The canonical Wnt/β-catenin signaling pathway is essential for the proper regulation of cell-fate decisions throughout embryogenesis and in adult issues. Activation of the Wnt signaling pathway allows for nuclear localization of the cell adhesion protein β-catenin, which then interacts primarily with members of the T-Cell Factor/Lymphoid Enhancer Factor (TCF/LEF) transcription factor family to modulate gene activity. The TCF/LEF family includes TCF7, TCF7L1, TCF7L2, and LEF1. While all four family members share a common DNA binding consensus sequence, their expression throughout embryogenesis and adult stem cell populations is unique, with their misexpression commonly occurring in Wnt related cancers and correlating strongly with metastasis and poor patient outcomes.
TCF/LEF exchange at target gene loci is a key feature of mediating context-specific cellular responses to Wnt signaling and can be observed to occur in a variety of populations throughout development and in adult stem cell populations. To model TCF/LEF exchange in vitro we have optimized a micropatterning fabrication and culture protocol capable of identifying and isolating discrete LEF1-only and TCF7L1-only populations during gastrulation-like processes. To characterize how complements of TCF/LEFs change during cellular divisions we have developed a novel mitotic chromatin proteomic technique. This method identifies LEF1 as the only TCF/LEF to remain associated with mitotic chromatin in Wnt-activated conditions in mouse embryonic stem cells that are transitioning out of pluripotency as a consequence of removing leukemia inhibitory factor from their culture medium. Additionally, gene targeting techniques were used to label endogenous LEF1 and TCF7L1 with different fluorescent proteins in a single mouse embryonic stem cell line, allowing us to use TCF/LEF protein expression as a reporter of Wnt/β-catenin pathway status, which we found to be capable of identifying a unique set of compounds that are undetected by traditional Wnt activity (TOP-Flash) reporter screens.
By using gene editing technology, and novel applications of proteomic and cell culture techniques, we have been able to investigate the mechanisms driving TCF/LEF expression and exchange in mouse embryonic stem cells to identify potentially clinically relevant therapeutic targets for their potential use in addressing TCF/LEF dysregulation in cancer. We have identified a novel mechanism through which TCF/LEFs maintain cell fate over cellular division; presented a novel live-cell drug screening platform capable of identifying compounds missed by existing platforms; and presented an optimized cell culture technique for the isolation of TCF/LEF exchange events. Taken together, the work in this thesis provides new insights into the mechanisms through which TCF/LEFs regulate their gene targets during cell fate transitions and throughout mitosis. / Thesis / Doctor of Science (PhD) / Throughout development and adult life cells are in constant communication, using a variety of cell signaling pathways to maintain adult stem cell populations and to pattern tissues throughout the body. Communication between cells often requires one cell to release a protein molecule (called a ligand) that is recognized by a receptor molecule on the surface of another cell. These cell surface receptors, when bound by the signaling ligand become activated and often set of a cascade of internal cellular events that ultimately result in changes in gene transcription in the nucleus. These transcriptional changes are toggled by proteins known as sequence-specific transcription factors that are able to selectively regulate expression of target genes. The net effect of combinations of extracellular ligands binding cell surface receptors determines the selective recruitment of specific transcription factors that activate a cell’s transcriptional program, in turn defining its fate and function.
A very important developmental signaling pathway is the Wnt signaling pathway, which employs a family of secreted Wnt molecules as ligands. The Wnt pathway is critical at all stages of organismal development and plays an essential role in tissue maintenance in mature animals. However, due to its critical role in stem cell maintenance, when mutations occur in Wnt signaling components it can have dire consequences. Wnt signaling has been found to be disrupted in more than 70-80% of all cancers. One major feature among these Wnt-related cancers is the inappropriate expression and mobilization of Wnt transcription factors. While the expression and activity of Wnt transcription factors – known as T-Cell Factor/Lymphoid Enhancer Factors (TCF/LEFs) – changes throughout development and stem cell maintenance, their inappropriate expression is frequently associated with metastasis and poor patient outcomes.
We have used mouse embryonic stem cells (mESCs) as a model system with which to study the mechanisms employed by TCF/LEFs to regulate their target genes. Through a number of approaches, which include adding fluorescent tags to TCF/LEF factors to track their intercellular locations and expression levels or enzymatic tags to identify proteins that interact with individual TCF/LEFs during a snapshot of cell activity, we have gained new knowledge about how these critical transcription factors regulate Wnt-regulated transcriptional programs. We also describe a method for generating micropatterned growth surfaces for mESCs that forces clusters of cells to grow within small circular shapes with a diameter of 1 mm or less. We show that mESCs confined to circular micropatterns differentiate in a highly reproducible manner that allows us to study the cell populations undergoing differentiation with a focus on cell fate determination mechanisms.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/25916 |
| Date | January 2020 |
| Creators | Gordon, Victor |
| Contributors | Doble, Brad, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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