Guo RJ , Funakoshi S , Lee HH , Kong J , Lynch JP .

DK068366 NIDDK NIH HHS , P01 DE12467 NIDCR NIH HHS, P30-DK50306 NIDDK NIH HHS , R01 DK068366 NIDDK NIH HHS

	Fig. 1. Comparison of TOPFLASH inhibition with SI promoter activation in DLD1 and 293T cells. ( A and B ) Luciferase activity was determined after Flag-Cdx1 (X1; gray bar) or Flag-Cdx2 (X2; white bar) expression vectors or an empty vector control (E; black bar), were co-transfected with TOPFLASH and SI reporters; 2 μg or 400 ng of the expression vectors were transfected into DLD1 or 293T cells (400 ng of expression vectors). a, Significantly differs from empty control and Cdx1, P < 0.01. b, Significantly differs from empty control, P < 0.05. ( C and D ) Protein levels of Flag-Cdx1 and Flag-Cdx2 after transfection of DLD1 and 293T cells.
	Fig. 2. The inhibition of TOPFLASH by Cdx2 requires distinct domains in the N-terminus. The Cdx2-responsive reporter SI-Luc or the β-catenin/TCF reporter TOPFLASH were transfected into 293T cells to determine the effect of Cdx2 mutations upon their transcriptional activity. ( A) Luciferase activity after co-transfection with Cdx2 wild-type and truncation mutants. ( B ) Additional Cdx2 truncation mutants were generated and tested for their ability to activate SI-Luc or inhibit TOPFLASH. In Cdx2 diagram, HD: homeodomain; A, B,C: conserved domains. Insets: Western blots showing equal protein levels of new mutants and wild-type Cdx2 after transfection. Blots were probed for Cdx2 with tubulin as a loading control. Cdx2ND and Cdx2 S56,57,58,60 were probed using a polyclonal antibody against the Cdx2 C-terminus. a, significantly differs from Cdx2 wild-type, P < 0.001; b, significantly differs from Cdx2 wild-type and Empty vector, P < 0.05; c, Differs from Empty vector and Cdx2Δ50, P < 0.01. * Significantly differs from empty vector control, P < 0.001.
	Fig. 3. The inhibition of TOPFLASH by Cdx2 correlates with disruption of the β-catenin–TCF complex. (A) Constitutively activated chimeric LEF/TCF proteins VP16-Lef1 (17) and CatCLef ( 26 ) or the stabilized β-catenin mutant (S33Y) were transfected into 293T cells along with TOPFLASH and an expression vector for Cdx2 (white bar) or an empty control vector (gray bar). Luciferase activity was determined as before; a, differs from all other transfections, P < 0.001; b, differs from CatCLef and empty vector, P < 0.05; ( B ) A myc-tagged TCF4 and the stabilized β-catenin mutant were co-transfected into 293T cells along with an empty vector control or increasing amounts of a Cdx2 expression vector. Then myc-TCF4 was immunoprecipitated, and the products were analyzed for the presence of β-catenin. ( C ) Similar study except it was carried out in DLD1 colon cancer cells without the addition of the β-catenin mutant S33Y. ( D ) Wild-type Cdx1 and Cdx2 truncation mutants are tested for their ability to disrupt the β-catenin–TCF co-immunoprecipitation complex in 293T cells; 50 ng of each Cdx expression vector was transfected for this study.
	Fig. 4. Cdx2 protein binds β-catenin in vivo but not in vitro . ( A ) A Flag-tagged Cdx2 or the empty Flag vector were co-transfected along with β-catenin (S33Y) into 293T cells, and FLAG or β-catenin immunoprecipitations were performed. ( B ) β-catenin co-immunoprecipitates with Cdx2 in human Caco2 cells. ( C)35 S-labeled Cdx2 was incubated with a full-length GST–β-catenin, or with the control Cdx2-binding GST–UBC9 protein, and then GST pull-down was performed. As a control, the GST–β-catenin construct was incubated with an 35 S-labeled TCF4 protein. ( D) Increasing amounts of an in vitro translated Cdx2 protein were pre-incubated with GST–β-catenin before the addition of 35 S-TCF4.
