Elucidating Wnt signalling mechanisms in pluripotent stem cells

背景：Wnt/β-连环蛋白信号通路与干细胞生物学调控和细胞命运决定密切相关。我的研究计划的总体目标是阐明Wnt信号在发育过程中和体干细胞中调节基因活性的核心机制。 Wnt/β-连环蛋白信号传导的转录介质是TCF/LEF家族的四种高迁移率族转录因子。在小鼠胚胎干细胞（mESC）中，TCF 7 L1在转录和蛋白质水平上占主导地位，而LEF 1因子在这些细胞中几乎检测不到。当诱导mESC退出多能状态时，TCF 7 L1水平降低，LEF 1显著上调。我的实验室已经产生了mESC细胞系，这些细胞系经过工程改造，使内源性TCF 7 L1和LEF 1基因的N-末端被不同的荧光蛋白标记。通过在支持多能性或促进早期分化的条件下对TCF 7 L1/LEF 1进行荧光可视化，我们已经确定了它们在定位上的显著差异，我们建议进一步询问这些差异，以更好地描述Wnt信号调节细胞命运决定的机制。 我们已经观察到，LEF 1在有丝分裂的所有阶段都与有丝分裂染色体相关联。但TCF 7 L1的情况并非如此。我们试图确定LEF 1是否是一个小的转录因子子集，有能力“有丝分裂书签”的基因活性区域之一。 我们已经优化了微模式化条件，分离高度可重复的mESC群体经历原肠胚样事件，与TCF 7 L1/LEF 1的表达模式可通过荧光显微镜检测。我们假设，原肠胚形成过程，在体外模拟，需要TCF 7 L1活性的变化，以及TCF 7 L1和LEF 1之间的Wnt-反应元件的交换。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/项目学生在五年内完成： 1)确定LEF 1在有丝分裂各阶段的有丝分裂染色质保留的功能意义。 2)阐明mESCs三系分化过程中靶基因TCF 7 L1/LEF 1募集/交换发生的机制。 影响：调节转录的机制和细胞基因组的表观遗传状态是理解DNA中编码的细胞和发育程序如何执行的关键。拟议的研究将为TCF/LEF转录因子如何在Wnt/β-catenin信号通路下游进行调节提供重要的新见解，该通路对正常干细胞行为和适当的生物体发育至关重要。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。

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.