Beta-catenin-mediated activation of Wnt target genes utilizes a biomolecular condensate-dependent mechanism

Abstract

The Wnt/{beta}-catenin signaling pathway plays numerous, essential roles in animal development and tissue/stem cell maintenance. The activation of genes regulated by Wnt/{beta}-catenin signaling requires the nuclear accumulation of {beta}-catenin, a transcriptional co-activator. {beta}-catenin is recruited to many Wnt-regulated enhancers through direct binding to T-cell factor/Lymphoid enhancer factor (TCF/LEF) family transcription factors. {beta}-catenin has previously been reported to form phase-separated biomolecular condensates (BMCs), which was implicated as a component of {beta}-catenins 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 {beta}-catenin BMCs in Wnt target gene regulation. We find that {beta}-catenin BMCs are miscible with LEF1 BMCs in vitro. We characterized a panel of {beta}-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 in vitro and signaling activity in vivo. Although different Wnt targets have different sensitivities to loss of {beta}-catenins aromatic residues, the activation of every target examined was compromised by aromatic substitution. These mutants are not defective in nuclear import, and 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 {beta}-catenin to form biomolecular condensates in the nucleus is tightly linked to its function as a transcriptional co-regulator.