Expression of CD44 in Apc and Tcf mutant mice implies regulation by the WNT pathway.
(1/898)
Overexpression of cell surface glycoproteins of the CD44 family is an early event in the colorectal adenoma-carcinoma sequence. This suggests a link with disruption of APC tumor suppressor protein-mediated regulation of beta-catenin/Tcf-4 signaling, which is crucial in initiating tumorigenesis. To explore this hypothesis, we analyzed CD44 expression in the intestinal mucosa of mice and humans with genetic defects in either APC or Tcf-4, leading to constitutive activation or blockade of the beta-catenin/Tcf-4 pathway, respectively. We show that CD44 expression in the non-neoplastic intestinal mucosa of Apc mutant mice is confined to the crypt epithelium but that CD44 is strongly overexpressed in adenomas as well as in invasive carcinomas. This overexpression includes the standard part of the CD44 (CD44s) as well as variant exons (CD44v). Interestingly, deregulated CD44 expression is already present in aberrant crypt foci with dysplasia (ACFs), the earliest detectable lesions of colorectal neoplasia. Like ACFs of Apc-mutant mice, ACFs of familial adenomatous polyposis (FAP) patients also overexpress CD44. In sharp contrast, Tcf-4 mutant mice show a complete absence of CD44 in the epithelium of the small intestine. This loss of CD44 concurs with loss of stem cell characteristics, shared with adenoma cells. Our results indicate that CD44 expression is part of a genetic program controlled by the beta-catenin/Tcf-4 signaling pathway and suggest a role for CD44 in the generation and turnover of epithelial cells. (+info)
Myogenic basic helix-loop-helix proteins and Sp1 interact as components of a multiprotein transcriptional complex required for activity of the human cardiac alpha-actin promoter.
(2/898)
Activation of the human cardiac alpha-actin (HCA) promoter in skeletal muscle cells requires the integrity of DNA binding sites for the serum response factor (SRF), Sp1, and the myogenic basic helix-loop-helix (bHLH) family. In this study we report that activation of the HCA correlates with formation of a muscle-specific multiprotein complex on the promoter. We provide evidence that proteins eluted from the multiprotein complex specifically react with antibodies directed against myogenin, Sp1, and SRF and that the complex can be assembled in vitro by using the HCA promoter and purified MyoD, E12, SRF, and Sp1. In vitro and in vivo assays revealed a direct association of Sp1 and myogenin-MyoD mediated by the DNA-binding domain of Sp1 and the HLH motif of myogenin. The results obtained in this study indicate that protein-protein interactions and the cooperative DNA binding of transcriptional activators are critical steps in the formation of a transcriptionally productive multiprotein complex on the HCA promoter and suggest that the same mechanisms might be utilized to regulate the transcription of muscle-specific and other genes. (+info)
Evolutionary conservation of MyoD function and differential utilization of E proteins.
(3/898)
The formation of striated muscle in both vertebrates and invertebrates involves the activity of the MyoD family of basic-helix-loop-helix (bHLH) transcription factors. The high degree of evolutionary conservation of MyoD-related proteins, both in the sequence of their bHLH domains and in their general developmental expression patterns, suggests that these factors are also conserved at the level of function. We have addressed this directly using MyoD and E protein factors from vertebrates, Drosophila, and Caenorhabditis elegans. Various MyoD and E factor combinations were tested for their ability to interact in vitro and to function in vivo in the myogenic conversion of 10T12 mouse fibroblasts. We found that the ability of different homo- and heterodimers to bind DNA in vitro was an accurate measure of biological activity in vivo. A second assessment of conserved function comes from the ability of these factors to rescue a C. elegans hlh-1 (CeMyoD) null mutation. We found that both Drosophila and chicken MyoD-related factors were able to rescue a C. elegans CeMyoD loss-of-function mutation. These results demonstrate a remarkable degree of functional conservation of these myogenic factors despite differences in E-protein interactions. (+info)
Activation of somatostatin receptor II expression by transcription factors MIBP1 and SEF-2 in the murine brain.
(4/898)
Somatostatin receptor type II expression in the mammalian brain displays a spatially and temporally very restricted pattern. In an investigation of the molecular mechanisms controlling these patterns, we have recently shown that binding of the transcription factor SEF-2 to a novel initiator element in the SSTR-2 promoter is essential for SSTR-2 gene expression. Further characterization of the promoter identified a species-conserved TC-rich enhancer element. By screening a mouse brain cDNA expression library, we cloned a cDNA encoding the transcription factor MIBP1. MIBP1 interacts specifically with both the TC box in the SSTR-2 promoter and with the SEF-2 initiator-binding protein to enhance transcription from the basal SSTR-2 promoter. We then investigated SSTR-2, SEF-2, and MIBP1 mRNA expression patterns in the developing and adult murine brain by Northern blotting and in situ hybridization. While SEF-2 is widely expressed in many neuronal and nonneuronal tissues, MIBP1 expression overlapped precisely with expression of SSTR-2 in the frontal cortex and hippocampus. In summary, our data for the first time define a regulatory role for the transcription factor MIBP1 in mediating spatially and temporally regulated SSTR-2 expression in the brain. (+info)
Tcf-1-mediated transcription in T lymphocytes: differential role for glycogen synthase kinase-3 in fibroblasts and T cells.
