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At least 127 records · Page 7Linked to original sources
PubMed2003

Lymphoid enhancer factor-1 and beta-catenin inhibit Runx2-dependent transcriptional activation of the osteocalcin promoter.

Functional control of the transcription factor Runx2 is crucial for normal bone formation. Runx2 is detectable throughout osteoblast development and maturation and temporally regulates several bone-specific genes. In this study, we identified a novel post-translational mechanism regulating Runx2-dependent activation of the osteocalcin promoter. A functional binding site for the high mobility group protein lymphoid enhancer-binding factor 1 (LEF1) was found adjacent to the proximal Runx2-binding site in the osteocalcin promoter. In transcription assays, LEF1 repressed Runx2-induced activation of the mouse osteocalcin 2 promoter in several osteoblast lineage cell lines. Mutations in the LEF1-binding site increased the basal activity of the osteocalcin promoter; however, the LEF1 recognition site in the osteocalcin promoter was surprisingly not required for LEF1 repression. A novel interaction between the DNA-binding domains of Runx2 and LEF1 was identified and found crucial for LEF1-mediated repression of Runx2. LEF1 is a nuclear effector of the Wnt/LRP5/beta-catenin signaling pathway, which is also essential for osteoblast proliferation and normal skeletal development. A constitutively active beta-catenin enhanced LEF1-dependent repression of Runx2. These data identify a novel mechanism of regulating Runx2 activity in osteoblasts and link Runx2 transcriptional activity to beta-catenin signaling.

3T3 Cells↗
PubMed2003

CK2 phosphorylation of the armadillo repeat region of beta-catenin potentiates Wnt signaling.

Protein kinase CK2 is a ubiquitous serine/threonine kinase involved in many biological processes. It is overexpressed in many malignancies including rodent and human breast cancer, and is up-regulated in Wnt-transfected mammary epithelial cells, where it can be found in a complex with dishevelled and beta-catenin. beta-Catenin is a substrate for CK2 and inhibition of CK2 reduces levels of beta-catenin and dishevelled. Here we report that inhibition of CK2 using pharmacologic agents or expression of kinase inactive subunits reduces beta-catenin-dependent transcription and protein levels in a proteasome-dependent fashion. The major region of phosphorylation of beta-catenin by CK2 is the central armadillo repeat domain, where carrier proteins like axin and the adenomatous polyposis coli gene product APC interact with beta-catenin. The major CK2 phosphorylation site in this domain is Thr393, a solvent-accessible residue in a key hinge region of the molecule. Mutation of this single amino acid reduces beta-catenin phosphorylation, cotranscriptional activity, and stability. Thus, CK2 is a positive regulator of Wnt signaling through phosphorylation of beta-catenin at Thr393, leading to proteasome resistance and increased protein and co-transcriptional activity.

Animals↗
PubMed2006

Role of the N-terminal activation domain of the coiled-coil coactivator in mediating transcriptional activation by beta-catenin.

The coiled-coil coactivator (CoCoA) is involved in transcriptional activation of target genes by nuclear receptors and the xenobiotic aryl hydrocarbon receptor, as well as target genes of the Wnt signaling pathway, which is mediated by the lymphocyte enhancer factor (LEF)/T cell factor transcription factors and the coactivator beta-catenin. The recruitment of CoCoA by nuclear receptors is accomplished by the interaction of the central coiled-coiled domain of CoCoA with p160 coactivators; the C-terminal activation domain (AD) of CoCoA is used for downstream signaling, whereas the function of the N-terminal region is undefined. Here we report that the N terminus of CoCoA contains another AD, which is necessary and sufficient for synergistic activation of LEF1-mediated transcription by CoCoA and beta-catenin. The N-terminal AD contains a p300 binding motif, which is important for synergistic cooperation of CoCoA and p300 as coactivators for LEF1 and beta-catenin. p300 contributes to the function of the CoCoA N-terminal AD primarily through its histone acetyltransferase activity. Moreover, in cultured cells, endogenous p300 is recruited to the promoter of an integrated reporter gene by the N terminus of CoCoA. Thus, the coactivator function of CoCoA for nuclear receptors and LEF1/beta-catenin involves differential utilization of two different CoCoA ADs.

