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Biomedical subjects

C Murre

Publications and source records attributed to C Murre.

At least 37 records · Page 2Linked to original sources
PubMed1999

Ectopic expression of E47 or E12 promotes the death of E2A-deficient lymphomas.

Mice with null mutations in the E2A gene are highly susceptible to the spontaneous development of thymic lymphomas. To understand better how E2A deficiency may contribute to lymphomagenesis, we have observed the consequences of enforced expression of the E2A gene products E12 and E47 in cell lines derived from lymphomas that arose spontaneously in E2A-deficient mice. E2A-expressing cells are steadily eliminated from lymphoma cultures into which E47 or E12 was introduced. The mechanism underlying the loss of E2A-expressing cells does not involve an arrest in cell-cycle progression. Rather, the E2A proteins activate a programmed cell death pathway in these lymphomas. This E2A-mediated cell death appears to be preceded by a loss of mitochondrial transmembrane potential. These data provide direct evidence that E2A gene products can act as tumor suppressors.

Adenovirus E2 Proteins↗
PubMed1999

Positive and negative regulation of V(D)J recombination by the E2A proteins.

A key feature of B and T lymphocyte development is the generation of antigen receptors through the rearrangement and assembly of the germline variable (V), diversity (D), and joining (J) gene segments. However, the mechanisms responsible for regulating developmentally ordered gene rearrangements are largely unknown. Here we show that the E2A gene products are essential for the proper coordinated temporal regulation of V(D)J rearrangements within the T cell receptor (TCR) gamma and delta loci. Specifically, we show that E2A is required during adult thymocyte development to inhibit rearrangements to the gamma and delta V regions that normally recombine almost exclusively during fetal thymocyte development. The continued rearrangement of the fetal Vgamma3 gene segment in E2A-deficient adult thymocytes correlates with increased levels of Vgamma3 germline transcripts and increased levels of double-stranded DNA breaks at the recombination signal sequence bordering Vgamma3. Additionally, rearrangements to a number of Vgamma and Vdelta gene segments used predominantly during adult development are significantly reduced in E2A-deficient thymocytes. Interestingly, at distinct stages of T lineage development, both the increased and decreased rearrangement of particular Vdelta gene segments is highly sensitive to the dosage of the E2A gene products, suggesting that the concentration of the E2A proteins is rate limiting for the recombination reaction involving these Vdelta regions.

Animals↗
PubMed1999

Activity regulation of a Hox protein and a role for the homeodomain in inhibiting transcriptional activation.

Hox proteins are transcription factors that assign positional identities along the body axis of animal embryos. Different Hox proteins have similar DNA-binding functions in vitro and require cofactors to achieve their biological functions. Cofactors can function by enhancement of the DNA-binding specificity of Hox proteins, as has been shown for Extradenticle (Exd). We present results supporting a novel mechanism for Hox cofactor function: regulation of transcriptional activation function. First, we provide evidence that the Hox protein Deformed (Dfd) can interact with simple DNA-binding sites in Drosophila embryos in the absence of Exd, but this binding is not sufficient for transcriptional activation of reporter genes. Secondly, either Dfd or a Dfd-VP16 hybrid mediate much stronger activation in embryos on a Dfd-Exd composite site than on a simple Dfd-binding site, even though the two sites possess similar Dfd-binding affinities. This suggests that Exd is required to release the transcriptional activation function of Dfd independently of Exd enhancement of Dfd-binding affinity on the composite site. Thirdly, transfection assays confirmed that Dfd possesses an activation domain, which is suppressed in a manner dependent on the presence of the homeodomain. The regulation of Hox transcriptional activation functions may underlie the different functional specificities of proteins belonging to this developmental patterning family.

3T3 Cells↗
PubMed1999

Transcription factors in hematopoiesis.

The advent of gene targeting in the mouse has led to rapid advances in the identification of factors controlling gene expression that are essential for normal hematopoietic development. Recent work has also uncovered roles for some of these factors in leukemogenesis and in the global regulation of chromatin structure.

Animals↗
PubMed1999

A conserved motif present in a class of helix-loop-helix proteins activates transcription by direct recruitment of the SAGA complex.

The class I helix-loop-helix (HLH) proteins, which include E2A, HEB, and E2-2, have been shown to be required for lineage-specific gene expression during T and B lymphocyte development. Additionally, the E2A proteins function to regulate V(D)J recombination, possibly by allowing access of variable region segments to the recombination machinery. The mechanisms by which E2A regulates transcription and recombination, however, are largely unknown. Here, we identify a novel motif, LDFS, present in the vertebrate class I HLH proteins as well as in a yeast HLH protein that is essential for transactivation. We provide both genetic and biochemical evidence that the highly conserved LDFS motif stimulates transcription by direct recruitment of the SAGA histone acetyltransferase complex.

