Wnt3a-dependent and -independent Protein Interaction Networks of Chromatin-bound β-catenin in Mouse Embryonic Stem Cells

Abstract

Canonical Wnt signaling is repeatedly used during development to control cell fate, and it is often implicated in human cancer. ␤-catenin, the effector of Wnt signaling, has a dual function in the cell and is involved in both cell adhesion and transcription. Nuclear ␤-catenin controls transcription through association with transcription factors of the TCF family and the recruitment of epigenetic modifiers. In this study, we used a strategy combining the genetic manipulation of mouse embryonic stem cells with affinity purification and quantitative mass spectroscopy utilizing stable isotope labeling with amino acids in cell culture to study the interactome of chromatin-bound ␤-catenin with and without Wnt3a stimulation. We uncovered previously unknown interactions of ␤-catenin with transcription factors and chromatin-modifying complexes. The canonical Wnt signaling pathway plays multiple roles in development and is often dysregulated in human cancers (1). The key event in the canonical Wnt signaling is the regulation of ␤-catenin. Under normal conditions, the levels of ␤-catenin are low as a result of its phosphorylation by the destruction complex and subsequent ubiquitination and degradation by the proteasome (1). The destruction complex contains the scaffold proteins APC and Axin1, protein phosphatase 2a, and the kinases glycogen synthase kinase (GSK)-3␤ and casein kinase I alpha. Upon binding of Wnt ligands to the receptors Frizzled and LRP5/6, the destruction complex is turned off via translocation to the plasma membrane, where it binds Dishevelled. Thus, ␤-catenin is stabilized, translocates to the nucleus, and associates with TCF factors and with various proteins that are implicated in chromatin structure and RNA polymerase II regulation (1, 2).The interactions of ␤-catenin with chromatin effector proteins are concentrated at the C-terminal domain of ␤-catenin, as shown for the histone acetyltransferases CBP and p300, the histone methyltransferase MLL, the nucleosome repositioning SWI/SNF family member protein Brg1, the Mediator component MED12, and the BCL-9/Pygo complex, which has the ability to bind methylated H3K4 (2). It is not probable that all these bulky multiprotein assemblies simultaneously occupy the C-terminal domain of ␤-catenin. Other transcription factors use the same repertoire of co-activators via sequential or cycling recruitment (3). It is likely that ␤-catenin serves as a platform which orchestrates the sequential recruitment of chromatin remodeling factors in a stimulus-dependent manner.In mES 1 cells, the epigenetic regulation of gene expression has been shown to take place at the levels of DNA methylation, histone modification, nucleosome packaging and rearrangement, and higher order chromatin organization (4). Unique to key developmental genes in mES cells are bivalent chromatin domains with both activating and repressing marks, which contribute to the pluripotency potential of mES cells (5). Although Wnt/␤-catenin signaling has been shown to play a role in the self-renewal and plurip...

The Role of Pontin and Reptin in Cellular Physiology and Cancer Etiology

“…The pathway is often found activated in a variety of cancers, either through activating mutations in β-catenin itself or in proteins involved in Wnt-signaling (Morin, 1999 ; Macdonald et al, 2009 ). Pontin and Reptin possess opposing regulatory functions in this pathway (Bauer et al, 1998 , 2000 ; Yakulov et al, 2013 ) (Table 2 ). The ATPases were shown in vitro to directly interact with β-catenin in the same region, and thus might exhibit competitive binding (Bauer et al, 1998 , 2000 ).…”

A Protein Interaction between β-Catenin and Dnmt1 Regulates Wnt Signaling and DNA Methylation in Colorectal Cancer Cells

“…β-catenin is one of the best studied oncoproteins and diverse β-catenin protein-protein interactions have been identified. Recent studies, for example, have revealed the association of β-catenin with chromatin and epigenetic modifying complexes (4,5), and it is clear that our knowledge of β-catenin protein function is quite incomplete.…”

The Wnt/β-Catenin Pathway Regulates the Expression of the miR-302 Cluster in Mouse ESCs and P19 Cells

“…Mouse ESCs [28,29] were cultured in DMEM supplemented with 15% FCS, penicillin, streptomycin, glutamine, non-essential amino acids, sodium pyruvate, β-mercaptoethanol and LIF (in-house produced). P19 cells [30] were cultured in DMEM supplemented with 10% FBS, penicillin, streptomycin, glutamine and non-essential amino acid.…”