Amino Acid Sequence↗
PubMed2000

Hot spots in beta-catenin for interactions with LEF-1, conductin and APC.

Interactions between beta-catenin and LEF-1/TCF, APC and conductin/axin are essential for wnt-controlled stabilization of beta-catenin and transcriptional activation. The wnt signal transduction pathway is important in both embryonic development and tumor progression. We identify here amino acid residues in beta-catenin that distinctly affect its binding to LEF-1/TCF, APC and conductin. These residues form separate surface clusters, termed hot spots, along the armadillo superhelix of beta-catenin. We also show that complementary charged and hydrophobic amino acids are required for formation of the bipartite beta-catenin-LEF-1 transcription factor. Moreover, we demonstrate that conductin/axin binding to beta-catenin is essential for beta-catenin degradation, and that APC acts as a cofactor of conductin/axin in this process. Binding of APC to conductin/axin activates the latter and occurs between their SAMP and RGS domains, respectively.

Adenomatous Polyposis Coli Protein↗
PubMed2001

Alx4 binding to LEF-1 regulates N-CAM promoter activity.

During murine embryogenesis, expression of the paired-like homeodomain protein Alx4 is restricted to tissues whose development depends on the expression of lymphoid enhancer factor-1 (LEF-1). Given the defects seen in hair follicle development in both LEF-1 and Alx4 knockout and mutant animals and the overlapping expression patterns, we predicted that LEF-1 and Alx4 might form physical complexes. We demonstrate here the interaction between LEF-1 and Alx4. This interaction is mediated through a specific proline-rich domain in the N-terminal region of Alx4 and requires the DNA-binding domain (HMG-box) of LEF-1. We also demonstrate that LEF-1 and Alx4 can bind simultaneously to adjacent sites on the neural cell adhesion molecule (N-CAM) promoter and that this binding alters N-CAM promoter activity. Furthermore, when expressed in primary mammary stromal cells, Alx4 decreases the expression of endogenous N-CAM protein. These results reveal a potential mechanism that gives rise to mesenchymal-specific activities of LEF-1.

Adenoviridae↗
PubMed2005

PITX2, beta-catenin and LEF-1 interact to synergistically regulate the LEF-1 promoter.

PITX2, beta-catenin and lymphoid enhancer factor (LEF-1) are required for the inductive formation of several epithelial-derived organs, including teeth. Lef-1 is expressed in the dental epithelium after Pitx2, and both factors have overlapping expression patterns in the tooth bud and cap stages. Our analysis of Pitx2-/- mutant mice showed reduced Lef-1 expression in facial tissues by RT-PCR and quantitative RT-PCR. Consistent with these results we show that the human 2.5 kb LEF-1 promoter is activated by PITX2. Furthermore, the LEF-1 promoter is differentially activated by PITX2 isoforms, which are co-expressed in dental epithelium. The 2.5 kb LEF-1 promoter contains two regions that act to inhibit its transcription in concert with PITX2. The proximal region contains a Wnt-responsive element (WRE) that attenuates PITX2 activation. LEF-1 cannot autoregulate LEF-1 expression; however co-transfection of PITX2 and LEF-1 result in a synergistic activation of the 2.5 kb LEF-1 promoter. LEF-1 specifically interacts with the PITX2 C-terminal tail. Deletion of a distal 800 bp segment of the LEF-1 promoter resulted in enhanced PITX2 activation, and increased synergistic activation in the presence of LEF-1. Furthermore, beta-catenin in combination with PITX2 synergistically activates the LEF-1 promoter and this activation is independent of the Wnt-responsive element. beta-catenin directly interacts with PITX2 to synergistically regulate LEF-1 expression. We show a new mechanism where LEF-1 expression is regulated through PITX2, LEF-1 and beta-catenin direct physical interactions. LEF-1 and beta-catenin interactions with PITX2 provide new mechanisms for the regulation of PITX2 transcriptional activity.