Amino Acid Sequence↗
PubMed1998

Induction of early B cell factor (EBF) and multiple B lineage genes by the basic helix-loop-helix transcription factor E12.

The transcription factors encoded by the E2A and early B cell factor (EBF) genes are required for the proper development of B lymphocytes. However, the absence of B lineage cells in E2A- and EBF-deficient mice has made it difficult to determine the function or relationship between these proteins. We report the identification of a novel model system in which the role of E2A and EBF in the regulation of multiple B lineage traits can be studied. We found that the conversion of 70Z/3 pre-B lymphocytes to cells with a macrophage-like phenotype is associated with the loss of E2A and EBF. Moreover, we show that ectopic expression of the E2A protein E12 in this macrophage line results in the induction of many B lineage genes, including EBF, IL7Ralpha, lambda5, and Rag-1, and the ability to induce kappa light chain in response to mitogen. Activation of EBF may be one of the critical functions of E12 in regulating the B lineage phenotype since expression of EBF alone leads to the activation of a subset of E12-inducible traits. Our data demonstrate that, in the context of this macrophage line, E12 induces expression of EBF and together these transcription factors coordinately regulate numerous B lineage-associated genes.

Animals↗
PubMed1998

The role of E-proteins in B- and T-lymphocyte development.

Department of lymphocytes from hematopoietic stem cells is controlled, in part, by the activity of transcriptional regulatory proteins. In particular, one class of helix-loop-helix proteins, termed E-proteins, have been implicated in the regulation of gene expression during B-cell development. Recent analysis of gene-targeted mice has allowed a direct assessment of the functional roles of several E-protein family members in hematopoiesis. In this review we describe the defects in B- and T-lymphocyte development in mice carrying targeted mutations in the E-protein genes and discuss our current understanding of the role of these proteins in lymphoid development.

Animals↗
PubMed1998

Characterization of ABF-1, a novel basic helix-loop-helix transcription factor expressed in activated B lymphocytes.

Proteins of the basic helix-loop-helix (bHLH) family are required for a number of different developmental pathways, including neurogenesis, lymphopoiesis, myogenesis, and sex determination. Using a yeast two-hybrid screen, we have identified a new bHLH transcription factor, ABF-1, from a human B-cell cDNA library. Within the bHLH region, ABF-1 shows a remarkable conservation with other HLH proteins, including tal-1, NeuroD, and paraxis. Its expression pattern is restricted to a subset of lymphoid tissues, Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines, and activated human B cells. ABF-1 is capable of binding an E-box element either as a homodimer or as a heterodimer with E2A. Furthermore, a heterodimeric complex containing ABF-1 and E2A can be detected in EBV-immortalized lymphoblastoid cell lines. ABF-1 contains a transcriptional repression domain and is capable of inhibiting the transactivation capability of E47 in mammalian cells. ABF-1 represents the first example of a B-cell-restricted bHLH protein, and its expression pattern suggests that ABF-1 may play a role in regulating antigen-dependent B-cell differentiation.

Adenovirus E2 Proteins↗
PubMed1997

Both E12 and E47 allow commitment to the B cell lineage.

The E2A gene products, E12 and E47, are required for proper B cell development. Mice lacking the E2A gene products generate only a very small number of B220+ cells, which lack immunoglobulin DJ(H) rearrangements. We have now generated mice expressing either E12 or E47. B cell development in mice expressing E12 but lacking E47 is perturbed at the pro-B cell stage, and these mice lack IgM+B220+ B cells in both bone marrow and spleen. IgM+B220+ B cells can be detected, albeit at significantly reduced levels, in the bone marrow and spleen of mice lacking E12. Ectopic expression of both E12 and E47 in a null mutant background shows that E12 and E47 act in concert to promote B lineage development. Taken together, the data indicate that both E12 and E47 allow commitment to the B cell lineage and act synergistically to promote B lymphocyte maturation.

Animals↗
PubMed1997

Pbx raises the DNA binding specificity but not the selectivity of antennapedia Hox proteins.

We have used a binding site selection strategy to determine the optimal binding sites for Pbx proteins by themselves and as heterodimeric partners with various Hox gene products. Among the Pbx proteins by themselves, only Pbx3 binds with high affinity, as a monomer or as a homodimer, to an optimal binding site, TGATTGATTTGAT. An inhibitory domain located N terminal of the Pbx1 homeodomain prevents intrinsic Pbx1 binding to this sequence. When complexed with Hoxc-6, each of the Pbx gene products binds the same consensus sequence, TGATTTAT, which differs from the site bound by Pbx3 alone. Three members of the Antennapedia family, Hoxc-6, Hoxb-7, and Hoxb-8, select the same binding site in conjunction with Pbx1. The affinities of these proteins as heterodimeric partners with Pbx1 for the selected optimal binding site are similar. However, the binding specificity of Hox proteins for optimal binding sites is increased, compared to nonspecific DNA, in the presence of Pbx proteins. Thus, while cooperative DNA binding involving heterodimers of Pbx and Hox gene products derived from members within the Antennapedia family does not increase binding site selectivity, DNA binding specificity of the Hox gene products is significantly enhanced in the presence of Pbx.

