Dissecting nuclear Wingless signalling: recruitment of the transcriptional co-activator Pygopus by a chain of adaptor proteins.

Members of the Wingless (Wg)/Wnt family of secreted glycoproteins control cell fate during embryonic development and adult homeostasis. Wnt signals regulate the expression of target genes by activating a conserved signal transduction pathway. Upon receptor activation, the signal is transmitted intracellularly by stabilization of Armadillo (Arm)/beta-catenin. Arm/beta-catenin translocates to the nucleus, interacts with DNA-binding factors of the Pangolin (Pan)/TCF/LEF class and activates transcription of target genes in cooperation with the recently identified proteins Legless/BCL9 (Lgs) and Pygopus (Pygo). Here, we analyse the mode of action of Pan, Arm, Lgs, and Pygo in Drosophila cultured cells. We provide evidence that together these four proteins form a 'chain of adaptors' linking the NH2-terminal homology domain (NHD) of Pygo to the DNA-binding domain of Pan. We show that the NHD has potent transcriptional activation capacity, which differs from that of acidic activator domains and depends on a conserved NPF tripeptide. A single point mutation within this NPF motif abolishes the transcriptional activity of the Pygo NHD in vitro and strongly reduces Wg signalling in vivo. Together, our results suggest that the transcriptional output of Wg pathway activity largely relies on a 'chain of adaptors' design to direct the Pygo NHD to Wg target promoters in an Arm-dependent manner

IRS1 Regulation by Wnt/β-Catenin Signaling and Varied Contribution of IRS1 to the Neoplastic Phenotype*

Dysregulation of β-catenin levels and localization and constitutive activation of β-catenin/TCF (T cell factor)-regulated gene expression occur in many cancers, including the majority of colorectal carcinomas and a subset of ovarian endometrioid adenocarcinomas. Based on the results of microarray-based gene expression profiling we found the insulin receptor substrate 1 (IRS1) gene as one of the most highly up-regulated genes upon ectopic expression of a mutant, constitutively active form of β-catenin in the rat kidney epithelial cell line RK3E. We demonstrate expression of IRS1 can be directly activated by β-catenin, likely in part via β-catenin/TCF binding to TCF consensus binding elements located in the first intron and downstream of the IRS1 transcriptional start site. Consistent with the proposal that β-catenin is an important regulator of IRS1 expression in vivo, we observed that IRS1 is highly expressed in many cancers with constitutive stabilization of β-catenin, such as colorectal carcinomas and ovarian endometrioid adenocarcinomas. Using a short hairpin RNA approach to abrogate IRS1 expression and function, we found that IRS1 function is required for efficient de novo neoplastic transformation by β-catenin in RK3E cells. Our findings add to the growing body of data implicating IRS1 as a critical signaling component in cancer development and progression

Requirement of Cell Cycle and Apoptosis Regulator 1 for Target Gene Activation by Wnt and β-Catenin and for Anchorage-independent Growth of Human Colon Carcinoma Cells*

Aberrant Wnt signaling promotes oncogenesis by increasing cellular levels of β-catenin, which associates with DNA-bound transcription factors and activates Wnt target genes. However, the molecular mechanism by which β-catenin mediates gene expression is still poorly understood. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1), which was recently shown to function as a transcriptional coactivator for nuclear receptors, also interacts with β-catenin and enhances the ability of β-catenin to activate expression of transiently transfected reporter genes. Furthermore, association of CCAR1 with the promoter of an endogenous Wnt/β-catenin target gene in a colon cancer cell line depends on the presence of β-catenin. Depletion of CCAR1 inhibits expression of several Wnt/β-catenin target genes and suppresses anchorage-independent growth of the colon cancer cell line. Thus, CCAR1 is a novel component of Wnt/β-catenin signaling that plays an important role in transcriptional regulation by β-catenin and that, therefore, may represent a novel target for therapeutic intervention in cancers involving aberrantly activated Wnt/β-catenin signaling

Beta-catenin hits chromatin: regulation of Wnt target gene activation

The canonical Wnt pathway has gathered much attention in recent years owing to its fundamental contribution to metazoan development, tissue homeostasis and human malignancies. Wnt target gene transcription is regulated by nuclear beta-catenin, and genetic assays have revealed various collaborating protein cofactors. Their daunting number and diverse nature, however, make it difficult to arrange an orderly picture of the nuclear Wnt transduction events. Yet, these findings emphasize that beta-catenin-mediated transcription affects chromatin. How does beta-catenin cope with chromatin regulation to turn on Wnt target genes