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R Grosschedl

Publications and source records attributed to R Grosschedl.

At least 37 records · Page 2Linked to original sources
PubMed1998

Redundant regulation of T cell differentiation and TCRalpha gene expression by the transcription factors LEF-1 and TCF-1.

Lymphoid enhancer factor 1 (LEF-1) and T cell factor 1 (TCF-1) are closely related transcription factors that are both expressed during murine T cell differentiation and that regulate the T cell receptor alpha (TCRalpha) enhancer in transfection assays. Targeted gene disruption of either the Tcf1 or Lef1 gene in mice did not affect TCRalpha gene expression and resulted in an incomplete defect or no defect in thymocyte differentiation. Here, we examine a potential redundancy of these transcription factors by analyzing double-mutant mice. In fetal thymic organ cultures from Lef1-/- Tcf1-/- mice, alpha/beta T cell differentiation is completely arrested at the immature CD8+ single-positive (CD8+ ISP) stage and is markedly impaired at an earlier stage. In addition, we find that sorted CD8+ ISP cells from Lef1-/- Tcf1-/- mice express TCRbeta but show a severely reduced level of TCRalpha gene transcription. Together, these data show that LEF-1 and TCF-1 are redundant in the regulation of T cell differentiation and gene expression.

Adoptive Transfer↗
PubMed1998

Modulation of transcriptional regulation by LEF-1 in response to Wnt-1 signaling and association with beta-catenin.

Wnt signaling is thought to be mediated via interactions between beta-catenin and members of the LEF-1/TCF family of transcription factors. Here we study the mechanism of transcriptional regulation by LEF-1 in response to a Wnt-1 signal under conditions of endogenous beta-catenin in NIH 3T3 cells, and we examine whether association with beta-catenin is obligatory for the function of LEF-1. We find that Wnt-1 signaling confers transcriptional activation potential upon LEF-1 by association with beta-catenin in the nucleus. By mutagenesis, we identified specific residues in LEF-1 important for interaction with beta-catenin, and we delineated two transcriptional activation domains in beta-catenin whose function is augmented in specific association with LEF-1. Finally, we show that a Wnt-1 signal and beta-catenin association are not required for the architectural function of LEF-1 in the regulation of the T-cell receptor alpha enhancer, which involves association of LEF-1 with a different cofactor, ALY. Thus, LEF-1 can assume diverse regulatory functions by association with different proteins.

3T3 Cells↗
PubMed1998

A mouse mandibular culture model permits the study of neural crest cell migration and tooth development.

A major issue in developmental biology is to determine how time and position-restricted instructions are signaled and received during morphogenesis of different phenotypes, of which tooth, Meckel's cartilage and tongue formation are classical examples. It is now evident that a hierarchy of growth factors and their downstream transcription factors regulate the timing, sequence and position of cells and tissues in forming different phenotypes during embryogenesis. Here we report the development of an early mandibular organ culture model. Explants of E8 and E9 first branchial arch were cultured and produced mandibular processes with cap stage tooth formation, Meckel's cartilage and tongue development. In tandem, vital dye (Dil) labeling studies confirmed that rhombomeres 1-4 give rise to craneal neural crest (CNC) cells which emigrate from the neural fold to the forming maxillary and mandibular arches. Furthermore, we have tested the feasibility of investigating the regulation of different phenotypes within the first branchial arch by a transcription factor using this early mandibular organ culture model. Lymphoid enhancing factor 1 (Lef1), a transcription factor, has been implicated to regulate tooth formation in vivo. We have analyzed the expression of Lef1 and studied the biological effects of Lef1 on E8 embryonic mouse first branchial arch explants in organ culture. Collectively, these results demonstrate that first branchial arch explant model is suitable for studies of rhombencephalic crest cell fate during mandibular morphogenesis and can be used as a model with direct access to investigate the molecular mechanism in regulating first branchial arch morphogenesis.

Animals↗
PubMed1997

LEF-1/TCF proteins mediate wnt-inducible transcription from the Xenopus nodal-related 3 promoter.

