Table of contents
Nuclear activities of TCF
Transcriptional activation of Wnt/Wg-responsive genes requires the stabilization and nuclear accumulation of ß-catenin, a dedicated coactivator of LEF/TCF enhancer-binding proteins. Recombinant ß-catenin strongly enhances binding and transactivation by LEF-1 on chromatin templates in vitro. Interestingly, different LEF-1 isoforms vary in their ability to bind nucleosomal templates in the absence of ß-catenin, owing to N-terminal residues that repress binding to chromatin, but not nonchromatin, templates. Transcriptional activation in vitro requires both the armadillo (ARM) repeats and the C terminus of ß-catenin, whereas the phosphorylated N terminus is inhibitory to transcription. A fragment spanning the C terminus (CT) and ARM repeats 11 and 12 (CT-ARM), but not the CT alone, functions as a dominant negative inhibitor of LEF-1-ß-cat activity in vitro and can block ATP-dependent binding of the complex to chromatin. LEF-1-ß-cat transactivation in vitro is repressed by inhibitor of ß-catenin and Tcf-4 (ICAT), a physiological inhibitor of Wnt/Wg signaling that interacts with ARM repeats 11 and 12, and by the nonsteroidal anti-inflammatory compound, sulindac. None of these transcription inhibitors (CT-ARM, ICAT, or sulindac) can disrupt the LEF-1-ß-cat complex after it is stably bound to chromatin. It is concluded that the CT-ARM region of ß-catenin functions as a chromatin-specific activation domain, and that several inhibitors of the Wnt/Wg pathway directly modulate LEF-1-ß-cat activity on chromatin (Tutter, 2001).
Although some DNA-binding proteins recognize their binding sites in chromatin efficiently in vitro, others must be incubated with specific chromatin remodeling complexes or chromatin-modifying enzymes to activate transcription from fully-assembled chromatin templates. Therefore, it was important to assess whether the recombinant LEF-1-ß-cat complex can activate transcription from a preassembled nucleosomal template. LEF-1-ß-cat transactivation is very inefficient when the complex is incubated with the pBRE template after nucleosome assembly, but transcription is enhanced significantly in the presence of purified recombinant p300. Activation by p300 is specific because it does not enhance transcription without enhancer factors, or when incubated with LEF-1-ß-catDeltaC. It was also asked whether LEF-1-ß-cat activity could be enhanced by ATP-dependent chromatin remodeling complexes. Although the complex could not be activated with a purified SWI/SNF fraction, LEF-1-ß-cat activity is stimulated by a partially-purified chromatin remodeling fraction (RMF), which contains the hSWI/SNF and hACF/ISWI remodeling complexes and is devoid of p300. The effect of RMF is similar to that observed with recombinant p300, and in combination the two fractions function synergistically. LEF-1-ß-cat activation under these conditions can still be repressed selectively by CT-ARM, and not by the CT fragment of ß-catenin. Enhanced binding in the presence of the RMF fraction is more pronounced with LEF-1-ß-cat than with LEF-1 alone. Thus LEF-1-ß-cat can strongly activate transcription from fully assembled chromatin templates when incubated with p300 and chromatin remodeling enzymes (Tutter, 2001).
DNase I footprint analysis of these transcription reactions revealed that the LEF-1-ß-cat complex also binds very poorly on its own to the pBRE enhancer when added to the template after the chromatin template has been fully assembled, and under these conditions binding of the complex is enhanced considerably by the addition of RMF. Enhanced binding of LEF-1-ß-cat to chromatin in the presence of RMF is completely inhibited by apyrase, indicating that an ATP-dependent chromatin remodeling step is required. Interestingly, RMF-enhanced binding can also be competed by the CT-ARM fragment and not by the CT fragment. In these experiments the CT and CT-ARM inhibitors, or apyrase, were added together with the LEF-1-ß-cat complex and the RMF fraction after the completion of nucleosome assembly. In contrast, purified recombinant p300 does not affect the binding of LEF-1-ß-cat to chromatin. Taken together, these data indicate that LEF-1-ß-cat transactivation requires p300 and chromatin remodeling, and that the CT-ARM fragment can block both ATP-dependent binding of the LEF-1-ß-cat complex to preassembled chromatin, as well as the transcriptional activity of the complex after it has bound stably to chromatin (Tutter, 2001).
