Determining the effects of Sox9 on the cell cycle of intestinal stem cells

项目摘要/摘要 肠隐窝内有多种增殖细胞，包括高增殖的运输放大细胞，较少 增殖活跃的肠干细胞(ISCs)，以及不常分裂的静止ISCs。有证据表明 “塑料”特性体现在这些细胞类型中，但目前人们对这些细胞是如何 相互转换或决定完全血统承诺。Magness实验室的研究发现了转录因子 Sox9在基于隐窝的细胞中不同水平表达，范围从非常高的非分裂或缓慢分裂 细胞(Sox9高)，AISCs中间(Sox9低)，TA细胞最低(Sox9低)。Sox9影响 增殖率：Sox9基因敲除(Sox9KO)小鼠在隐窝中过度增殖，Sox9过表达 (Sox9OE)抑制增殖，当Sox9水平恢复到内源性水平时，这种情况可以逆转。 初步数据表明，Sox9OE的增殖减少并不是由于细胞死亡或 相反，在Sox9OE上显示更多的细胞处于G1期。中心假说 这一建议的核心是Sox9水平通过调节G1的长度来调节ISCs的增殖 细胞周期时相。如果得到经验的支持，这个概念对于理解 ISC用来决定细胞命运和在活动状态和储备状态之间相互转换的机制。 所有AIMS将使用一种新的策略，通过诱导剂将培养的Sox9KO小鼠和人类ISCs Sox9OE等位基因允许在其他相同的情况下重新引入Sox9表达的生理水平 SOX9KO ISCS。目的1将探索不同Sox9水平对参与基因转录的影响 细胞周期。Aim 2将使用新的双报告PIP-Fucci结构来精确量化细胞周期的变化 通过活体成像在自由生长的细胞中诱导不同Sox9水平的相长度。AIM 3将测试 SOX9通过延长到达细胞周期限制点的时间来延长G1期，并测试这是否导致 SOX9诱导Rb1的表达，初步数据表明。这些目标将对之前的 揭示了Sox9如何调节细胞周期的影响，并将为我们提供一个可能的机制 ISCs如何在动态平衡和损伤中保持其不同的增殖率。 这项工作将在胃肠道生物学和疾病中心的Magness实验室进行 (CGIBD)在北卡罗来纳大学教堂山分校。CGIBD是一个久负盛名的NIH资助的 以培养年轻的GI调查人员的职业生涯而闻名的计划。在这个出色的培训环境中，我 将获得肠道干细胞生物学方面的专业知识，并学习原代细胞/器官培养的新技术， 单细胞生物学/基因组学和生物信息学。我的职业发展培训计划侧重于导师制 技能、勇气、与更大的胃肠道、干细胞和细胞周期社区的网络，以及 教育机会，这将促进我作为一名学术科学家的独立之路。

Project Summary/Abstract The intestinal crypt houses diverse proliferative cells including highly proliferative Transit Amplifying cells, less proliferative active intestinal stem cells (ISCs), and infrequently dividing quiescent ISCs. Evidence indicates ‘plastic’ properties are embodied in these cell types, but little is currently known about how these cells interconvert or decide to fully lineage commit. Studies from the Magness Lab found the transcription factor Sox9 to be expressed at different levels in crypt-based cells ranging from very high in non- or slowly-dividing cells (Sox9high), intermediate in aISCs (Sox9low), and lowest in TA cells (Sox9sublow). Sox9 affects proliferation rate: Sox9 knockout (Sox9KO) mice have hyperproliferation in crypts, and Sox9 over-expression (Sox9OE) blocks proliferation, which can be reversed when Sox9-levels return to endogenous levels. Preliminary data indicates that decreased proliferation upon Sox9OE does not a result from cell death or differentiation, instead showing more cells in the G1 cell cycle phase upon Sox9OE. The central hypothesis of this proposal is that Sox9 levels regulate proliferation in ISCs by modulating the length of the G1 cell cycle phase. If empirically supported, this concept has important implications for understanding mechanisms ISCs employ to make cell fate decisions and interconvert between active and reserve states. All aims will use a novel strategy for transfecting cultured Sox9KO mouse and human ISCs with an inducible Sox9OE allele to allow for reintroduction of physiological levels of Sox9 expression in otherwise identical Sox9KO ISCs. Aim 1 will explore the transcriptomic effects different Sox9 levels have on genes involved in the cell cycle. Aim 2 will use the new two-reporter PIP-FUCCI construct to precisely quantify changes in cell cycle phase lengths induced by different Sox9 levels via live-imaging in freely growing cells. Aim 3 will test whether Sox9 lengthens G1 phase by increasing time to the cell cycle restriction point and test whether this results from Sox9 inducing Rb1 expression, as indicated by preliminary data. These aims will shed light on previously uncovered effects of how Sox9 regulates the cell cycle and will also give insight into a possible mechanism for how ISCs maintain their varied proliferation rates in homeostasis and injury. This work will be performed in the Magness Lab in the Center for Gastrointestinal Biology and Disease (CGIBD) at the University of North Carolina at Chapel Hill. The CGIBD is a well-established NIH-funded program known for cultivating the careers of young GI investigators. In this outstanding training environment, I will gain expertise in intestinal stem cell biology and learn new techniques in primary cell/organoid culture, single-cell biology/genomics, and bioinformatics. My career development training plan focuses on mentorship skills, grantsmanship, networking with the greater gastrointestinal, stem cell, and cell cycle communities, and didactic opportunities that will promote my path to independence as an academic scientist.