Antennapedia Homeodomain Protein↗
PubMed1997

E2A deficiency leads to abnormalities in alphabeta T-cell development and to rapid development of T-cell lymphomas.

The E2A gene products, E12 and E47, are critical for proper early B-cell development and commitment to the B-cell lineage. Here we reveal a new role for E2A in T-lymphocyte development. Loss of E2A activity results in a partial block at the earliest stage of T-lineage development. This early T-cell phenotype precedes the development of a T-cell lymphoma which occurs between 3 and 9 months of age. The thymomas are monoclonal and highly malignant and display a cell surface phenotype similar to that of immature thymocytes. In addition, the thymomas generally express high levels of c-myc. As assayed by comparative genomic hybridization, each of the tumor populations analyzed showed a nonrandom gain of chromosome 15, which contains the c-myc gene. Taken together, the data suggest that the E2A gene products play a role early in thymocyte development that is similar to their function in B-lineage determination. Furthermore, the lack of E2A results in development of T-cell malignancies, and we propose that E2A inactivation is a common feature of a wide variety of human T-cell proliferative disorders, including those involving the E2A heterodimeric partners tal-1 and lyl-1.

Animals↗
PubMed1997

Conformational changes induced in Hoxb-8/Pbx-1 heterodimers in solution and upon interaction with specific DNA.

Two classes of homeodomain proteins, Hox and Pbx gene products, have the ability to bind cooperatively to DNA. In Hox proteins, the homeodomain and a highly conserved hexapeptide are required for cooperative DNA binding. In Pbx, the homeodomain and a region immediately C terminal of the homeodomain are essential for cooperativity. Using fluorescence and circular dichroism spectroscopy, we demonstrated that Hox and Pbx proteins interact in the absence of DNA. The interaction in solution is accompanied by conformational changes. Furthermore, upon interaction with specific DNA, additional conformational changes are induced in the Pbx-1/Hoxb-8 heterodimer. These data indicate that prior to DNA binding, Hox-Pbx interaction in solution is accompanied by structural alterations. We propose that these conformational changes modulate the DNA binding properties of these proteins, ultimately resulting in cooperative DNA binding.

DNA↗
PubMed1997

A novel fibroblast growth factor gene expressed in the developing nervous system is a downstream target of the chimeric homeodomain oncoprotein E2A-Pbx1.

Pbx1 is a homeodomain transcription factor that has the ability to form heterodimers with homeodomain proteins encoded by the homeotic selector (Hox) gene complexes and increase their DNA-binding affinity and specificity. A current hypothesis proposes that interactions with Pbx1 are necessary for Hox proteins to regulate downstream target genes that in turn control growth, differentiation and morphogenesis during development. In pre B cell leukemias containing the t(1;19) chromosome translocation, Pbx1 is converted into a strong transactivator by fusion to the activation domain of the bHLH transcription factor E2A. The E2A-Pbx1 fusion protein should therefore activate transcription of genes normally regulated by Pbx1. We have used the subtractive process of representational difference analysis to identify targets of E2A-Pbx1. We show that E2A-Pbx1 can directly activate transcription of a novel member of the fibroblast growth factor family of intercellular signalling molecules, FGF-15. The FGF-15 gene is expressed in a regionally restricted pattern in the developing nervous system, suggesting that FGF-15 may play an important role in regulating cell division and patterning within specific regions of the embryonic brain, spinal cord and sensory organs.

Amino Acid Sequence↗
PubMed1997

Specific residues in the Pbx homeodomain differentially modulate the DNA-binding activity of Hox and Engrailed proteins.

