IOS: Interpretation of Stem Cell Promoting Transcription Factor Gradient

植物新梢的生长顶端形成所有的地上器官，如叶、花和果实。为了在一生中持续发育这些器官，植物必须保持干细胞池--即有能力适应无数可能的命运的细胞。尽管这些干细胞分化成独特的地上器官，但这些干细胞库保持相对稳定。确切地说，植物(和其他生物)如何管理干细胞维持和分化的微妙平衡，仍然是生物学领域的一个重要的悬而未决的问题。为了进一步解决这个问题，该项目将使用基因操作方法、生化分析和高分辨率实时成像来精确描述调节这种平衡的基因表达过程是如何在植物干细胞中控制的。了解干细胞维持和分化的微妙平衡将有助于培育具有最佳生物量和谷物产量的作物，并广泛地为基因调控提供新的范式。此外，基因表达的动态实时成像将被整合到一项外展活动中，旨在向来自低收入和拉丁裔占主导地位的当地学校的高中生解释基于探究的科学学习技术。(http://cybersam.ucr.edu/Plone/lab-outreach-2).该项目还将培训一名博士后研究员、一名研究生和几名本科生，学习活跃组织发育中基因调控的方法和原理。在拟南芥中，WUSCHEL(WUS)是一种干细胞促进转录因子，它从肋分生组织(RM)进入干细胞结构域/中心区(CZ)，在那里它抑制促进分化转录因子的转录，并通过直接结合启动子激活自己的负调控因子CLAVATA3(CLV3)的转录。WUS如何在距离RM较远的地方激活CLV3，以及它如何激活某些基因并抑制其他基因，目前尚不清楚。Gonehal实验室未发表的工作揭示，WUS在CLV3增强子区域以单体和二聚体的形式分别在较低和较高的蛋白质浓度下与几个间距较小、具有不同亲和力的顺式元件(顺式调节模块[CRM])结合。体内顺式元件操作和对WUS蛋白水平的操作表明，WUS利用CRM在Rm中较高浓度的WUS抑制CLV3，在CZ中在较低浓度下激活CLV3。这项工作旨在通过了解CLV3 CRM的作用模式和机制来了解CLV3表达的时空精确度的调节，鉴定受WUS抑制的基因中WUS结合的顺式元件，并通过对可能与WUS相互作用的辅助因子的功能分析来理解依赖浓度的转录歧视机制。这一活动将导致植物转录调控的新范式，以及总体上浓度依赖的转录调控。从这项工作中获得的见解将有助于对茎和根干细胞生态位中转录调控的比较分析。

The growing tips of plant shoots give rise to all aboveground organs, such as leaves, flowers, and fruits. To continuously develop these organs throughout their lives, plants must maintain pools of stem cells - i.e., cells with the capacity to adopt numerous possible fates. Despite the differentiation of these stem cells into unique above-ground organs, these stem cell pools remain relatively constant. Precisely how plants (and other organisms) manage this delicate balance of stem cell maintenance and differentiation remains an important outstanding question in the field of biology. To further address this question, this project will use gene manipulation methods, biochemical analyses, and high-resolution live imaging to precisely describe how the gene expression processes that regulate this balance are controlled in plant stem cells. Understanding this delicate balance of stem cell maintenance and differentiation will facilitate the development of crop plants with optimal biomass and grain yield, as well as broadly provide new paradigms for gene regulation. Additionally, the dynamic live imaging of gene expression will be integrated into an outreach campaign aimed at explaining inquiry-based scientific learning techniques to high school students from lower income and Latino-dominated local area schools (http://cybersam.ucr.edu/Plone/lab-outreach-2). The project will additionally train a postdoctoral fellow, a graduate student and several undergraduate students in methods and principles of gene regulation in actively developing tissues. In Arabidopsis, WUSCHEL (WUS), a stem cell promoting transcription factor, moves from the rib meristem (RM) into the stem cell domain/central zone (CZ) where it represses transcription of differentiation promoting transcription factors and activates transcription of its own negative regulator-CLAVATA3 (CLV3) by directly binding the promoters. How WUS manages to activate CLV3 at a distance from the RM, and how it activates some genes and represses others is not understood. Unpublished work from the Gonehal Lab reveals that WUS binds several closely spaced cis-elements with different affinities (a cis-regulatory module [CRM]) in the CLV3 enhancer region as monomer and as dimer at lower and higher protein concentrations, respectively. The in vivo cis-element manipulations and manipulation of the WUS protein levels reveal that WUS utilizes the CRM to repress CLV3 at higher WUS concentration in the RM and activate at lower concentration in the CZ. This work aims to understand the regulation of spatio-temporal precision of CLV3 expression by understanding the mode and mechanism of action of the CLV3 CRM, characterize WUS binding cis-elements in a gene repressed by WUS and understand mechanisms of concentration-dependent transcriptional discrimination through functional analysis of putative co-factors that interact with WUS. This activity will lead to a new paradigm in transcriptional regulation in plants and in concentration-dependent transcriptional regulation in general. Insights obtained from this work will facilitate comparative analysis of transcriptional regulation in shoot and root stem cell niches.