(5/898)
Beta-catenin is the vertebrate homolog of the Drosophila segment polarity gene Armadillo and plays roles in both cell-cell adhesion and transduction of the Wnt signaling cascade. Recently, members of the Lef/Tcf transcription factor family have been identified as protein partners of beta-catenin, explaining how beta-catenin alters gene expression. Here we report that in T cells, Tcf-1 also becomes transcriptionally active through interaction with beta-catenin, suggesting that the Wnt signal transduction pathway is operational in T lymphocytes as well. However, although Wnt signals are known to inhibit the activity of the negative regulatory protein kinase glycogen synthase kinase-3beta (GSK-3beta), resulting in increased levels of beta-catenin, we find no evidence for involvement of GSK-3beta in Tcf-mediated transcription in T cells. That is, a dominant negative GSK-3beta does not specifically activate Tcf transcription and stimuli (lithium or phytohemagglutinin) that inhibit GSK-3beta activity also do not activate Tcf reporter genes. Thus, inhibition of GSK-3beta is insufficient to activate Tcf-dependent transcription in T lymphocytes. In contrast, in C57MG fibroblast cells, lithium inactivates GSK-3beta and induces Tcf-controlled transcription. This is the first demonstration that lithium can alter gene expression of Tcf-responsive genes, and points to a difference in regulation of Wnt signaling between fibroblasts and lymphocytes. (+info)
Different sequence requirements for expression in erythroid and megakaryocytic cells within a regulatory element upstream of the GATA-1 gene.
(6/898)
The lineage-restricted transcription factor GATA-1 is required for differentiation of erythroid and megakaryocytic cells. We have localized a 317 base pair cis-acting regulatory element, HS I, associated with a hematopoietic-specific DNase I hypersensitive site, which lies approx. 3.7 kilobases upstream of the murine hematopoietic-specific GATA-1 IE promoter. HS I directs high-level expression of reporter GATA-1/lacZ genes to primitive and definitive erythroid cells and megakaryocytes in transgenic mice. Comparative sequence analysis of HS I between human and mouse shows approx. 63% nucleotide identity with a more conserved core of 169 base pairs (86% identity). This core contains a GATA site separated by 10 base pairs from an E-box motif. The composite motif binds a multi-protein hematopoietic-specific transcription factor complex which includes GATA-1, SCL/tal-1, E2A, Lmo2 and Ldb-1. Point mutations of the GATA site abolishes HS I function, whereas mutation of the E-box motif still allows reporter gene expression in both lineages. Strict dependence of HS I activity on a GATA site implies that assembly of a protein complex containing a GATA-factor, presumably GATA-1 or GATA-2, is critical to activating or maintaining its function. Further dissection of the 317 base pair region demonstrates that, whereas all 317 base pairs are required for expression in megakaryocytes, only the 5' 62 base pairs are needed for erythroid-specific reporter expression. These findings demonstrate differential lineage requirements for expression within the HS I element. (+info)
XCtBP is a XTcf-3 co-repressor with roles throughout Xenopus development.
(7/898)
XTcf-3 is an HMG box transcription factor that mediates Xenopus dorsal-ventral axis formation. As a Wnt pathway effector, XTcf-3 interacts with beta-catenin and activates the expression of the dorsal organizing gene siamois, while in the absence of beta-catenin, XTcf-3 functions as a transcriptional repressor. We show that XTcf-3 contains amino- and carboxy-terminal repressor domains and have identified a Xenopus member of the C-terminal Binding Protein family of transcriptional co-repressors (XCtBP) as the C-terminal co-repressor. We show that two XCtBP binding sites near the XTcf-3 carboxy-terminus are required for the interaction of XTcf-3 and XCtBP and for the transcriptional repression mediated by the XTcf-3 carboxy-terminal domain. By fusing the GAL4 activation domain to XCtBP we have generated an antimorphic protein, XCtBP/G4A, that activates siamois transcription through an interaction with endogenous XTcf-3. Ectopic expression of XCtBP/G4A demonstrates that XCtBP functions in the regulation of head and notochord development. Our data support a role for XCtBP as a co-repressor throughout Xenopus development and indicate that XCtBP/G4A will be a useful tool in determining how XCtBP functions in various developmental processes. (+info)
Beta- and gamma-catenin mutations, but not E-cadherin inactivation, underlie T-cell factor/lymphoid enhancer factor transcriptional deregulation in gastric and pancreatic cancer.
(8/898)
Adenomatous polyposis coli (APC) mutations are present in >70% of colon cancers. The APC protein binds to beta-catenin (beta-cat), a protein first identified because of its role in E-cadherin (E-cad) cell adhesion. In some colon cancers lacking APC defects, mutations in presumptive glycogen synthase kinase 3beta phosphorylation sites near the beta-cat NH2 terminus appear to render beta-cat resistant to regulation by APC and glycogen synthase kinase 3beta. In cells with APC or beta-cat defects, beta-cat is stabilized and, in turn, binds to and activates T-cell factor (Tcf)/lymphoid enhancer factor (Lef) transcription factors. To further explore the role of APC, beta-cat, Tcf, and E-cad defects in gastrointestinal cancers, we assessed gastric and pancreatic cancers for constitutive Tcf transcriptional activity (CTTA). Two of four gastric and two of eight pancreatic cancer lines showed CTTA. One gastric and one pancreatic cancer had mutations in the NH2-terminal phosphorylation sites of beta-cat. The other gastric cancer with CTTA had a missense mutation at serine 28 of gamma-cat, a potential phosphorylation site in this beta-cat-related protein. Although E-cad is an important binding partner for beta-cat and gamma-cat, E-cad inactivation did not result in CTTA. The beta-cat and gamma-cat mutant proteins identified in our studies strongly activated Tcf transcription in vitro, whereas beta-cat mutant proteins with large NH2-terminal deletions had only modest effects on Tcf. Our results suggest a role for Tcf deregulation in gastric and pancreatic cancer, resulting from beta-cat and gamma-cat mutations in some cases and, in others, from yet to be defined defects. Furthermore, these data imply that the consequences of APC and beta-cat mutations are distinct from the effects of E-cad inactivation. (+info)