	Fig. 5. A subdomain located in the N-terminus is required for Cdx2 transcriptional activity and TOPFLASH inhibition. ( A ) N-terminal amino acid sequences of five Cdx2 mutants generated for this study. Mutant sequences in bold and underlined. ( B ) Cdx2 mutants were tested for their ability to transactivate an SI reporter or block β-catenin/TCF-mediated TOPFLASH activation as before. S33Y-β-catenin was co-transfected into 293T cells along with wild-type Cdx2 (white bar), mutant Cdx2s (gray bars), or the empty vector control (black bar); a, significantly different from wild-type Cdx2 and empty vector controls, P < 0.005; b, differs from all other expression vectors, P < 0.001; c, significantly differs from wild-type Cdx2 and empty vector controls, P < 0.05. ( C ) Alignment of murine Cdx2 with other Cdx sequences using MacVector (Oxford Molecular). Genebank accession numbers indicated. Conserved subdomain indicated by box.
	Fig. 6. Inhibition of cancer cell proliferation by Cdx2 requires a conserved subdomain in the N-terminus. (A) HCT116 cells were transfected with a GFP expression vector as well as wild-type Cdx2, the Cdx2 truncation mutants, with Cdx2Mut4, or the empty vector as control. At 48 h, the cells were stained with propidium iodide and DNA content quantitated in the GFP+ cells. *Significantly differs from empty vector, Cdx2ND, and Cdx2Δ50, P < 0.05 by. ( B) Model of Cdx2 inhibition of Wnt/β-catenin transcriptional activity. β-catenin translocates to the nucleus where it partners with a TCF family member, bind DNA and activates target genes. In the presence of Cdx2 expression and an as yet unidentified factor or post-translational modification (X), β-catenin associates with Cdx2. This interaction prevents β-catenin/TCF transcriptional activity.

Aoki, Colonic polyposis caused by mTOR-mediated chromosomal instability in Apc+/Delta716 Cdx2+/- compound mutant mice. 2003, Pubmed

Aoki, Colonic polyposis caused by mTOR-mediated chromosomal instability in Apc+/Delta716 Cdx2+/- compound mutant mice. 2003, Pubmed
Arcinas, Molecular mechanisms of transcriptional control of bcl-2 and c-myc in follicular and transformed lymphoma. 2001, Pubmed
Bonhomme, The Cdx2 homeobox gene has a tumour suppressor function in the distal colon in addition to a homeotic role during gut development. 2003, Pubmed
Boudreau, Hepatocyte nuclear factor-1 alpha, GATA-4, and caudal related homeodomain protein Cdx2 interact functionally to modulate intestinal gene transcription. Implication for the developmental regulation of the sucrase-isomaltase gene. 2002, Pubmed
Boudreau, Sucrase-isomaltase gene transcription requires the hepatocyte nuclear factor-1 (HNF-1) regulatory element and is regulated by the ratio of HNF-1 alpha to HNF-1 beta. 2001, Pubmed
Boulanger, Cdk2-dependent phosphorylation of homeobox transcription factor CDX2 regulates its nuclear translocation and proteasome-mediated degradation in human intestinal epithelial cells. 2005, Pubmed
Clevers, Wnt/beta-catenin signaling in development and disease. 2006, Pubmed
Crissey, The homeodomain transcription factor Cdx1 does not behave as an oncogene in normal mouse intestine. 2008, Pubmed
Dang, CDX2 has tumorigenic potential in the human colon cancer cell lines LOVO and SW48. 2006, Pubmed
Dang, Expression of the gut-enriched Krüppel-like factor (Krüppel-like factor 4) gene in the human colon cancer cell line RKO is dependent on CDX2. 2001, Pubmed
Ee, Cdx-2 homeodomain protein expression in human and rat colorectal adenoma and carcinoma. 1995, Pubmed
Ezaki, The homeodomain transcription factors Cdx1 and Cdx2 induce E-cadherin adhesion activity by reducing beta- and p120-catenin tyrosine phosphorylation. 2007, Pubmed
Funakoshi, Repression of the desmocollin 2 gene expression in human colon cancer cells is relieved by the homeodomain transcription factors Cdx1 and Cdx2. 2008, Pubmed
Galceran, Rescue of a Wnt mutation by an activated form of LEF-1: regulation of maintenance but not initiation of Brachyury expression. 2001, Pubmed
Gao, Establishment of intestinal identity and epithelial-mesenchymal signaling by Cdx2. 2009, Pubmed
