Elucidating Wnt signalling mechanisms in pluripotent stem cells

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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.