Transcription Factors and Where to Find Them : Mechanisms and Methods of Mapping Transcription Factor Regulation

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.