Stem cell regulation by the transcription factor WOX5 in the Arabidopsis thaliana root meristem

位于植物分生组织中的多能干细胞支持新组织和器官的终生生产，如根、茎和叶。因此，破译调节干细胞的机制对于了解植物的生长和发育至关重要。根分生组织的小柱干细胞生态位结构简单，是研究干细胞调控的理想模型：静止的中心细胞维持一层小柱干细胞(CSCs)，其子代直接分化为感觉根帽中重力的小柱细胞。我们已经为这一提议做了大量的准备工作，以解决模式植物拟南芥在遗传和分子水平上的CSC调控机制。我们发现可移动转录因子WOX5(WUSCHEL相关Homeobox 5)是CSC规范的主要调节因子。异位表达WOX5产生足够数量和纯度的诱导小柱干细胞(ICSCs)，用于分子分析。使用细胞分选策略，我们发现WOX5直接调控iCSCs中的数百个基因，这表明干细胞状态是复杂遗传网络平衡表达的结果。基于我们的准备数据，我们假设对于其大多数靶点，WOX5通过诱导抑制性组蛋白修饰来抑制基因转录，从而塑造干细胞的表观遗传格局。此外，WOX5通过一种未知的机制转录激活一组基因，包括一种新的、重要的CSC规范调节因子HANABA Taranu(HAN)。该提案的目的是阐明构成柱状杆菌干细胞规范的WOX5调控的转录和信号网络。首先，使用分类的细胞特异性核，我们将研究WOX5在将分化细胞重新编程为iCSCs和真正的小柱干细胞过程中诱导的全基因组表观遗传修饰。其次，我们将通过遗传学和分子研究以及活体成像，探索编码干细胞抑制因子CDF4和阳性干细胞调节因子HAN的两个WOX5靶基因如何有助于平衡干细胞维持和细胞分化。所有必要的方法和大部分材料已经建立，使我们能够立即开始实验。将全基因组和特定基因的功能研究结合起来，提供了一个独特的机会来实现干细胞规范的综合机制图景。由于WOX蛋白是干细胞的保守调节者，在其他植物分生组织中也是如此，我们希望我们的结果将为深入了解植物干细胞调控的一般机制提供帮助。此外，这项建议不仅将解决干细胞生物学中的基本问题，而且还将产生对作物的持续生长和生产力具有重要农学意义的结果。

The pluripotent stem cells located in plant meristems sustain the lifelong production of new tissues and organs such as roots, stems and leaves. Deciphering the mechanisms that regulate stem cells is therefore central to understanding plant growth and development. The columella stem cell niche of the root meristem has a simple organization that makes it an ideal model to study stem cell regulation: the quiescent center cells maintain a single layer of columella stem cells (CSCs), whose daughters differentiate directly into columella cells that sense gravity in the root cap. We have done extensive preparatory work for this proposal to address the mechanisms of CSC regulation in the model plant Arabidopsis at the genetic and molecular levels. We discovered that the mobile transcription factor WOX5 (WUSCHEL-RELATED HOMEOBOX 5) is a major regulator of CSC specification. Ectopic WOX5 expression generates induced columella stem cells (iCSCs) in sufficient amounts and purity for molecular analysis. Using a cell sorting strategy, we find that WOX5 directly regulates several hundred genes in iCSCs, suggesting that the stem cell state is the result of the balanced expression of a complex genetic network. Based on our preparatory data, we hypothesize that for the majority of its targets, WOX5 represses gene transcription by inducing repressive histone modifications, thus shaping the epigenetic landscape of the stem cells. In addition, WOX5 transcriptionally activates a group of genes by a yet unknown mechanism, including a novel, important regulator of CSC specification, the transcription factor HANABA TARANU (HAN). The goal of this proposal is to elucidate the WOX5-regulated transcriptional and signaling networks that underlie columella stem cell specification. First, using sorted cell-specific nuclei, we will investigate the genome-wide epigenetic modifications induced by WOX5 during reprogramming of differentiated cells into iCSCs and in genuine columella stem cells. Second, we will explore how two WOX5 target genes, coding for the stem cell inhibitor CDF4 and the positive stem cell regulator HAN, contribute to balancing stem cell maintenance and cell differentiation by genetic and molecular studies and by live imaging. All necessary methods and most materials have already been established, enabling us to start the experiments without delay. Combining genome-wide with gene specific functional studies provide a unique opportunity to achieve an integrative mechanistic picture of stem cell specification. Because WOX proteins are conserved regulators of stem cells also in other plant meristems, we expect that our results will provide insight into general mechanisms of plant stem cell regulation. Furthermore, this proposal will not only address fundamental questions in stem cell biology, but also will yield results of agronomic importance for sustained growth and productivity of crops.