LEF-1 (lymphoid enhancer-binding factor 1) is a cell type-specific member of the family of high mobility group (HMG) domain proteins that recognizes a specific nucleotide sequence in the T cell receptor (TCR) alpha enhancer. In this study, the analysis of the DNA-binding properties of LEF-1 is extended; the contribution of these properties to the regulation of gene expression is examined. LEF-1, like nonspecific HMG-domain proteins, can interact with irregular DNA structures such as four-way junctions, albeit with lower efficiency than with specific duplex DNA. The LEF-induced DNA bend is directed toward the major groove. In addition, the interaction of LEF-1 with a specific binding site in circular DNA changes the linking number of DNA and unwinds the double helix. Two nucleotides in the LEF-1-binding site have been identified that are important for protein-induced DNA bending. Mutations of these nucleotides decrease both the extent of DNA bending and the transactivation of the TCR alpha enhancer by LEF-1, suggesting a contribution of protein-induced DNA bending to the function of TCR alpha enhancer (Giese, 1997).
The interaction between beta-catenin and LEF-1/TCF transcription factors plays a pivotal role in the Wnt-1 signaling pathway. The level of beta-catenin is regulated by partner proteins, including glycogen synthase kinase-3beta (GSK-3beta) and the adenomatous polyposis coli (APC) tumor suppressor protein. Genetic defects in APC are responsible for a heritable predisposition to colon cancer. APC protein and GSK-3beta bind beta-catenin, retain it in the cytoplasm, and facilitate the proteolytic degradation of beta-catenin. Abrogation of this negative regulation allows beta-catenin to translocate to the nucleus and to form a transcriptional activator complex with the DNA-binding protein lymphoid-enhancing factor 1 (LEF-1). This complex is thought to be involved in tumorigenesis. Covalent linkage of LEF-1 to beta-catenin and to transcriptional activation domains derived from the estrogen receptor or the herpes simplex virus protein VP16 generates transcriptional regulators that induce oncogenic transformation of chicken embryo fibroblasts. The chimeras between LEF-1 and beta-catenin or VP16 are constitutively active, whereas fusions of LEF-1 to the estrogen receptor are regulatable by estrogen. These experiments document the oncogenicity of transactivating LEF-1 and show that the transactivation domain normally provided by beta-catenin can be replaced by heterologous activation domains. These results suggest that the transactivating function of the LEF-1/beta-catenin complex is critical for tumorigenesis and that this complex transforms cells by activating specific LEF-1 target genes (Aoki, 1999).
The mammalian AML/CBFalpha runt domain (RD) transcription factors regulate hematopoiesis and osteoblast differentiation. Like their Drosophila counterparts, most mammalian RD proteins terminate in a common pentapeptide, VWRPY, which serves to recruit the corepressor Groucho (Gro). Using a yeast two-hybrid assay, in vitro association and pull-down experiments, it has been demonstrated that Gro and its mammalian homolog TLE1 specifically interact with AML1 and AML2. In addition to the VWRPY motif, other C-terminal sequences are required for these interactions with Gro/TLE1. TLE1 inhibits AML1-dependent transactivation of the T cell receptor (TCR) enhancers alpha and beta, which, in transfected Jurkat T cells, contain functional AML binding sites. LEF-1 is an additional transcription factor that mediates transactivation of TCR enhancers. LEF-1 and its Drosophila homolog Pangolin (Pan) are involved in the Wnt/Wg signaling pathway through interactions with the coactivator beta-catenin and its highly conserved fly homolog Armadillo (Arm). TLE/Gro interacts with LEF-1 and Pan, and inhibits LEF-1:beta-catenin-dependent transcription. These data indicate that, in addition to their activity as transcriptional activators, AML1 and LEF-1 can act, through recruitment of the corepressor TLE1, as transcriptional repressors in TCR regulation and Wnt/Wg signaling (Levanon, 1998).
A prominent feature of cell differentiation is the initiation and maintenance of an irreversible cell cycle arrest with the complex involvement of the retinoblastoma (See Drosophila Retinoblastoma-family protein) family (RB, p130, p107). The HBP1 transcriptional repressor has been isolated as a potential target of the RB family in differentiated cells. By homology, HBP1 is a sequence-specific HMG transcription factor, of which LEF-1 is the best-characterized family member. Several features of HBP1 suggest an intriguing role as a transcriptional and cell cycle regulator in differentiated cells:
Taken together, the results suggest that HBP1 may represent a unique transcriptional repressor with a role in initiation and establishment of cell cycle arrest during differentiation (Tevosian. 1997).
The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. The c-MYC oncogene has been identified as a target gene in this signaling pathway. Expression of c-MYC is repressed by wild-type APC and activated by beta-catenin; these effects are mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers (He, 1998).
