Wnt signaling regulates critical cellular interactions throughout normal development and directs cell fate decisions of stem cells. Previous work by our lab implicates -catenin as an essential modulator of embryonic stem cell self-renewal and differentiation. Genetic studies in mice have demonstrated broad functional redundancies between the most downstream effectors of the Wnt signaling cascade, the T-cell factor / Lymphoid enhancer factor (TCF/LEF) family of transcription factors. Despite this, loss-of-function experiments suggest that -catenin reinforces the pluripotent state by mediating a TCF switch in which repressive TCF7L1 is replaced with activating TCF7. However, these experiments do not account for potential confounding functional compensation by other TCF/LEF factors. As such, I hypothesized that TCF7 and TCF7L1 are functionally redundant in mouse embryonic stem cells and bind a largely overlapping set of target genes and interacting proteins.
In support of this notion, we demonstrated that both TCF7 and TCF7L1 were similarly able to restore the altered transcriptomic profile and differentiation deficits observed in mouse embryonic stem cells (mESCs) lacking all full-length TCF/LEFs. With the expectation that TCF7 and TCF7L1 recruit similar transcriptional co-regulators to a broadly overlapping set of target genes, we employed the unbiased techniques, ChIP-seq and BioID to test our hypothesis. We observed that regardless of the degree of Wnt signaling activity, TCF7L1 was more abundantly associated with chromatin than TCF7, and TCF7 and TCF7L1 regulate distinct target genes. We demonstrated that Wnt stimulation, simulated by GSK-3 inhibition, facilitates TCF7L1 interactions with transcriptional modulators such as the BAF and nuclear receptor co-repressor complexes, despite a reduction in TCF7L1 levels. Taken together, the work in this thesis provides new insights into the mechanisms of Wnt target gene regulation by the TCF/LEF factors. / Thesis / Doctor of Philosophy (PhD) / Stem cells are capable of giving rise to multiple different cell types and thus are able to generate all adult tissues. The identity of a cell is controlled by external signals that regulate internal programs encoded by our genes. The execution of the instructions in genetic programs is conducted by proteins called transcription factors that can turn different genes on or off, giving rise to distinct cell types. The T-Cell Factors and Lymphoid Enhancer Factor (TCF/LEFs) are a family of four transcription factors regulated by external signaling molecules called Wnts. By using the TCF/LEFs, Wnts establish gene outputs that determine the identity of cells throughout embryonic development and in adult tissues. However, the mechanisms used by this family of transcription factors to establish the programs controlling cellular identity remain poorly understood. Using genetically engineered mouse embryonic stem cells, we have uncovered new information about the mechanisms TCF/LEFs use to regulate gene function, identified programs controlled by TCF/LEFs, and discovered potential protein partners that work with TCF/LEFs to implement genetic programs. This thesis provides novel insights into the control of cell identity by the TCF/LEFs, which has implications for the numerous human diseases linked to abnormal Wnt-mediated signaling.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/24307 |
| Date | January 2019 |
| Creators | Moreira, Steven |
| Contributors | Doble, Bradley, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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