The Wnt signaling pathway is a biological mechanism for cell-cell communication found across all species of the animal kingdom. This pathway plays a major role in virtually all stages of embryonic development, and it governs central aspects of stem cell biology, regeneration, and tissue homeostasis. In addition, dysregulation of the pathway is associated with developmental malformations and several forms of sever cancer. However, it is still not fully understood how Wnt signaling can mediate such a variety of processes and outcomes. How is a single pathway, which according to the current models is described as a mostly linear cascade of events, able to induce diverging responses in different biological contexts? Finding an answer to this question would not only satisfy scientific curiosity but could also have clinical significance. Given the importance of Wnt signaling in normal tissue function, therapeutically targeting the pathway has historically proven to be difficult. Thus, a better understanding of the tissue-specific properties of the pathway could help us uncover a way to distinguish disease-related cells from healthy cells and identify new targets whose inhibition could impair disease while avoiding detrimental effects on normal tissue function.       

This thesis represents four years of research that aims to address the knowledge gaps outlined above. Specifically, the work has been focusing on exploring the time- and tissue-specific properties of Wnt signaling by assessing the genome-wide consequences of perturbing this pathway in different model systems. Through this work, we have revealed further instances of disconnection between classical Wnt components, challenging the current established models of how Wnt signaling operates. Furthermore, we demonstrate that the cellular response to Wnt activation occur in a time-dependent manner, with different responsive patterns in different cell types, and even heterogeneously across cells in an otherwise homogenous cell population, contributing to the emerging notion of context-specific Wnt signaling. Finally, we identify a new tissue-specific player in Wnt-mediated transcriptional regulation, which holds promise as a possible therapeutic target in the continuing battle against cancer. 

In summary, the scientific results presented in this thesis extend our current knowledge of the Wnt signaling pathway by highlighting context-specific aspects that could help explain how this fundamental process adopts different regulatory avenues. This, in turn, could prove important for our ability to identify and ultimately combat disease-specific traits, including finding the Achilles heel of cancer.    

Gene regulation is a fundamental process in development and disease. Transcription factors (TFs) play a pivotal role by binding specific genomic regions to regulate target genes. In this thesis, we explored the landscape of TF binding through CUT&RUN, with a particular focus on Wnt signaling and its key mediator, β-catenin.

Paper I introduced CUT&RUN Low Volume Urea (LoV-U), optimized for co-factors like β-catenin. This method allowed for high-quality profiling of diverse targets in both cell lines and mouse tissues. Paper II identified common artifacts in CUT&RUN data, establishing a list of "Suspect" regions for data filtration. In Paper III, we used these tools to examine time-resolved β-catenin binding in two cell types and discovered that binding is dynamic over time and cell-type specific. Paper IV addressed one of the central challenges in TF and chromatin research — signal reproducibility. We developed ICEBERG (Increased Capture of Enrichment by Exhaustive Replicate aGgregation), a pipeline to improve the detection of TF binding across the genome. ICEBERG classified binding sites based on detection probability and uncovered previously missed, rare regulatory associations. In Paper V, we shifted focus to a broader landscape of mouse development. We created a CUT&RUN resource dataset of twelve targets in four embryonic tissues and identified "popular regions" bound by multiple TFs, enriched for essential developmental genes. Finally, in Paper VI, we identified a set of genomic regions where CTCF binding changes in response to Wnt activation. These regions overlap with β-catenin and are associated with changes in 3D genome architecture. By disrupting CTCF binding, we demonstrated that CTCF contributes to the regulation of key Wnt target genes.

Together, these studies represent a methodological and conceptual advance in the study of gene regulation, shedding new light on the nuclear mechanisms of Wnt/β-catenin signaling, and providing tools and methods for future research.