Investigating TCF-Independent β-catenin Signalling and Its Function in Mouse ES Cell Biology

Abstract

The Wnt/β-catenin pathway is a fundamental signalling pathway involved in a communicative process that regulates cellular function during development, immune responses, tissue homeostasis, cell fate determination and repair. It is a crucial pathway, with a multifunctional protein, β-catenin, acting as one of its key components. β-catenin’s interaction with the TCF/LEF family of proteins is very well characterised, however little is known about its TCF/LEF independent roles in the nucleus. To investigate the TCF/LEF independent signalling roles of β-catenin, we employed previously generated TCF/LEF quadruple knockout (QKO) and newly developed, TCF/LEF, β-catenin knockout (TLB) cell lines (i.e. QKO lines that also lack β-catenin expression). In vitro, similarly to QKO cells, the TLB line displayed a clear neuroectodermal differentiation bias and also displayed surprising upregulation of Wnt responsive genes. The neuroectodermal bias in the absence of β catenin reveals that the neuroectodermal differentiation program does not require β- catenin for its initiation. We describe a novel phenotype observed in embryoid body (EB) assays, where we observe that lack of β-catenin results in shedding of nonadherent cells from EBs. Based on our RNA seq data analyses of WT, QKO and TLB lines, the AIRE transcription factor was found to be a potential β-catenin target that is regulated in a TCF/LEF-independent manner. We observed an upregulation in AIRE expression in the QKO cell line, which was not observed in the TLB cell line. We postulate that AIRE plays a role in a β-catenin-regulated pluripotency regulatory network and describe its importance in maintaining self-renewal and cell pluripotency.