Animals↗
PubMed2002

Dynamics of a developmental switch: recursive intracellular and intranuclear redistribution of Caenorhabditis elegans POP-1 parallels Wnt-inhibited transcriptional repression.

POP-1, a Tcf/Lef factor, functions throughout Caenorhabditis elegans development as a Wnt-dependent reiterative switch to generate nonequivalent sister cells that are born by anterior-posterior cell divisions. We have observed the interaction between POP-1 and a target gene that it represses as it responds to Wnt signaling. Dynamic observations in living embryos reveal that POP-1 undergoes Wnt-dependent nucleocytoplasmic redistribution immediately following cytokinesis, explaining the differential nuclear POP-1 levels in nonequivalent sister cells. In unsignaled (anterior) but not Wnt-signaled (posterior) sister cells, POP-1 progressively coalesces into subnuclear domains during interphase, coincident with its action as a repressor. While the asymmetric distribution of POP-1 in nonequivalent sisters apparently requires a 124-amino-acid internal domain, neither the HMG box nor beta-catenin interaction domains are required. We find that a transcriptional activator, MED-1, associates in vivo with the end-1 and end-3 target genes in the mesoderm (anterior sister) and in the endoderm (posterior sister) following the asymmetric cell division that subdivides the mesendoderm. However, in the anterior sister, binding of POP-1 to the end-1 and end-3 genes blocks their expression. In vivo, binding of POP-1 to the end-1 and end-3 targets (in the posterior sister) is blocked by Wnt/MAPK signaling. Thus, a Tcf/Lef factor represses transactivation of genes in an unsignaled daughter cell by abrogating the function of a bound activator.

Animals↗
PubMed2005

Differential use of functional domains by coiled-coil coactivator in its synergistic coactivator function with beta-catenin or GRIP1.

beta-Catenin, a pivotal component of the Wnt-signaling pathway, binds to and serves as a transcriptional coactivator for the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcriptional activator proteins and for the androgen receptor (AR), a nuclear receptor. Three components of the p160 nuclear receptor coactivator complex, including CARM1, p300/CBP, and GRIP1 (one of the p160 coactivators), bind to and cooperate with beta-catenin to enhance transcriptional activation by TCF/LEF and AR. Here we report that another component of the p160 nuclear receptor coactivator complex, the coiled-coil coactivator (CoCoA), directly binds to and cooperates synergistically with beta-catenin as a coactivator for AR and TCF/LEF. CoCoA uses different domains to bind GRIP1 and beta-catenin, and it uses different domains to transmit the activating signal to the transcription machinery, depending on whether it is bound to GRIP1 or beta-catenin. CoCoA associated specifically with the promoters of transiently transfected and endogenous target genes of TCF/LEF, and reduction of the endogenous CoCoA level decreased the ability of TCF/LEF and beta-catenin to activate transcription of transient and endogenous target genes. Thus, CoCoA uses different combinations of functional domains to serve as a physiologically relevant component of the Wnt/beta-catenin signaling pathway and the androgen signaling pathway.

Animals↗
PubMed2005

Recognition complex between the HMG domain of LEF-1 and its cognate DNA studied by molecular dynamics simulations with explicit solvation.

