The Wnt/β-catenin signaling pathway plays numerous essential roles in animal development and tissue/stem cell maintenance. The activation of genes regulated by Wnt/β-catenin signaling requires the nuclear accumulation of β-catenin, a transcriptional co-activator. β-catenin is recruited to many Wnt-regulated enhancers through direct binding to T-cell factor/lymphoid enhancer factor (TCF/LEF) family transcription factors. β-catenin has previously been reported to form phase-separated biomolecular condensates (BMCs), which was implicated as a component of β-catenin's mechanism of action. This function required aromatic amino acid residues in the intrinsically disordered regions (IDRs) at the N- and C-termini of the protein. In this report, we further explore a role for β-catenin BMCs in Wnt target gene regulation. We find that β-catenin BMCs are miscible with LEF1 BMCs in vitro and in cultured cells. We characterized a panel of β-catenin mutants with different combinations of aromatic residue mutations in human cell culture and Drosophila melanogaster. Our data support a model in which aromatic residues across both IDRs contribute to BMC formation and signaling activity. Although different Wnt targets have different sensitivities to loss of β-catenin's aromatic residues, the activation of every target examined was compromised by aromatic substitution. These mutants are not defective in nuclear import or co-immunoprecipitation with several β-catenin binding partners. In addition, residues in the N-terminal IDR with no previously known role in signaling are clearly required for the activation of various Wnt readouts. Consistent with this, deletion of the N-terminal IDR results in a loss of signaling activity, which can be rescued by the addition of heterologous IDRs enriched in aromatic residues. Overall, our work supports a model in which the ability of β-catenin to form biomolecular condensates in the nucleus is tightly linked to its function as a transcriptional co-regulator.
