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维持新组织和器官如根、茎和叶的终身产生。因此，破译调节干细胞的机制对于理解植物生长和发育至关重要。根分生组织的柱干细胞小生境具有简单的组织结构，使其成为研究干细胞调节的理想模型：静止中心细胞维持单层柱干细胞（CSC），其子代直接分化为在根冠中感知重力的柱细胞。我们已经做了大量的准备工作，这一建议，以解决在模式植物拟南芥CSC的调控机制在遗传和分子水平。我们发现移动的转录因子WOX 5（WUSCHEL相关的HOMEOBOX 5）是CSC特化的主要调节因子。异位WOX5表达产生足够量和纯度的诱导的鼻小柱干细胞（iCSC）用于分子分析。使用细胞分选策略，我们发现WOX5直接调节iCSC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。基于我们的预备数据，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景观。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特异性调节因子，转录因子HANABA TARANU（HAN）。该提案的目标是阐明WOX5调控的转录和信号网络，其基础是小柱干细胞的规范。首先，使用分选的细胞特异性核，我们将研究在分化细胞重编程为iCSC和真正的小柱干细胞期间由WOX5诱导的全基因组表观遗传修饰。其次，我们将探索两个WOX5靶基因，编码干细胞抑制剂CDF4和阳性干细胞调节剂HAN，如何通过遗传和分子研究以及活体成像来平衡干细胞维持和细胞分化。所有必要的方法和大多数材料都已经确定，使我们能够立即开始实验。将全基因组与基因特异性功能研究相结合提供了一个独特的机会，以实现干细胞特化的综合机制。由于WOX蛋白也是其他植物分生组织中干细胞的保守调节因子，我们期望我们的研究结果将为植物干细胞调节的一般机制提供见解。此外，该提案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持续生长和生产力具有农学重要性的结果。

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.