Mutations in the adenomatous polyposis coli (APC) tumor-suppressor gene occur in most human colon cancers. Loss of functional APC protein results in the accumulation of beta-catenin. Mutant forms of beta-catenin have been discovered in colon cancers that retain wild-type APC genes, and also in melanomas, medulloblastomas, prostate cancer and gastric and hepatocellular carcinomas. The accumulation of beta-catenin activates genes that are responsive to transcription factors of the TCF/LEF family, with which beta-catenin interacts. Beta-catenin activates transcription from the cyclin D1 promoter, and sequences within the promoter that are related to consensus TCF/LEF-binding sites are necessary for activation. The oncoprotein p21ras further activates transcription of the cyclin D1 gene, through sites within the promoter that bind the transcriptional regulators Ets or CREB. Cells expressing mutant beta-catenin produce high levels of cyclin D1 messenger RNA and protein constitutively. Furthermore, expression of a dominant-negative form of TCF in colon-cancer cells strongly inhibits expression of cyclin D1 without affecting expression of cyclin D2, cyclin E, or cyclin-dependent kinases 2, 4 or 6. This dominant-negative TCF causes cells to arrest in the G1 phase of the cell cycle; this phenotype can be rescued by expression of cyclin D1 under the cytomegalovirus promoter. Abnormal levels of beta-catenin may therefore contribute to neoplastic transformation by causing accumulation of cyclin D1 (Tetsu, 1999).
beta-Catenin plays a dual role in the cell: it links the cytoplasmic side of cadherin-mediated cell-cell contacts to the actin cytoskeleton and it acts in signaling that involves transactivation in complex with transcription factors of the lymphoid enhancing factor (LEF-1) family. Elevated beta-catenin levels in colorectal cancer caused by mutations in beta-catenin or by the adenomatous polyposis coli molecule, which regulates beta-catenin degradation, result in the binding of beta-catenin to LEF-1 and increased transcriptional activation of mostly unknown target genes. The cyclin D1 gene is a direct target for transactivation by the beta-catenin/LEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter. Three inhibitors of beta-catenin activation, wild-type adenomatous polyposis coli, axin, and the cytoplasmic tail of cadherin, suppress cyclin D1 promoter activity in colon cancer cells. Cyclin D1 protein levels are augmented by beta-catenin overexpression and reduced in cells overexpressing the cadherin cytoplasmic domain. Increased beta-catenin levels may thus promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression into the cell cycle (Shtutman, 1999).
Lymphoid enhancer-binding factor 1 (LEF-1) is a regulatory high mobility group protein that activates the T cell receptor alpha (TCR alpha) enhancer in a context-restricted manner in T cells. The distal region of the human immunodeficiency virus-1 (HIV-1) enhancer, which contains DNA-binding sites for LEF-1 and Ets-1, also provides a functional context for activation by LEF-1. Mutations in the LEF-1-binding site inhibit the activity of multimerized copies of the HIV-1 enhancer. LEF-1/GAL4 can activate a GAL4-substituted HIV-1 enhancer 80- to 100-fold in vivo. Recombinant LEF-1 activates HIV-1 transcription on chromatin-assembled DNA in vitro. The packaging of DNA into chromatin in vitro strongly represses HIV-1 transcription and repression can be counteracted efficiently by preincubation of the DNA with LEF-1 (or LEF-1 and Ets-1) supplemented with fractions containing the promoter-binding protein, Sp1. Addition of TFE-3, which binds to an E-box motif upstream of the LEF-1 and Ets-1 sites, further augments transcription in this system. A truncation mutant of LEF-1 (HMG-88) containing the HMG box but lacking the trans-activation domain, does not activate transcription from nucleosomal DNA, indicating that bending of DNA by the HMG domain is not sufficient to activate transcription in vitro. Therefore, transcription activation by LEF-1 in vitro appears to be a chromatin-dependent process that requires a functional trans-activation domain in addition to the HMG domain (Sheridan, 1995).
The distal enhancer of the T-cell receptor (TCR) alpha chain gene has become a paradigm for studies of the assembly and activity of architectural enhancer complexes. Regulated TCRalpha enhancer activity has been reconstituted in vitro on chromatin templates using purified T-cell transcription factors (LEF-1, AML1, and Ets-1) and the cyclic AMP-responsive transcription factor CREB (See Drosophila dCREB2). When added in combination, these factors activate the TCRalpha enhancer in a highly synergistic manner. Alternatively, the enhancer could also be activated in vitro by high levels of either CREB or a complex containing all of the T-cell proteins (LEF-1, AML1, and Ets-1). Phosphorylation of CREB by protein kinase A enhances transcription 10-fold in vitro, and this effect is abolished by a point mutation affecting the CREB PKA phosphorylation site (Ser-133). Interestingly, LEF-1 strongly enhances the binding of the AML1/Ets-1 complex on chromatin, but not nonchromatin, templates. A LEF-1 mutant containing only the HMG DNA-binding domain is sufficient to form a higher-order complex with AML1/Ets-1, but exhibits only partial activity in transcription. It is concluded that the T cell-enriched proteins assemble on the enhancer independently of CREB and function synergistically with CREB to activate the TCRalpha enhancer in a chromatin environment (Mayall, 1997).
