Allele-specific endogenous tagging and quantitative analysis of beta-catenin in colorectal cancer cells

Abstract

Wnt signaling plays important roles in development, homeostasis, and tumorigenesis. Mutations in {beta}-catenin that activate Wnt signaling have been found in colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of {beta}-catenin are not fully understood. Here, we genome-engineered fluorescently tagged alleles of the endogenous {beta}-catenin in a colorectal cancer cell line. Wild-type and oncogenic mutant alleles were tagged with different fluorescent proteins, enabling the analysis of both variants in the same cell. We analyzed the properties of both {beta}-catenin alleles using immunoprecipitation, immunofluorescence and fluorescence correlation spectroscopy approaches, revealing distinctly different biophysical properties. In addition, activation of Wnt signaling by treatment with a GSK3{beta} inhibitor or a truncating APC mutation modulated the wild-type allele to mimic the properties of the mutant {beta}-catenin allele. The one-step tagging strategy demonstrates how genome engineering can be employed for the parallel functional analysis of different genetic variants.