Investigating Novel Beta-Catenin Interactions in Wnt Target Gene Regulation in Human and Drosophila Cells

Abstract

Activation of Wnt target genes requires the nuclear localization of β-catenin, a transcriptional co-regulator, to Wnt-regulated enhancers (WREs). β-catenin is recruited to WREs through direct binding to TCF-family transcription factors and mediates transcriptional activation by binding additional transcriptional co-activators. This process has traditionally been conceptualized through traditional mechanisms of protein-protein interaction. A recent study suggests that human β-catenin can form biomolecular condensates, which implies an alternative mechanism of protein-protein interaction and suggests that these condensates may be important for β-catenin’s function as a transcriptional co-regulator. The primary focus of this thesis is to further examine the requirement of β-catenin condensate formation in regulating Wnt target genes by utilizing a variety of experimental readouts in Drosophila and human tissue.

I characterized the transcriptional activity of a panel of β-catenin mutants which are defective in the ability to form biomolecular condensates. These mutants had different combinations of aromatic amino acid residues within β-catenin’s terminal, intrinsically disordered regions (IDRs) mutated to alanine. The results of the experiments with these mutants support a model in which the ability of β-catenin to form biomolecular condensates is tightly linked to its ability to regulate Wnt target genes.

A significant portion of my thesis work also focused on changes in the histone modification profile of Wnt target genes in response to Wnt activity. It has been previously observed that in response to Wnt signaling, Wnt target gene loci exhibit widespread histone acetylation. This is different from the general histone acetylation profile of active genes, which exhibit histone acetylation at cis-regulatory elements, such as enhancers and promoters. I sought to determine whether the widespread histone acetylation pattern emanated from a single WRE. To that end, I generated and characterized a Drosophila strain that had a WRE that regulates notum, a Wnt target gene, deleted with CRISPR/Cas9. These flies still expressed notum, suggesting that there are additional WREs that regulate notum’s expression. Additionally, we observed that histone acetylation is sufficient for transcription of the Wnt target gene, naked cuticle. Our results illustrate a complicated relationship between histone acetylation, WRE activity, and Wnt target gene transcription.

In total, my dissertation research characterizes the role of β-catenin-containing biomolecular condensates and histone acetylation in Wnt target gene regulation. My work provides a strong functional characterization of biomolecular condensates in Wnt target gene regulation and foundational studies for the sufficiency of histone acetylation in Wnt target gene expression. Future studies aimed at elucidating a direct link between biomolecular condensates and Wnt target gene transcription, in addition to characterizing a biomolecular condensate ‘grammar’ with β-catenin and additional transcriptional co-regulators, will be important next steps for this work.