An examination was made of the molecular basis for the synergistic regulation of the minimal TCR alpha enhancer by multiple proteins was examined. Reconstitution of TCR alpha enhancer function in nonlymphoid cells requires expression of the lymphoid-specific proteins LEF-1, Ets-1 and PEBP2 alpha (CBF alpha), and a specific arrangement of their binding sites in the enhancer. Ets-1 cooperates with PEBP2 alpha to bind adjacent sites at one end of the enhancer, forming a ternary complex that is unstable by itself. Stable occupancy of the Ets-1- and PEBP2 alpha-binding sites in a DNase I protection assay was found to depend on both a specific helical phasing relationship with a nonadjacent ATF/CREB-binding site at the other end of the enhancer and on LEF-1. The HMG domain of LEF-1 bends the DNA helix in the center of the TCR alpha enhancer. The HMG domain of the distantly related SRY protein, which also bends DNA, can partially replace LEF-1 in stimulating enhancer function in transfection assays. Taken together with the observation that Ets-1 and members of the ATF/CREB family have the potential to associate in vitro, these data suggest that LEF-1 can coordinate the assembly of a specific higher-order enhancer complex by facilitating interactions between proteins bound at nonadjacent sites (Giese, 1995).
Tcf transcription factors interact with beta-catenin and Armadillo to mediate Wnt/Wingless signaling. Two murine members of the Tcf family, mTcf-3 and mTcf-4 have been characterized. mTcf-3 mRNA is ubiquitously present in embryonic day 6.5 (E6.5) mouse embryos but gradually disappears over the next 3 to 4 days. mTcf-4 expression occurs first at E10.5 and is restricted to di- and mesencephalon and the intestinal epithelium during embryogenesis. The mTcf-3 and mTcf-4 proteins bind a canonical Tcf DNA motif and can complex with the transcriptional coactivator beta-catenin. Overexpression of Wnt-1 in a mammary epithelial cell line leads to the formation of a nuclear complex between beta-catenin and Tcf proteins and to Tcf reporter gene transcription. These data demonstrate a direct link between Wnt stimulation and beta-catenin/Tcf transcriptional activation and imply a role for mTcf-3 and -4 in early Wnt-driven developmental decisions in the mouse embryo (Korinek, 1998).
Genetic evidence suggests that regulation of beta-catenin and regulation of Tcf/Lef family transcription factors are downstream events in the Wnt signal transduction pathway. However, a direct link between Wnt activity and Tcf/Lef transcriptional activation has yet to be established. In this study, it has been shown that Wnt-1 induces a growth response in a cultured mammalian cell line: Rat-1 fibroblasts. Wnt-1 induces serum-independent cellular proliferation of Rat-1 fibroblasts and changes in morphology. Rat-1 cells stably expressing Wnt-1 (Rat-1/Wnt-1) show a constitutive up-regulation of cytosolic beta-catenin, while membrane-associated beta-catenin remains unaffected. Induction of cytosolic beta-catenin in Rat-1/Wnt-1 cells is correlated with activation of a Tcf-responsive transcriptional element. Thus evidence is provided that Wnt-1 induces Tcf/Lef transcriptional activation in a mammalian system. Expression of a mutant beta-catenin (beta-CatS37A) in Rat-1 cells does not result in a proliferative response or a detectable change in the cytosolic beta-catenin protein level. However, beta-CatS37A expression in Rat-1 cells results in strong Tcf/Lef transcriptional activation, comparable to that seen in Wnt-1-expressing cells. These results suggest that Wnt-1 induction of cytosolic beta-catenin may have functions in addition to Tcf/Lef transcriptional activation (Young, 1998).
Human Tcf-4, a Tcf family member that is expressed in colonic epithelium, transactivates transcription of an artifical promoter only when associated with ß-catenin. Nuclei of cells mutant for Adenomatous polyposis coli tumor suppressor protein are found to contain a stable ß-catenin hTcf-4 complex that is constitutively active. Reintroduction of APC removes ß-catenin from hTcf-4 and abrogates the transcriptional transactivation. Constitutive transcription of Tcf target genes, caused by loss of APC function, may be a crucial event in the early transformation of colonic epithelium (Korinek, 1997).