Molecular dynamics simulations of the complex formed between the HMG box of the lymphoid enhancer-binding factor (LEF-1) and its cognate DNA duplex were carried out with explicit inclusion of water. The simulation started with an NMR-based model (pdb code 2LEF) and the dynamics was pursued for 10 nanoseconds without constraints. It revealed that water intervenes in many ionic/polar interactions, establishing in particular local equilibria between direct and water-mediated hydrogen bonds, and thus increasing the entropy of the complex. Quite unexpectedly, the simulation indicated that a binding pocket for a specific water molecule may be reversibly formed at the apex of the bend induced in the DNA helix by LEF-1 binding, where a methionine side chain intercalates between two destacked adenines. We observed that the specific water molecule can temporarily replace the intercalated S-CH(3) group, acting as a sort of "extension" of the side chain. The residence time of this water molecule was about 3.5 ns. Simulations of the cognate DNA alone showed that this sequence has no intrinsic tendency to bend; therefore, the bending occurs solely as a consequence of the recognition, following the "induced-fit" mechanism.

Adenine↗
PubMed2003

Genomics, transcriptomics, proteomics, and numbers.

OBJECTIVE: To review the advances in clinically useful molecular biologic techniques and to identify their applications in clinical practice, as presented at the 11th Annual William Beaumont Hospital DNA Symposium. DATA SOURCES: The 8 manuscripts submitted were reviewed, and their major findings were compared with literature on the same or related topics. STUDY SELECTION: Manuscripts address the use of molecular techniques in microbiology to evaluate infectious disease and epidemiology; molecular microbiology methods, including rapid-cycle real-time polymerase chain reaction; peroxisome proliferator-activated receptor gamma as a potential therapeutic target in inflammatory bowel disease or colon cancer; the effect of nonapoptotic doses of the bisbenizamide dye Hoechst 33342 on luciferase expression in plasmid-transfected BC3H-1 myocytes; the routine use of cystic fibrosis screening and its challenges; and the use of flow cytometry and/or chromosomal translocation in the diagnostic evaluation of hematopoietic malignancies. DATA SYNTHESIS: Three current issues related to the use of molecular tests in clinical laboratories are (1) the restriction on introducing new tests secondary to existing patents or licenses; (2) the preanalytic variables for the different specimen types currently in use, including whole blood, plasma, serum, fresh or frozen tissues, and free-circulating DNA; and (3) the interpretation of studies evaluating the association of complex diseases with a single mutation or single-nucleotide polymorphism. Molecular methods have had a major impact on infectious disease through the rapid identification of organisms, the evaluation of outbreaks, and the characterization of drug resistance when compared with standard culture techniques. The activation of peroxisome proliferator-activated receptor gamma stimulated by thiazolidinedione is useful in the treatment of type II diabetes mellitus and may have value in preventing inflammatory bowel disease or colon cancer. Hoechst 33342 binding to adenine-thymine-rich regions in the minor groove of DNA is a fluorescent stain for DNA and initiates apoptosis at >10 microg/mL. Lower doses of Hoechst 33342 promote luciferase expression by a mechanism that may involve binding to cryptic promoters facilitated by dye-associated misalignment of the tertiary structure of DNA. The routine use of cystic fibrosis screening is complicated by the more than 1000 mutations associated with the disease. The use of 4-color flow cytometry and the detection of chromosomal translocation are both invaluable aids in establishing the diagnosis of lymphoid or myeloid hematopoietic malignancies. CONCLUSIONS: The current postgenomic era will continue to emphasize the use of microarrays and database software for genomic, transcriptomic, and proteomic screening in the search for useful clinical assays. The number of molecular pathologic techniques will expand as additional disease-associated mutations are defined.

Cystic Fibrosis↗
PubMed2003

Regulation of NF-kappaB-dependent lymphocyte activation and development by paracaspase.

Paracaspase (MALT1), a member of an evolutionarily conserved superfamily of caspase-like proteins, has been shown to bind and colocalize with the protein Bcl10 in vitro and, because of this association, has been suggested to be involved in the CARMA1-Bcl10 pathway of antigen-induced nuclear factor kappaB (NF-kappaB) activation. We demonstrate that primary T and B lymphocytes from paracaspase-deficient mice are defective in antigen-receptor-induced NF-kappaB activation, cytokine production, and proliferation. Paracaspase acts downstream of Bcl10 to induce NF-kappaB activation and is required for the normal development of B cells, indicating that paracaspase provides the missing link between Bcl10 and activation of the IkappaB kinase complex.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Ternary complex factor-serum response factor complex-regulated gene activity is required for cellular proliferation and inhibition of apoptotic cell death.