The Xenopus nodal-related 3 gene (Xnr3) is expressed in the Spemann organizer of the embryo and encodes a member of the transforming growth factor beta family that mediates some activities of the organizer. Xnr3 is transcriptionally activated by wnt signaling during gastrulation in the Xenopus embryo. Here we show that a small region of the Xnr3 promoter is sufficient to confer wnt-inducible transcription. By mutational analysis of the promoter, we have identified two distinct sequence elements required for the response to wnt signals. One regulatory sequence interacts with a factor which accumulates in Xenopus gastrulae independent of wnt signaling. The other functionally important site can bind mammalian LEF-1 protein, a member of the LEF-1/TCF family of transcription factors. In addition, misexpression of LEF-1 in embryo explants induces transcription of the endogenous Xnr3 gene. Taken together, these data provide further evidence for a role of LEF-1/TCF proteins in wnt signaling and identify the Spemann organizer-specific gene Xnr3 as a direct target of these transcription factors in vertebrates.

Animals↗
PubMed1997

Functional analysis of DNA bending and unwinding by the high mobility group domain of LEF-1.

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, we extend the analysis of the DNA-binding properties of LEF-1 and examine their contributions to the regulation of gene expression. We find that 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. We also show by a phasing analysis that the LEF-induced DNA bend is directed toward the major groove. In addition, we find that the interaction of LEF-1 with a specific binding site in circular DNA changes the linking number of DNA and unwinds the double helix. Finally, we identified two nucleotides in the LEF-1-binding site 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.

DNA, Superhelical↗
PubMed1997

LEF-1, a nuclear factor coordinating signaling inputs from wingless and decapentaplegic.

wingless and decapentaplegic signal during endoderm induction in Drosophila to regulate expression of the homeotic gene Ultrabithorax. Here, we define a minimal wingless response sequence in the midgut enhancer of Ultrabithorax. We show that this sequence is recognized by the murine transcription factor LEF-1 (lymphocyte enhancer binding factor 1) in a ternary complex with armadillo protein, the cytoplasmic target of the wingless signaling pathway. In stable transformants, transcriptional stimulation of the Ultrabithorax enhancer by LEF-1 depends on armadillo. Furthermore, overexpression of LEF-1 bypasses the need for wingless signaling and causes phenotypes in the midgut, notum, and wing that mimic wingless hyperstimulation. Finally, efficient transcriptional stimulation by LEF-1 in the midgut depends also on the decapentaplegic response sequence and is limited spatially by decapentaplegic signaling. Thus, LEF-1 coordinates inputs from multiple positional signals, consistent with its architectural role in regulating the assembly of multiprotein enhancer complexes.

Animals↗
PubMed1997

ALY, a context-dependent coactivator of LEF-1 and AML-1, is required for TCRalpha enhancer function.

LEF-1 is a transcription factor that participates in the regulation of the T-cell receptor alpha (TCR alpha) 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. With the aim of gaining insight into the function of context-dependent activation domains, we cloned ALY, a novel LEF-1-interacting protein. ALY is a ubiquitously expressed, nuclear protein that specifically associates with the activation domains of LEF-1 and AML-1 (CBF alpha2, PEBP2 alpha(B), which is another protein component of the TCR alpha enhancer complex. In addition, ALY can increase DNA binding by both LEF-1 and AML proteins. Overexpression of ALY stimulates the activity of the TCR alpha enhancer complex reconstituted in transfected nonlymphoid HeLa cells, whereas down-regulation of ALY by anti-sense oligonucleotides virtually eliminates TCR alpha enhancer activity in T cells. Similar to LEF-1, ALY can stimulate transcription in the context of the TCR alpha enhancer but apparently not when tethered to DNA through an heterologous DNA-binding domain. We propose that ALY mediates context-dependent transcriptional activation by facilitating the functional collaboration of multiple proteins in the TCR alpha enhancer complex.

Amino Acid Sequence↗
PubMed1997

Extension of chromatin accessibility by nuclear matrix attachment regions.