The distal 3' enhancer of the T cell receptor alpha-chain (TCRalpha) gene directs the tissue- and stage-specific expression and V(D)J recombination of this gene locus. Using an in vitro system that efficiently reproduces TCRalpha enhancer activity, it can be shown that long-range promoter-enhancer regulation requires a T cell-specific repressor complex that operates on the proximal promoter and is sensitive to the DNA topology of the proximal promoter. In this system, the distal enhancer (regulated by ATF/CREB, LEF-1 [a homolog of Drosophila Pangolin], Ets-1 and GATA-3) functions to derepress the promoter on supercoiled, but not relaxed, templates. The TCRalpha promoter is inactivated by a repressor complex that contains the architectural protein HMG I/Y. HMG I/Y binds DNA through contacts in the minor groove, in contrast to both NF-kappaB (see Drosophila homologs Dorsal and Dif) and ATF-2, which both interact with the same sequences in the major groove. In the absence of this repressor complex, expression of the TCRalpha gene is completely independent of the 3' enhancer and DNA topology. The interaction of the T cell-restricted protein LEF-1 with the TCRalpha enhancer is required for promoter derepression. In this system, the TCRalpha enhancer increases the number of active promoters rather than the rate of transcription. Thus, long-range enhancers function in a distinct manner from promoters and provide the regulatory link between repressors, DNA topology, and gene activity (Bagga, 1997).
LEF-1 is a transcription factor that participates in the regulation of the T-cell receptor alpha (TCRalpha) enhancer by facilitating the assembly of multiple proteins into a higher order nucleoprotein complex. The function of LEF-1 is dependent, in part, on the HMG domain that induces a sharp bend in the DNA helix, and on an activation domain that stimulates transcription only in a specific context of other enhancer-binding proteins. ALY, a novel LEF-1-interacting protein was cloned in order to gain insight into the function of context-dependent activation domains. ALY is a ubiquitously expressed, nuclear protein that specifically associates with the activation domains of LEF-1 and AML-1 (see Drosophila Lozenge and Runt), which is another protein component of the TCRalpha enhancer complex. In addition, ALY can increase DNA binding by both LEF-1 and AML proteins. Overexpression of ALY stimulates the activity of the TCRalpha enhancer complex reconstituted in transfected nonlymphoid HeLa cells, whereas down-regulation of ALY by anti-sense oligonucleotides virtually eliminates TCRalpha enhancer activity in T cells. Similar to LEF-1, ALY can stimulate transcription in the context of the TCRalpha enhancer but apparently not when tethered to DNA through an heterologous DNA-binding domain. It is proposed that ALY mediates context-dependent transcriptional activation by facilitating the functional collaboration of multiple proteins in the TCRalpha enhancer complex (Bruhn, 1997).
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. XTcf-3 contains amino- and carboxy-terminal repressor domains and a Xenopus member of the C-terminal Binding Protein family of transcriptional co-repressors (XCtBP) has been identified as the C-terminal co-repressor. 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, an antimorphic protein termed XCtBP/G4A is generated 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. These 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 (Brannon, 1999).
XCtBP is expressed throughout Xenopus development. Maternal XCtBP transcripts are present before fertilization through the start of zygotic transcription, but are not localized prior to the start of gastrulation. XCtBP transcripts begin to accumulate with the onset of neurulation (stage 13), eventually peak in expression in the tailbud embryo (stage 27), and persist into the tadpole. The maternal expression of XCtBP indicates that it could function with maternal XTcf-3 to regulate siamois expression. At the beginning of gastrulation XCtBP transcripts are not spatially restricted. Localized XCtBP transcripts first appear in a broad region of the anterior neural plate and in two stripes lateral to the midline at the early neurula stage (stage 13). Diffuse caudal XCtBP expression is also seen at stage 13 in the region of the future tailbud. At the end of neurulation, XCtBP transcripts are expressed in the eye placodes, along the branchial arches and in the developing brain (stage 20). This neural XCtBP expression extends the length of the embryo along the dorsal midline. Transverse sections of stage 20 embryos reveal that midline XCtBP transcripts are localized to the dorsal and lateral neural tube, a region of the embryo that also expresses two Wnts; Wnt-1 and Wnt-3a. XCtBP transcripts are also seen along the lateral surface of the somites. In dorsal and lateral views of a stage 20 embryo, XCtBP expression clearly outlines the somite borders and appears more intense in the tailbud. This pattern of somite and tailbud expression continues into the early tadpole (stage 33). XCtBP transcripts show continued expression throughout the head in early tadpoles, labeling the eyes, otic vesicles, branchial arches and brain, while more posteriorly XCtBP labels the pronephros. Curiously, XCtBP is excluded from the cement gland. Transverse sections of tadpoles show that XCtBP continues to be expressed in the dorsal and lateral neural tube. These results suggest that there are transcription factor targets of XCtBP, in addition to XTcf-3 and downstream of siamois, that participate in dorsoanterior structure formation. Furthermore, XCtBP is also likely to regulate non-Wnt pathways, since CtBP has been shown to bind a number of transcription factors, including bHLH, zinc-finger and nuclear receptor family members (Brannon, 1999).