Two classes of homeodomain proteins, Hox and Engrailed, have been shown to act in concert with the atypical homeodomain proteins Pbx and extradenticle. We now show that specific residues located within the Pbx homeodomain are essential for cooperative DNA binding with Hox and Engrailed gene products. Within the N-terminal region of the Pbx homeodomain, we have identified a residue that is required for cooperative DNA binding with three Hox gene products but not for cooperativity with Engrailed-2 (En-2). Furthermore, there are similarities between heterodimeric interactions involving the yeast mating type proteins MATa1 and MATalpha2 and those that allow the formation of Pbx/Hox and Pbx/En-2 heterodimers. Specifically, residues located in the a1 homeodomain that were previously shown to form a hydrophobic pocket allowing the alpha2 C-terminal tail to bind, are also required for Pbx/Hox and Pbx/En-2 cooperativity. Furthermore, we show that three residues located in the turn between helix 1 and helix 2, characteristic of many atypical homeodomain proteins, are required for cooperative DNA binding involving both Hox and En-2. Replacement of the three residues located in the turn between helix 1 and helix 2 of the Pbx homeodomain with those of the atypical homeodomain proteins controlling cell fate in the basidiomycete Ustilago maydis, bE5 and bE6, allows cooperative DNA binding with three Hox members but abolishes interactions with En-2. The data suggest that the molecular mechanism of homeodomain protein interactions that control cell fate in Saccharomyces cerevisiae and in the basidiomycetes may well be conserved in part in multicellular organisms.

Amino Acid Sequence↗
PubMed1996

Engrailed and Hox homeodomain proteins contain a related Pbx interaction motif that recognizes a common structure present in Pbx.

Hox gene products have the ability to interact with either extradenticle or pbx gene products to bind cooperatively to DNA. The region in Hox proteins that is required for this interaction is located N-terminal of the homeodomain and contains a highly conserved hexapeptide. We now show that the engrailed gene products also contain a Pbx interaction motif positioned within a previously conserved region, the EH2 domain. The EH2 domain is located N-terminal of the homeodomain. Two tryptophan residues present in the Drosophila and murine Engrailed EH2 domain are required for cooperativity with extradenticle and Pbx, respectively. A second conserved domain, EH3, is required as well for cooperativity with Pbx, since deletions or an insertion in this region reduce cooperative DNA binding. Peptides containing the Pbx interaction motif of either Engrailed or Hox are capable of destabilizing Engrailed-Pbx and Hox-Pbx cooperative DNA binding. These data indicate that the Pbx interaction motifs present in Hox and engrailed gene products recognize a common structure present in the Pbx family of homeodomain proteins.

Amino Acid Sequence↗
PubMed1996

The AD1 transactivation domain of E2A contains a highly conserved helix which is required for its activity in both Saccharomyces cerevisiae and mammalian cells.

A conserved region, designated the AD1 domain, is present in a class of helix-loop-helix (HLH) proteins, E proteins, that includes E12, E47, HEB, E2-2, and a Xenopus laevis HLH protein closely related to E12. We demonstrate that the AD1 domain in E2A and the conserved region of E2-2 activate transcription in both yeast and mammalian cells. The AD1 domain contains a highly conserved putative helix that is crucial for its transactivation properties. Circular dichroism spectroscopy data show that AD1 is structured and contains distinctive helical properties. In addition, we show that a synthetic peptide corresponding to the conserved region is unstructured in aqueous solution at neutral pH but can adopt an alpha-helical conformation in the presence of the hydrophobic solvent trifluoroethanol. Amino acid substitutions that destabilize the helix abolish the transactivation ability of the AD1 domain. Both structural and functional analyses of AD1 reveal striking similarities to the acidic class of activators. Remarkably, when wild-type and mutant proteins are expressed in mammalian cells and Saccharomyces cerevisiae, identical patterns of transactivation are observed, suggesting that the target molecule is conserved between S. cerevisiae and mammals. These data show that transactivation by E proteins is mediated, in part, by a strikingly conserved peptide that has the ability to form a helix in a hydrophobic solvent. We propose that the unstructured domain may become helical upon interaction with its cellular target molecule.

Amino Acid Sequence↗
PubMed1996

E2A gene products are not required for insulin gene expression.

Transcripts for E2A gene products, ubiquitous basic helix-loop-helix transactivating proteins, are expressed at high levels in the pancreatic epithelium. E2A proteins have been shown to bind the cognate E box sequence (CANNTG) of the insulin promoter/enhancer. E2A gene products dimerize with cell-specific basic helix-loop-helix proteins and synergize with the homeodomain transcription factor, PDX-1, in insulin gene transactivation. PDX-1 is also required for normal pancreatic development in mice. We investigated whether pancreatic development and insulin production could occur in the absence of E2A gene products by studying mice with a null mutation for the gene. E2A(-/-) mice demonstrated normal formation of pancreatic endocrine and exocrine tissue in histochemical sections as well as positive and distinct immunostaining for insulin and glucagon in islet tissue, signifying development of mature beta- and alpha-cells. Moreover, E2A(-/-) mice displayed no significant difference in blood glucose levels or pancreatic insulin content compared with wild-type littermates. These data show that although E2A gene products probably play an important role in insulin gene expression, pancreatic development and insulin production can proceed in their absence.

Adenovirus E2 Proteins↗