Elucidating Wnt signalling mechanisms in pluripotent stem cells

背景：Wnt/β-catenin信号通路与干细胞生物学的调控和细胞命运的决定密切相关。我的研究计划的首要目标是阐明Wnt信号在发育过程中和在体细胞中调节基因活性的核心机制。Wnt/β-catenin信号的转录介体是Tcf/Lef家族的四个高迁移率族转录因子。在小鼠胚胎干细胞(MESCs)中，TCF7L1在转录本和蛋白质水平上占主导地位，而LEF1因子在这些细胞中几乎检测不到。当mESCs被诱导退出多能性状态时，TCF7L1水平降低，LEF1显著上调。我的实验室已经产生了mESC系，这样内源TCF7L1和LEF1基因的N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TCF7L1/LEF1进行荧光可视化，我们发现了它们在定位上的显著差异，我们建议进一步询问这些差异，以试图更好地描述Wnt信号调节细胞命运决定的机制。我们观察到LEF1在有丝分裂的所有阶段都与有丝分裂染色体相关。但TCF7L1并非如此。我们试图确定LEF1是否是能够以有丝分裂方式标记基因活动区的转录因子的一小部分。我们优化了微图案化条件，以分离经历原肠形成样事件的高度可重复性的mESC群体，并通过荧光显微镜检测到TCF7L1/LEF1的表达模式。我们假设，正如在体外模拟的那样，原肠形成过程需要TCF7L1活性的变化以及TCF7L1和LEF1之间在Wnt反应元件上的交换。我们提出了以下短期目标，将由两名研究生水平的HQP和本科生暑期/项目学生在五年的过程中完成：1)确定LEF1有丝分裂染色质保留在有丝分裂所有阶段的功能意义。2)阐明小鼠胚胎干细胞三系分化过程中TCF7L1/LEF1在靶基因上募集/交换的机制。影响：调控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是如何执行的关键。这项拟议的研究将为如何在Wnt/β-catenin信号通路下游调控TCF/Lef转录因子提供重要的新见解，这一通路对正常的干细胞行为和正常的生物发育至关重要。上述两个目标将为有机会实现这些目标的总部基地提供丰富的学习经验，其中将使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.