Wnt signals regulate differentiation of neural crest cells through the ß-catenin associated with a nuclear mediator of the lymphoid-enhancing factor 1 (LEF-1)/T-cell factors (TCFs) family. An interaction has been demonstrated between the basic helix-loop-helix and leucine-zipper region of microphthalmia-associated transcription factor (MITF) and LEF-1. MITF is essential for melanocyte differentiation and its heterozygous mutations cause auditory-pigmentary syndromes. Functional cooperation of MITF with LEF-1 results in synergistic transactivation of the dopachrome tautomerase (DCT) gene promoter, an early melanoblast marker. This activation depends on the separate cis-acting elements, which are also responsible for the induction of the DCT promoter by lithium chloride (which mimics Wnt signaling). ß-catenin is required for efficient transactivation, but is dispensable for the interaction between MITF and LEF-1. The interaction with MITF is unique to LEF-1 and not detectable with TCF-1. LEF-1 also cooperates with the MITF-related proteins, such as TFE3, to transactivate the DCT promoter. Therefore, this study suggests that the MITF/TFE3 family is a new class of nuclear modulators for LEF-1. The MITF/TFE3 family may ensure efficient propagation of Wnt signals in many types of cells (Yasumoto, 2002).
The transactivation of TCF target genes induced by Wnt pathway mutations constitutes the primary transforming event in colorectal cancer (CRC). Disruption of ß-catenin/TCF-4 activity in CRC cells induces a rapid G1 arrest and blocks a genetic program that is physiologically active in the proliferative compartment of colon crypts. Coincidently, an intestinal differentiation program is induced. The TCF-4 target gene c-MYC plays a central role in this switch by direct repression of the p21CIP1/WAF1 promoter. Following disruption of ß-catenin/TCF-4 activity, the decreased expression of c-MYC releases p21CIP1/WAF1 transcription, which in turn mediates G1 arrest and differentiation. Thus, the ß-catenin/TCF-4 complex constitutes the master switch that controls proliferation versus differentiation in healthy and malignant intestinal epithelial cells (van de Wetering, 2002).
c-MYC plays a central role in the proliferative capacity of many cancers, including CRC. tHE data imply that c-MYC blocks the expression of the cell cycle inhibitor p21CIP1/WAF1. The region responsible for p21CIP1/WAF1 regulation has been mapped to a 200 bp fragment of the proximal promoter. The presence of MIZ-1 and c-MYC on this promoter suggests that c-MYC-mediated repression of p21CIP1/WAF1 occurs by a mechanism resembling c-MYC control of p15INK4b, i.e., through preventing promoter activation by the transcription factor MIZ-1. Decreased expression of c-MYC would allow MIZ-1 to activate p21CIP1/WAF1 transcription. The complementarity in the expression of c-MYC and p21CIP1/WAF1 in the intestine supports this mechanism (van de Wetering, 2002).
In the small intestine, the progeny of stem cells migrate in precise patterns. Absorptive, enteroendocrine, and goblet cells migrate toward the villus while Paneth cells occupy the bottom of the crypts. Here it has been shown that ß-catenin and TCF inversely control the expression of the EphB2/EphB3 receptors and their ligand ephrin-B1 in colorectal cancer and along the crypt-villus axis. Disruption of EphB2 and EphB3 genes reveals that their gene products restrict cell intermingling and allocate cell populations within the intestinal epithelium. In EphB2/EphB3 null mice, the proliferative and differentiated populations intermingle. In adult EphB3-/- mice, Paneth cells do not follow their downward migratory path, but scatter along crypt and villus. It is concluded that in the intestinal epithelium ß-catenin and TCF couple proliferation and differentiation to the sorting of cell populations through the EphB/ephrin-B system (Batlle, 2002).
Early neural patterning along the anteroposterior (AP) axis appears to involve a number of signal transducing pathways, but the precise role of each of these pathways for AP patterning and how they are integrated with signals that govern neural induction step is not well understood. The nature of Fgf response element (FRE) has been investigated in a posterior neural gene, Xcad3 (Xenopus caudal homolog), which plays a crucial role of posterior neural development. Evidence suggests that FREs of Xcad3 are widely dispersed in its intronic sequence and that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs that lie in juxtaposition. Functional and physical analyses indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on these FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox family transcription factors and negatively acting Tcf family transcription factor(s) (Haremaki, 2003).
