Elucidating Wnt signalling mechanisms in pluripotent stem cells

阐明多能干细胞中的 Wnt 信号传导机制

• 批准号: RGPIN-2020-06530
• 负责人: Doble, Bradley
• 金额: $ 2.33万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761988
• 关键词: Elucidating; Wnt; signalling; mechanisms; pluripotent

Background: The Wnt/beta-catenin signalling pathway is strongly linked to the regulation of stem cell biology and cell fate determination. The overarching goal of my research program is to elucidate the core mechanisms through which Wnt signaling regulates gene activity during development and in somatic stem cells. The transcriptional mediators of Wnt/beta-catenin signalling are four high mobility group transcription factors of the TCF/LEF family. In mouse embryonic stem cells (mESCs), TCF7L1 predominates at the transcript and protein levels, whereas the factor LEF1 is virtually undetectable in these cells. When mESCs are induced to exit the pluripotent state, TCF7L1 levels diminish and LEF1 is dramatically upregulated. My lab has generated mESC lines engineered such that endogenous TCF7L1 and LEF1 genes have been N-terminally tagged with different fluorescent proteins. Through fluorescence visualization of TCF7L1/LEF1 in conditions supporting pluripotency or promoting early differentiation, we have identified striking differences in their localization that we we propose to further interrogate in an attempt to better describe the mechanisms through which Wnt signaling regulates cell fate determination. We have observed that LEF1 stays associated with mitotic chromosomes throughout all stages of mitosis. This is not the case for TCF7L1. We seek to determine if LEF1 is one of a small subset of transcription factors that has the ability to 'mitotically bookmark' regions of gene activity.  We have optimized micropatterning conditions to isolate highly reproducible mESC populations undergoing gastrulation-like events, with TCF7L1/LEF1 expression patterns detectable by fluorescence microscopy. We hypothesize that the gastrulation process, as modelled in vitro, requires changes in TCF7L1 activity as well as an exchange between TCF7L1 and LEF1 at Wnt-responsive elements. We propose the following short-term objectives, to be carried out by two graduate-level HQP and undergraduate summer/project students over the course of five years: 1) To determine the functional significance of the mitotic chromatin retention of LEF1 during all stages of mitosis. 2) To elucidate the mechanisms through which TCF7L1/LEF1 recruitment/exchange occurs at target genes during tri-lineage differentiation of mESCs. Impact: The mechanisms regulating transcription and the epigenetic status of a cell's genome are key to understanding how cellular and developmental programs encoded in DNA are executed. The proposed research will provide important new insights into how the TCF/LEF transcription factors are regulated downstream of the Wnt/beta-catenin signalling pathway, a pathway that is critical for normal stem cell behaviour and proper organismal development. The two objectives described above will provide a rich learning experience, in which state-of-the-art equipment and methodologies will be employed, for the HQP that have the opportunity to undertake the objectives.

背景：Wnt/ β -连环蛋白信号通路与干细胞生物学和细胞命运决定的调控密切相关。我的研究计划的总体目标是阐明通过Wnt信号调节发育过程中的基因活性和体细胞干细胞的核心机制。Wnt/ β -catenin信号传导的转录介质是TCF/LEF家族的四种高迁移率组转录因子。在小鼠胚胎干细胞（mESCs）中，TCF7L1在转录物和蛋白水平上占主导地位，而LEF1因子在这些细胞中几乎检测不到。当mESCs被诱导退出多能状态时，TCF7L1水平降低，LEF1显著上调。我的实验室已经生成了mESC细胞系，使内源性TCF7L1和LEF1基因被不同的荧光蛋白n端标记。通过在支持多能性或促进早期分化的条件下对TCF7L1/LEF1的荧光可视化，我们发现了它们在定位上的显著差异，我们建议进一步研究这些差异，以更好地描述Wnt信号调节细胞命运决定的机制。我们观察到，在有丝分裂的所有阶段，LEF1都与有丝分裂染色体相关。TCF7L1的情况并非如此。我们试图确定LEF1是否是具有“有丝分裂书签”基因活性区域能力的转录因子的一个小子集。我们优化了微图条件，以分离高可重复性的mESC群体，经历类似原肠胚形成的事件，用荧光显微镜检测TCF7L1/LEF1表达模式。我们假设体外模拟的原肠胚形成过程需要TCF7L1活性的改变以及TCF7L1和LEF1在wnt响应元件上的交换。我们提出以下短期目标，由两名研究生水平的HQP和本科生暑期/项目学生在五年的课程中进行：1)确定有丝分裂所有阶段中LEF1染色质保留的功能意义。2)阐明mESCs三系分化过程中TCF7L1/LEF1在靶基因上的募集/交换机制。影响：调控细胞基因组转录和表观遗传状态的机制是理解DNA编码的细胞和发育程序如何执行的关键。这项研究将为了解TCF/LEF转录因子如何在Wnt/ β -连环蛋白信号通路下游受到调控提供重要的新见解，而Wnt/ β -连环蛋白信号通路对于正常的干细胞行为和适当的组织发育至关重要。上述两个目标将为有机会实现这些目标的HQP提供丰富的学习经验，其中将采用最先进的设备和方法。