4promoter and the known binding sequence of TCF4 for the promoter were used as positive controls, and as a negative control, we used an unbound region of the promoter. induce expression of known Wnt targets in animal caps. Moreover, Zic2 knockdown in transgenic Wnt reporter embryos led to ectopic Wnt signaling activity mainly at the midbrain-hindbrain boundary. Together, our results demonstrate a previously unknown role for ZIC2 as a transcriptional regulator of the -cateninTCF4 complex. genes (genes result in diverse phenotypes such as cerebellar malformations in mutants (1), HPE2 in mutants (2), and left-right asymmetry in mutants (3). Among members of the family, is unique in that it is the only maternally expressed gene, and it is the only one of the genes known to be associated with both major forms of HPE: classic HPE and midline interhemispheric HPE (4). The mechanisms by which defects impact brain development are largely unknown. In (genes are induced by is usually induced at the midbrain-hindbrain boundary (6), whereas and are induced at the forebrain-midbrain boundary and midbrain (7, 8). It has been suggested that genes function as activators of Wnt signaling by acting directly on the expression of the Wnt ligands (9). However, the role of Zic2 in transcriptional activity of the -catenin/Tcf complex, has not been studied in detail. Several studies have indicated a crucial role for in neuroectodermal differentiation (10C13), the process that is also known to be regulated by Wnt antagonists ((14, 15). Expression of and inhibition of Wnt signaling have been shown to be required for the specification of anterior neural fates within the neural plate (13, 16C19). When Wnt/-catenin signals are antagonized, neural progenitors are massively induced (10). These findings prompted us to explore the conversation of ZIC2 and Wnt signaling using and models. The results demonstrate a direct conversation of ZIC2 with TCF4 and identify ZIC2 as a negative regulator of canonical Wnt signaling. EXPERIMENTAL PROCEDURES Cell Culture The human embryonic kidney 293T cell collection and colon cancer cell lines Caco-2, SW480, HCT116, and DLD-1 were obtained from American Type Culture Collection and S-Ruxolitinib managed in Dulbecco’s altered Eagle’s medium supplemented with 10% fetal bovine serum. Plasmids Human full-length ZIC2, a kind gift from Dr. Y. Yang (20), and ZIC2 deletions were subcloned into pcDNA3.1 (Invitrogen). pFLAG-CMV4-TCF4 and its S-Ruxolitinib deletion mutants were kindly provided by Dr. M. Idogawa S-Ruxolitinib (21). pFLAG-TCF4-DN101 was generated by PCR. The pCS2-LEFN–cateninN53 S-Ruxolitinib construct made up Rabbit Polyclonal to PXMP2 of -catenin residues 53C781 fused to LEF1 with a deletion of residues 7C264 was a kind gift from Dr. P. Vogt (22). The TOPflash reporter, which contains three optimal TCF-binding sites upstream of a minimal c-promoter that drives expression of the luciferase gene, and the FOPflash reporter, which contains critical nucleotide replacements within the binding elements, were obtained from Upstate. The human pcDNA3-TCF4E expression vector was a kind gift from Dr. Osamu Tetsu. Myc-pCS2+Zic2 was generated by subcloning the ORF (a nice gift from Dr. D. W. Houston) into the Myc-pCS2+ vector. The pCS2-Myc-Zic2-Tcf plasmid, which lacks the Tcf-binding site (amino acids 339C501), was generated by PCR. Myc–catenin in the pCS2+ plasmid was explained previously (23). The sequences of all plasmids were verified by sequencing. Western Blotting Cells were lysed in lysis buffer (50 mm Tris HCl (pH 7.4), 150 mm NaCl, 1 mm EDTA, and 1% Triton X-100) supplemented with proteinase inhibitor combination (Roche Applied Science) and phosphatase inhibitor combination (Sigma). Protein samples were run on a 10% BisTris gel (Invitrogen) S-Ruxolitinib and electroblotted on Hybond-C nitrocellulose membranes (Amersham Biosciences). After incubation with antibody and washing actions, the blots were developed using ECL Western blotting detection reagent (Amersham Biosciences). The following antibodies were used: rabbit anti-ZIC2 polyclonal antibody (Sigma), mouse anti-TCF4 monoclonal antibody clone 6H5-3 (Millipore), anti-FLAG monoclonal antibody M2 and anti-Myc monoclonal antibody (Sigma), anti–catenin antibody (BD Transduction Laboratories), anti-GAPDH antibody (Abcam), and anti-cyclin D1 polyclonal antibody (Santa Cruz Biotechnology). For.