Members of the ternary complex factor (TCF) subfamily of the ETS-domain transcription factors are activated through phosphorylation by mitogen-activated protein kinases (MAPKs) in response to a variety of mitogenic and stress stimuli. The TCFs bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). The association of TCFs with SREs within immediate-early gene promoters is suggestive of a role for the ternary TCF-SRF complex in promoting cell cycle entry and proliferation in response to mitogenic signaling. Here we have investigated the downstream gene regulatory and phenotypic effects of inhibiting the activity of genes regulated by TCFs by expressing a dominantly acting repressive form of the TCF, Elk-1. Inhibition of ternary complex activity leads to the downregulation of several immediate-early genes. Furthermore, blocking TCF-mediated gene expression leads to growth arrest and triggers apoptosis. By using mutant Elk-1 alleles, we demonstrated that these effects are via an SRF-dependent mechanism. The antiapoptotic gene Mcl-1 is identified as a key target for the TCF-SRF complex in this system. Thus, our data confirm a role for TCF-SRF-regulated gene activity in regulating proliferation and provide further evidence to indicate a role in protecting cells from apoptotic cell death.

Alleles↗
PubMed2003

Ccd1, a novel protein with a DIX domain, is a positive regulator in the Wnt signaling during zebrafish neural patterning.

Wnt signaling plays a crucial role in directing cell differentiation, polarity, and growth. In the canonical pathway, Wnt receptors activate Dishevelled (Dvl), which then blocks the degradation of a key signal transducer, beta-catenin, leading to the nuclear accumulation of beta-catenin and induction of Wnt target genes through TCF/LEF family transcription factors. Here we identified a novel zebrafish gene encoding Ccd1, which possesses a DIX (Dishevelled-Axin) domain. DIX domains are essential for the signal transduction of two major Wnt downstream mediators, Dvl and Axin. Ccd1 formed homomeric and heteromeric complexes with Dvl and Axin and activated TCF-dependent transcription in vitro. In addition, overexpression of ccd1 in zebrafish embryos led to a reduction in the size of the eyes and forebrain (posteriorization), as seen with wnt8 overexpression, whereas a dominant-negative ccd1 (DN-ccd1) caused the opposite phenotype. Furthermore, the Wnt activation phenotype induced by ccd1 was inhibited by the expression of axin1 or DN-ccd1, and the wnt8 overexpression phenotype was rescued by DN-ccd1, suggesting that Ccd1 functions downstream of the Wnt receptor and upstream of Axin. These results indicate that Ccd1 is a novel positive regulator in this Wnt signaling pathway during zebrafish neural patterning.

Amino Acid Sequence↗
PubMed2001

Bcl10 and MALT1, independent targets of chromosomal translocation in malt lymphoma, cooperate in a novel NF-kappa B signaling pathway.

At least two distinct recurrent chromosomal translocations have been implicated in the pathogenesis of MALT lymphoma. The first, t(1;14), results in the transfer of the entire Bcl10 gene to chromosome 14 wherein Bcl10 expression is inappropriately stimulated by the neighboring Ig enhancer. The second, t(11;18), results in the synthesis of a novel fusion protein, API2-MALT1. Until now, no common mechanism of action has been proposed to explain how the products of these seemingly unrelated translocations may contribute to the same malignant process. We show here that Bcl10 and MALT1 form a strong and specific complex within the cell, and that these proteins synergize in the activation of NF-kappaB. The data support a mechanism of action whereby Bcl10 mediates the oligomerization and activation of the MALT1 caspase-like domain. This subsequently activates the IKK complex through an unknown mechanism, setting in motion a cascade of events leading to NF-kappaB induction. Furthermore, the API2-MALT1 fusion protein also strongly activates NF-kappaB and shows dependence upon the same downstream signaling factors. We propose a model whereby both the Bcl10.MALT1 complex and the API2-MALT1 fusion protein activate a common downstream signaling pathway that originates with the oligomerization-dependent activation of the MALT1 caspase-like domain.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Melanocyte-specific microphthalmia-associated transcription factor isoform activates its own gene promoter through physical interaction with lymphoid-enhancing factor 1.