Gottardi, E-cadherin suppresses cellular transformation by inhibiting beta-catenin signaling in an adhesion-independent manner. 2001, Pubmed
Gross, Phosphorylation of the homeotic tumor suppressor Cdx2 mediates its ubiquitin-dependent proteasome degradation. 2005, Pubmed
Guo, Cdx1 inhibits human colon cancer cell proliferation by reducing beta-catenin/T-cell factor transcriptional activity. 2004, Pubmed , Xenbase
Guo, The role of Cdx proteins in intestinal development and cancer. 2004, Pubmed
Hecht, Regulation of sucrase and lactase in developing rats: role of nuclear factors that bind to two gene regulatory elements. 1997, Pubmed
Hinoi, CDX2 regulates liver intestine-cadherin expression in normal and malignant colon epithelium and intestinal metaplasia. 2002, Pubmed
Kahler, Lymphoid enhancer factor-1 and beta-catenin inhibit Runx2-dependent transcriptional activation of the osteocalcin promoter. 2003, Pubmed
Keller, Cdx1 or Cdx2 expression activates E-cadherin-mediated cell-cell adhesion and compaction in human COLO 205 cells. 2004, Pubmed
Korinek, Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. 1998, Pubmed
Korinek, Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma. 1997, Pubmed
Kuhnert, Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1. 2004, Pubmed
Lynch, The caudal-related homeodomain protein Cdx1 inhibits proliferation of intestinal epithelial cells by down-regulation of D-type cyclins. 2000, Pubmed
Lynch, Cdx1 inhibits the proliferation of human colon cancer cells by reducing cyclin D1 gene expression. 2003, Pubmed
Lynch, The genetic pathogenesis of colorectal cancer. 2002, Pubmed
Mallo, Expression of the Cdx1 and Cdx2 homeotic genes leads to reduced malignancy in colon cancer-derived cells. 1998, Pubmed
McLin, Repression of Wnt/beta-catenin signaling in the anterior endoderm is essential for liver and pancreas development. 2007, Pubmed , Xenbase
Okubo, Hyperactive Wnt signaling changes the developmental potential of embryonic lung endoderm. 2004, Pubmed
Orsulic, E-cadherin binding prevents beta-catenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. 1999, Pubmed
Rawat, Ectopic expression of the homeobox gene Cdx2 is the transforming event in a mouse model of t(12;13)(p13;q12) acute myeloid leukemia. 2004, Pubmed
Rings, Phosphorylation of the serine 60 residue within the Cdx2 activation domain mediates its transactivation capacity. 2001, Pubmed
Saegusa, A functional role of Cdx2 in beta-catenin signaling during transdifferentiation in endometrial carcinomas. 2007, Pubmed
Sampson, Negative regulation of the Wnt-beta-catenin pathway by the transcriptional repressor HBP1. 2001, Pubmed
Schneikert, The canonical Wnt signalling pathway and its APC partner in colon cancer development. 2007, Pubmed
Silberg, CDX1 protein expression in normal, metaplastic, and neoplastic human alimentary tract epithelium. 1997, Pubmed
Soubeyran, Cdx1 promotes differentiation in a rat intestinal epithelial cell line. 1999, Pubmed
Stone, Gut-enriched Krüppel-like factor regulates colonic cell growth through APC/beta-catenin pathway. 2002, Pubmed
Suh, A homeodomain protein related to caudal regulates intestine-specific gene transcription. 1994, Pubmed
Suh, An intestine-specific homeobox gene regulates proliferation and differentiation. 1996, Pubmed
Suh, DNA methylation down-regulates CDX1 gene expression in colorectal cancer cell lines. 2002, Pubmed
Suzuki, Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer. 2004, Pubmed
Tago, Inhibition of Wnt signaling by ICAT, a novel beta-catenin-interacting protein. 2000, Pubmed , Xenbase
Van de Wetering, Extensive alternative splicing and dual promoter usage generate Tcf-1 protein isoforms with differential transcription control properties. 1996, Pubmed
Witek, The putative tumor suppressor Cdx2 is overexpressed by human colorectal adenocarcinomas. 2005, Pubmed
Wong, Loss of CDX1 expression in colorectal carcinoma: promoter methylation, mutation, and loss of heterozygosity analyses of 37 cell lines. 2004, Pubmed
Zorn, Regulation of Wnt signaling by Sox proteins: XSox17 alpha/beta and XSox3 physically interact with beta-catenin. 1999, Pubmed , Xenbase