The reporter constructs containing the FREs exhibit high dose dependence on Fgf similar to that shown for endogenous Xcad3, when examined in the embryonic cell culture assay. Sequence and mutagenesis analyses reveal that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs (EBMs and TLBMs respectively) that lie in juxtaposition. The EBM is known to serve as the binding site for Ets family transcription factors that are nuclear effectors of the Fgf/Ras/Mapk pathway. Indeed, functional and physical analyses show that Ets proteins are involved in the Fgf response of Xcad3 as transcriptional activators, and that Xcad3 is directly targeted by the Fgf signaling pathway. This conclusion is consistent with the previous observation that Fgf can induce Xcad3 expression in the animal cap assay within 2 hours of its addition and even in the presence of the protein synthesis inhibitor cycloheximide, which indicates that Xcad3 is an immediate early target of Fgf signaling (Haremaki, 2003 and references therein).
TLBMs could serve as the binding sites for Tcf/Lef family transcription factors that are nuclear effectors of the Wnt/ß-catenin pathway. It was anticipated that XTcf3 would functioned as a co-activator of Ets proteins, since Wnt signaling has been suggested as being involved in activation of posterior neural genes. Surprisingly, however, functional analysis reveals that XTcf3 acts as a repressor of Xcad3. The data suggest that the endogenous pool of ß-catenin in ectoderm cells is considerably smaller compared with that of XTcf3 co-repressors such as XCtBP and Groucho. This in turn implies that Wnt signaling could activate Xcad3 expression in embryonic cells, when they are provided with a larger pool of ß-catenin. Marginal zone cells of the early gastrula embryo, where Xcad3 is initially expressed, are among such candidate cells, since a relatively large amount of ß-catenin is translocated into the nucleus in these cells. Recently, a mutant function of Tcf3 as a repressor has revealed in the zebrafish headless mutant that carries a mutation in Tcf3. In this mutant, expression of midbrain-hindbrain boundary genes such as En2 and Pax2 is de-repressed in more anterior neural region, leading to severe head defects. It would be interesting to know whether similar anterior expansion is seen in Cdx gene expression in this mutant (Haremaki, 2003 and references therein).
Sox2 is de-repressed by Bmp antagonists in the neurogenic region of ectoderm during neural induction. Sox2, which shares a cognate DNA bindings motif with Tcf/Lef family members, is required as a co-activator for the Fgf response of Xcad3. Sox2 is likely to compete with XTcf3 for TLBMs in the composite FREs to cooperate with Ets proteins that bind to adjacent EBMs. Physical analysis supports this idea. Both Sox and Ets family transcription factors interact with specific partner factors to direct signals to target genes, but direct partnership between them has not been reported. Collectively, these results indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on the FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox proteins and negatively acting Tcf protein (Haremaki, 2003).
Notch signaling in the presomitic mesoderm (psm) is critical for somite formation and patterning. WNT signals regulate transcription of the Notch ligand Dll1 in the tailbud and psm. LEF/TCF factors cooperate with TBX6 to activate transcription from the Dll1 promoter in vitro. Mutating either T or LEF/TCF sites in the Dll1 promoter abolishes reporter gene expression in vitro as well as in the tail bud and psm of transgenic embryos. These results indicate that WNT activity, in synergy with TBX6, regulates Dll1 transcription and thereby controls Notch activity, somite formation, and patterning (Hofmann, 2004).
Wnt signaling, which is mediated by LEF1/TCF transcription factors, has been placed upstream of the Notch pathway in vertebrate somitogenesis. The molecular basis for this presumed hierarchy has been examined and it has been shown that a targeted mutation of Lef1, which abrogates LEF1 function and impairs the activity of coexpressed TCF factors, affects the patterning of somites and the expression of components of the Notch pathway. LEF1 was found to bind multiple sites in the Dll1 promoter in vitro and in vivo. Moreover, mutations of LEF1-binding sites in the Dll1 promoter impair expression of a Dll1-LacZ transgene in the presomitic mesoderm. Finally, the induced expression of LEF1-ß-catenin activates the expression of endogenous Dll1 in fibroblastic cells. Thus, Wnt signaling can affect the Notch pathway by a LEF1-mediated regulation of Dll1 (Galceran, 2004).