Transcription of the variable region of the rearranged immunoglobulin mu gene is dependent on an enhancer sequence situated within one of the introns of the gene. Experiments with transgenic mice have shown that activation of the promoter controlling this transcription also requires the matrix-attachment regions (MARs) that flank the intronic enhancer. As this mu gene enhancer can establish local areas of accessible chromatin, we investigated whether the MARs can extend accessibility to more distal positions. We eliminated interactions between enhancer- and promoter-bound factors by linking mu enhancer/MAR fragments to the binding sites for bacteriophage RNA polymerases that were either close to or one kilobase distal to the enhancer. The mu enhancer alone mediated chromatin accessibility at the proximal site but required a flanking MAR to confer accessibility upon the distal promoter. This long-range accessibility correlates with extended demethylation of the gene construct but not with whether it is being actively transcribed. MARs thus collaborate with the mu enhancer to generate an extended domain of accessible chromatin.

Animals↗
PubMed1997

EBF and E47 collaborate to induce expression of the endogenous immunoglobulin surrogate light chain genes.

Early B cell factor (EBF) and E47 participate in the transcriptional control of early B lymphocyte differentiation. With the aim of identifying genetic targets for these transcription factors, we stably transfected cDNAs encoding EBF or a covalent homodimer of E47, individually or together, into immature hematopoietic Ba/F3 cells, which lack both factors. In combination, EBF and E47 induce efficient expression of the endogenous immunoglobulin surrogate light chain genes, lambda5 and VpreB, whereas other pre-B cell-specific genes remain silent. Multiple functionally important EBF and E47 binding sites were identified in the lambda5 promoter/enhancer region, indicating that lambda5 is a direct genetic target for these transcription factors. Taken together, these data suggest that EBF and E47 synergize to activate expression of a subset of genes that define an early stage of the B cell lineage.

Animals↗
PubMed1996

Functional interaction of beta-catenin with the transcription factor LEF-1.

The cytoplasmic proteins beta-catenin of vertebrates and armadillo of Drosophila have two functions: they link the cadherin cell-adhesion molecules to the cytoskeleton, and they participate in the wnt/wingless signal pathway. Here we show, in a yeast two-hybrid screen, that the architectural transcription factor LEF-1 (for lymphoid enhancer-binding factor) interacts with beta-catenin. In mammalian cells, coexpressed LEF-1 and beta-catenin form a complex that is localized to the nucleus and can be detected by immunoprecipitation. Moreover, LEF-1 and beta-catenin form a ternary complex with DNA that splays an altered DNA bend. Microinjection of LEF-1 into XenoPus embryos induces axis duplication, which is augmented by interaction with beta-catenin. Thus beta-catenin regulates gene expression by direct interaction with transcription factors such as LEF-1, providing a molecular mechanism for the transmission of signals, from cell-adhesion components or wnt protein to the nucleus.

Animals↗
PubMed1996

Lef1 expression is activated by BMP-4 and regulates inductive tissue interactions in tooth and hair development.

Targeted inactivation of the murine gene encoding the transcription factor LEF-1 abrogates the formation of organs that depend on epithelial-mesenchymal tissue interactions. In this study we have recombined epithelial and mesenchymal tissues from normal and LEF-1-deficient embryos at different stages of development to define the LEF-1-dependent steps in tooth and whisker organogenesis. At the initiation of organ development, formation of the epithelial primordium of the whisker but not tooth is dependent on mesenchymal Lef1 gene expression. Subsequent formation of a whisker and tooth mesenchymal papilla and completion of organogenesis require transient expression of Lef1 in the epithelium. These experiments indicate that the effect of Lef1 expression is transmitted from one tissue to the other. In addition, the finding that the expression of Lef1 can be activated by bone morphogenetic protein 4 (BMP-4) suggests a regulatory role of this transcription factor in BMP-mediated inductive tissue interactions.

Animals↗
PubMed1995

Structural basis for DNA bending by the architectural transcription factor LEF-1.