Waardenburg syndrome type 2 (WS2) is associated with heterozygous mutations in the gene encoding microphthalmia-associated transcription factor (MITF) and characterized by deafness and hypopigmentation due to lack of melanocytes in the inner ear and skin. Melanocyte-specific MITF isoform (MITF-M) is essential for melanocyte differentiation and is transcriptionally induced by Wnt signaling that is mediated by beta-catenin and LEF-1. Here we show that MITF-M transactivates its own promoter (M promoter) by interacting with LEF-1, as judged by transient expression assays and in vitro protein-protein binding assays, whereas no transactivation of the M promoter was detected with MITF-M alone or with the combination of MITF-M and dominant-negative LEF1 that lacks the beta-catenin-binding domain. This synergy depends on the three LEF-1-binding sites that are clustered in the proximal M promoter. Importantly, MITF-M recruited on the M promoter could function as a non-DNA-binding cofactor for LEF-1. Thus, MITF-M may function as a self-regulator of its own expression to maintain a threshold level of MITF-M that is required for melanocyte development. We suggest that MITF-M haploinsufficiency may impair the dosage-sensitive role of MITF-M or the correct assembly of multiple transcription factors, involving MITF-M, on the M promoter, which could account for dominant inheritance of WS2.

Animals↗
PubMed2002

Microphthalmia-associated transcription factor interacts with LEF-1, a mediator of Wnt signaling.

Wnt signals regulate differentiation of neural crest cells through the beta-catenin associated with a nuclear mediator of the lymphoid-enhancing factor 1 (LEF-1)/T-cell factors (TCFs) family. Here we show the interaction 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 that mimics Wnt signaling. beta-catenin is required for efficient transactivation, but 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. This study therefore suggests that the MITF/TFE3 family is a new class of nuclear modulators for LEF-1, which may ensure efficient propagation of Wnt signals in many types of cells.

Amino Acid Sequence↗
PubMed2003

T-cell factor 4N (TCF-4N), a novel isoform of mouse TCF-4, synergizes with beta-catenin to coactivate C/EBPalpha and steroidogenic factor 1 transcription factors.

We have cloned T-cell factor 4N (TCF-4N), an alternative isoform of TCF-4, from developing pituitary and 3T3-L1 preadipocytes. This protein contains the N-terminal interaction domain for beta-catenin but lacks the DNA binding domain. While TCF-4N inhibited coactivation by beta-catenin of a TCF/lymphoid-enhancing factor (LEF)-dependent promoter, TCF-4N potentiated coactivation by beta-catenin of several non-TCF/LEF-dependent promoters. For example, TCF-4N synergized with beta-catenin to activate the alpha-inhibin promoter through functional and physical interactions with the orphan nuclear receptor steroidogenic factor 1 (SF-1). In addition, TCF-4N and beta-catenin synergized with the adipogenic transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) to induce leptin promoter activity. The mechanism by which beta-catenin and TCF-4N coactivated C/EBPalpha appeared to involve p300, based upon synergy between these important transcriptional regulators. Consistent with TCF-4N's redirecting the actions of beta-catenin in cells, ectopic expression of TCF-4N in 3T3-L1 preadipocytes partially relieved the block of adipogenesis caused by beta-catenin. Thus, we propose that TCF-4N inhibits coactivation by beta-catenin of TCF/LEF transcription factors and potentiates the coactivation by beta-catenin of other transcription factors, such as SF-1 and C/EBPalpha.

3T3 Cells↗