Renal dysplasia, the major cause of childhood renal failure in humans, arises from perturbed renal morphogenesis and molecular signaling during embryogenesis. Induction of molecular crosstalk between Smad1 and ß-catenin occurs in the TgAlk3QD mouse model of renal medullary cystic dysplasia. The finding that Myc, a Smad and ß-catenin transcriptional target and effector of renal epithelial dedifferentiation, is misexpressed in dedifferentiated epithelial tubules provides a basis for investigating coordinate transcriptional control by Smad1 and ß-catenin in disease. Enhanced interactions occur between a molecular complex consisting of Smad1, ß-catenin and Tcf4 and adjacent Tcf- and Smad-binding regions located within the Myc promoter in TgAlk3QD dysplastic renal tissue, and Bmp-dependent cooperative control of Myc transcription by Smad1, ß-catenin and Tcf4. Analysis of nuclear extracts derived from TgAlk3QD and wild-type renal tissue revealed increased levels of Smad1/ß-catenin molecular complexes, and de novo formation of chromatin-associated Tcf4/Smad1 molecular complexes in TgAlk3QD tissues. Analysis of a 476 nucleotide segment of the 1490 nucleotide Myc genomic region upstream of the transcription start site demonstrated interactions between Tcf4 and the Smad consensus binding region and associations of Smad1, ß-catenin and Tcf4 with oligo-duplexes that encode the adjacent Tcf- and Smad-binding elements only in TgAlk3QD tissues. In collecting duct cells that express luciferase under the control of the 1490 nucleotide Myc genomic region, Bmp2-dependent stimulation of Myc transcription is dependent on contributions by each of Tcf4, ß-catenin and Smad1. These results provide novel insights into mechanisms by which interacting signaling pathways control transcription during the genesis of renal dysplasia (Hu, 2005)
Transcriptional regulation of TCF
ß-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of ß-catenin is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis coli (APC) or mutations in ß-catenin itself. Another tumor suppressor, PTEN, is also involved in the regulation of nuclear ß-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. Nuclear ß-catenin expression is constitutively elevated in PTEN null cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a ß-catenin/TCF-regulated gene, is also suppressed upon reexpression of PTEN. Mechanistically, PTEN increases the phosphorylation of ß-catenin and enhances its rate of degradation. A pathway is defined that involves mainly integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of ß-catenin stability, nuclear ß-catenin expression, and transcriptional activity. These data indicate that ß-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression (Persad, 2001).
The canonical Wnt and sonic hedgehog pathways have been independently linked to cell proliferation in a variety of tissues and systems. However, interaction of these signals in the control of cell cycle progression has not been studied. This study demonstrates that in the developing vertebrate nervous system these pathways genetically interact to control progression of the G1 phase of the cell cycle. By in vivo loss-of-function experiments, the absolute requirement was demonstrated of an upstream Shh activity for the regulation of Tcf3/4 expression. In the absence of Tcf3/4, the canonical Wnt pathway cannot activate target gene expression, including that of cyclin D1, and the cell cycle is necessarily arrested at G1. In addition to the control of G1 progression, Shh activity controls the G2 phase through the regulation of cyclin E, cyclin A and cyclin B expression, and this is achieved independently of Wnt. Thus, in neural progenitors, cell cycle progression is co-ordinately regulated by Wnt and Shh activities (Alvarez-Medina, 2009).
The progressive loss of CNS myelin in patients with multiple sclerosis (MS) has been proposed to result from the combined effects of damage to oligodendrocytes and failure of remyelination. A common feature of demyelinated lesions is the presence of oligodendrocyte precursors (OLPs) blocked at a premyelinating stage. However, the mechanistic basis for inhibition of myelin repair is incompletely understood. To identify novel regulators of OLP differentiation, potentially dysregulated during repair, a genome-wide screen of 1040 transcription factor-encoding genes expressed in remyelinating rodent lesions was performed. Approximately 50 transcription factor-encoding genes show dynamic expression during repair, and expression of the Wnt pathway mediator Tcf4 (aka Tcf7l2) within OLPs is specific to lesioned-but not normal-adult white matter. Beta-catenin signaling is active during oligodendrocyte development and remyelination in vivo. Moreover, similar regulation is observed of Tcf4 in the developing human CNS and lesions of MS. Data mining revealed elevated levels of Wnt pathway mRNA transcripts and proteins within MS lesions, indicating activation of the pathway in this pathological context. Dysregulation of Wnt-beta-catenin signaling in OLPs results in profound delay of both developmental myelination and remyelination, based on (1) conditional activation of beta-catenin in the oligodendrocyte lineage in vivo and (2) findings from APC(Min) mice, which lack one functional copy of the endogenous Wnt pathway inhibitor APC. Together, these findings indicate that dysregulated Wnt-beta-catenin signaling inhibits myelination/remyelination in the mammalian CNS. Evidence of Wnt pathway activity in human MS lesions suggests that its dysregulation might contribute to inefficient myelin repair in human neurological disorders (Fancy, 2009).
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