Lymphoid enhancer-binding factor (LEF-1) and the closely related T-cell factor 1 (TCF-1) are sequence-specific and cell-type-specific DNA-binding proteins that play important regulatory roles in organogenesis and thymocyte differentiation. LEF-1 participates in regulation of the enhancer associated with the T cell receptor (TCR)-alpha gene by inducing a sharp bend in the DNA and facilitating interactions between Ets-1, PEBP2-alpha, and ATF/CREB, transcription factors bound at sites flanking the LEF-1 site. It seems that LEF-1 plays an architectural role in the assembly and function of this regulatory nucleoprotein complex. LEF-1 recognizes a specific nucleotide sequence through a high-mobility-group (HMG) domain. Proteins containing HMG domains bind DNA in the minor groove, bend the double helix, and recognize four-way junctions and other irregular DNA structures. Here we report the solution structure of a complex of the LEF-1 HMG domain and adjacent basic region with its cognate DNA. The structure reveals the HMG domain bound in the widened minor groove of a markedly distorted and bent double helix. The basic region binds across the narrowed major groove and contributes to DNA recognition.

Amino Acid Sequence↗
PubMed1995

Failure of B-cell differentiation in mice lacking the transcription factor EBF.

Early B-cell factor (EBF) is a cell type-specific transcription factor that is expressed at all antigen-independent stages of B-lymphocyte differentiation and participates in the regulation of the mb-1 gene. Here we show, by targeted gene disruption in mice, that EBF is necessary for the generation of immunoglobulin-expressing B cells. EBF-deficient mice lack B cells that have rearranged their immunoglobulin D and JH gene segments, but contain B220+CD43+ progenitor cells that express germline mu and IL-7 receptor transcripts. Various non-lymphoid tissues that express EBF are apparently normal in homozygous mutant mice, including olfactory neurons in which EBF was identified as Olf-1 (refs 5, 6). Together, these data suggest that EBF plays a specific and important role in the transcriptional control of B-cell differentiation at a stage before Ig (immunoglobulin) gene rearrangement but after commitment of cells to the B-lymphoid lineage.

Animals↗
PubMed1995

EBF contains a novel zinc coordination motif and multiple dimerization and transcriptional activation domains.

Early B cell factor (EBF) was identified and cloned as a transcription factor expressed specifically in B lymphocytes and adipocytes. This protein was also identified as olfactory factor 1 (Olf-1) in olfactory neurons. In this study, we analyzed the structural requirements for DNA binding, homodimerization and transcriptional activation by EBF. A carboxyl-terminal region, containing a repeat of alpha-helices related to the helix-loop-helix motif, is important for dimerization of EBF in solution and can confer dimerization upon a heterologous DNA binding protein. The amino-terminal DNA binding domain by itself is monomeric, but can mediate assembly of dimers on optimized and correctly spaced half-sites. Mutational analysis of the DNA binding domain of EBF indicated that a novel zinc coordination motif consisting of H-X3-C-X2-C-X5-C is important for DNA recognition. Deletion analysis and transfer of regions of EBF onto a heterologous DNA binding domain identified a serine/threonine-rich transcriptional activation domain. Moreover, the DNA binding domain of EBF can mediate transcriptional activation from optimized binding sites. Thus, EBF contains both a complex DNA binding domain that allows for dimerization and transcriptional activation, and additional dimerization and activation domains.

Amino Acid Sequence↗
PubMed1995

Assembly and function of a TCR alpha enhancer complex is dependent on LEF-1-induced DNA bending and multiple protein-protein interactions.

In this study we examine the molecular basis for the synergistic regulation of the minimal TCR alpha enhancer by multiple proteins. We find that 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. We show that 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 was found previously to bend the DNA helix in the center of the TCR alpha enhancer. We now show that 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.

Activating Transcription Factors↗
PubMed1995

Higher-order nucleoprotein complexes in transcription: analogies with site-specific recombination.

Regulation of transcription involves the assembly of multiprotein complexes at enhancers and promoters. Interactions between adjacent and non-adjacent DNA-binding proteins can augment the specificity and stability of multi-component nucleoprotein complexes. Recently, several proteins have been identified that can function as 'architectural' elements in the assembly of higher-order nucleoprotein structures reminiscent of those involved in site-specific recombination in prokaryotes.

Animals↗