Differential Wnt/β-catenin signaling via TCF7L2/LEF1 binding specificity shapes cellular and tumor phenotypes

Abstract The mechanisms by which Wnt/β-catenin signaling regulates gene expression in a tissue- and context-specific manner remain poorly understood, limiting our ability to target the aberrant cell growth typical of many Wnt-driven cancers. Here we focus on malignant liver tumors driven by activating CTNNB1 (β-catenin) mutations that nevertheless display distinct phenotypic states and Wnt outputs. By profiling patient-derived organoids via single-cell transcriptomics and chromatin dynamics, we identify subtype-specific transcriptional and epigenetic profiles. Using CUT&RUN, we show that β-catenin engages distinct genomic regions, dictated by differential association with TCF/LEF family transcription factors. Specifically, we define a novel sequence-specific regulatory element engaged by β-catenin only upon interaction with TCF7L2, revealing that partner choice, independent of CTNNB1 mutational status, ultimately determines cell fate. Our findings, validated across multiple tumor models and patient tissues, offer a framework for understanding how differential β-catenin-TCF/LEF interaction orchestrates context-specific Wnt signaling outcomes. Significance Wnt/β-catenin signaling is crucial for development and cancer, yet how it drives different gene programs across tissues is unclear. Using patient-derived liver tumor organoids, we show that β-catenin’s transcriptional output depends on its binding partner: LEF1 or TCF7L2. These factors guide β-catenin to distinct genomic regions, activating either stemness or differentiation genes. We identify a novel helper motif that directs β-catenin-TCF7L2 binding and target selection. By linking partner choice and motif specificity to context-dependent gene regulation, our work provides a unifying mechanism explaining how Wnt/β-catenin signaling produces diverse cellular outcomes.