功能测定以比较Δ Np63- GFPhi和Δ Np63-GFP低SG细胞以保留其祖细胞能力和其分化成 SG器官球中的成熟细胞谱系（Aim1A）。此外，我们将进行体内转录组学研究， 通过RNA测序（RNA-seq）来生成Δ Np63-GFPhi SG的纯群体中的mRNA表达谱 祖细胞和Δ Np63-GFP低SG细胞（Aim 1B）。这种体内研究是重要的，因为它们将 首次在广泛和动态范围内鉴定SG祖细胞的基因表达谱。 其次，我们将进行体内ChIP-seq研究，以确定关键的调控网络和调控途径。 通过SG细胞中的Δ Np63。总的来说，我们的方法使用遗传定义的模型系统和尖端的 下一代测序技术将更好地阐明SG祖细胞的转录组学景观 阐明了Δ Np63调控的转录调控网络和信号通路。因为这种 虽然SG细胞的基因组研究特别缺乏，但我们提出的实验可能会揭示新的 生物标志物和重要的调节剂和驱动器的干细胞功能在这个器官。长期来看，这样的知识 将对患有SG功能障碍疾病的人类患者具有临床和治疗意义。

PROJECT SUMMARY The delicate balance between proliferation and differentiation of stem/progenitor cells of the salivary gland (SG) must be tightly regulated to ensure proper morphogenesis, homeostasis and regeneration. Alterations to normal SG function, clinically often manifested with hyposalivation, are associated with diseases such as Sjögren’s Syndrome and with γ-irradiation therapy of Head and Neck Squamous Carcinoma patients. Currently, treatment options for hyposalivation and mechanisms for restoring normal SG function remain limited. Therefore, the development of new tools and strategies directed at identifying transcriptional and signaling networks underlying stem/progenitor cell function of the SG with the ultimate goal for use in stem cell regenerative-based therapies and tissue engineering approaches, are critical. Hence, our goal is to examine the transcriptome landscape of the stem/progenitor cells of the SG and identify and characterize the regulatory networks of this cell population. 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 the SG. However, our current knowledge of how ΔNp63 regulates gene expression in the stem/progenitor cells of the SG is lacking. Thus examining the p63-driven regulatory networks is a key step towards a better understanding of the biology of the SG progenitor cells and in directing new strategies in treating SG dysfunction. To address these knowledge gaps, we will utilize SG stem/progenitor cells obtained from novel ΔNp63-GFP transgenic mice to study two major areas of interest. First, we will perform functional assays to compare the abilities of ΔNp63- GFPhi and ΔNp63-GFPlow SG cells to retain both their progenitor capacity and their ability to differentiate into mature cell lineages in SG organospheres (Aim1A). Furthermore, we will perform in vivo transcriptomic profiling (RNA-seq) to generate the mRNA expression profile in a pure population of ΔNp63-GFPhi SG progenitor cells and ΔNp63-GFPlow SG cells (Aim 1B). Such in vivo studies are important, since they will identify for the first time the gene expression profile of SG progenitor cells on a broad and dynamic scale. Second, we will perform in vivo ChIP-seq studies to identify key regulatory networks and pathways regulated by ΔNp63 in SG cells. Collectively, our approach using a genetically-defined model system and cutting-edge next generation sequencing technology will better elucidate the transcriptomic landscape of SG progenitor cells and shed light on the ΔNp63-governed transcriptional regulatory network and signaling pathways. Since such genomic studies are particularly lacking for SG cells, our proposed experiments will likely reveal new biomarkers and important regulators and drivers of stem cell function in this organ. Long term, such knowledge will have clinical and therapeutic implications for human patients who suffer from SG dysfunction diseases.