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T Wirth

Publications and source records attributed to T Wirth.

At least 55 records · Page 3Linked to original sources
PubMed1998

Application of differential cDNA screening techniques to the identification of unique gene expression in tumours and lymphocytes.

Development, differentiation and cell death in all organisms are mediated by tightly regulated programs of differential gene expression. Furthermore, changes in gene expression profiles are responsible for tumour formation and tumour progression, as well as for many other human diseases. Thus, the definition of distinct patterns of gene expression is not only essential for understanding complex biological processes but also leads to the identification of novel targets for therapy of various diseases.

DNA, Complementary↗
PubMed1997

Glutamate-dependent activation of NF-kappaB during mouse cerebellum development.

NF-kappaB and activator protein 1 (AP-1) are dimeric transcription factors involved in transcriptional regulation in many cells, including neurons. We have examined their activity during mouse cerebellum development, a postnatal process starting just after birth and completed by the fourth postnatal (PN) week. The activity of these factors was analyzed by binding of nuclear extracts to a synthetic oligonucleotide representing the kappaB site of human immunodeficiency virus or the AP-1 site of the urokinase promoter. NF-kappaB activity was observed from 7 PN, was restricted to the developing cerebellum, and was not observed in the early postnatal neocortex and hippocampus. On the other hand, AP-1 activity was not found in cerebellum but was present in both neocortex and hippocampus. Moreover, a kappaB-driven transgene was found to be increasingly expressed in the cerebellum from 5 PN to 10 PN but not in the adult. The regulation of NF-kappaB activation in mouse cerebellum was analyzed by intraperitoneal injection of glutamate receptor antagonists to 9 PN mice, which abolished NF-kappaB-binding activity, suggesting an endogenous loop of glutamate receptor activation. Glutamate receptor agonists, on the other hand, induced NF-kappaB nuclear translocation in the cerebellum of 5 PN mice, which is a stage in which NF-kappaB is not yet endogenously activated. This effect was specific for NF-kappaB and not observed for AP-1. In adult mice, NF-kappaB activity was absent in the cerebellum and was not induced by intraperitoneal injection of glutamate receptor agonists. These data show that NF-kappaB is specifically activated during cerebellum development and indicate an important role of glutamate receptors in this process.

2-Amino-5-phosphonovalerate↗
PubMed1997

Inducible expression and phosphorylation of coactivator BOB.1/OBF.1 in T cells.

BOB.1/OBF.1 is a transcriptional coactivator that is constitutively expressed in B cells and interacts with the Oct1 and Oct2 transcription factors. Upon activation of Jurkat T cells and primary murine thymocytes with phorbol esters and ionomycin, BOB.1/OBF.1 expression and transactivation function were induced. BOB.1/OBF.1 was phosphorylated at Ser184 both in vivo and in vitro, and this modification was required for inducible activation. Mutation of Ser184 also diminished transactivation function in B cells, suggesting that the activating phosphorylation that is inducible in T cells is constitutively present in B cells. Thus, BOB.1/OBF.1 is a transcriptional coactivator that is critically regulated by posttranslational modifications to mediate cell type-specific gene expression.

Amino Acid Sequence↗
PubMed1997

RelB is a key player for both kappa B-dependent transcription and demethylation in B cells.

NF-kappa B was originally identified as a B cell-specific nuclear factor binding to the intronic kappa-light-chain enhancer element. It is found constitutively in the nucleus of mature B and plasma cells. In other cell types including pre-B cells, NF-kappa B is sequestered in the cytoplasm but can be induced by a variety of stimuli. In contrast to essentially all other mature B cells and plasma cell lines, the S107 plasmacytoma cell line lacks both constitutive and inducible kappa B-binding activity. A molecular characterization of the defect in these S107 cells suggests that the primary defect lies in the signal transduction pathway leading to NF-kappa B induction. Ectopic expression of RelB after stable transfection of these cells restores constitutive nuclear kappa B-binding activity. Moreover, kappa B-dependent transcription is also restored. Finally we demonstrate, that in contrast to parental S107 cells, the stable RelB transfectants have also regained the ability to specifically demethylate a transfected immunoglobulin kappa-locus. These data suggest that RelB is critically involved in both B cell-specific transcription and demethylation directed by the intronic kappa-enhancer element.

B-Lymphocytes↗
PubMed1997

Identification of target genes of the lymphoid-specific transcription factor Oct2.

The Oct2 transcription factor is expressed predominantly in B lymphocytes and plays an essential role during the terminal phase of B cell differentiation. The regulatory regions of several genes specifically expressed in B cells contain functional binding sites for Oct2. Nevertheless, none of the genes originally thought to be regulated by Oct2 were affected in their expression in Oct2-deficient B cells. In an attempt to find such elusive Oct2 target genes and to understand the molecular function of Oct2 in B cell development, we isolated cDNAs for Oct2 target genes. So far, we have identified five potential targets for Oct2: the membrane glycoprotein CD36, the cysteine-rich secreted protein 3 (CRISP-3), a mouse homolog of the human monocyte/neutrophil elastase inhibitor (mEI) and two unknown cDNA sequences Nov1 and Nov2. These target genes show quite distinct expression patterns demonstrating that transcription factors in addition to Oct2 are involved in their regulation. Whereas CD36 and mEI were expressed in all hematopoetic cell lines containing Oct2,. CRISP-3 is pre-B cell-specific, Nov1 is plasma B cell-specific and Nov2 is B cell-specifically expressed.

Animals↗
PubMed1996

CD40, but not lipopolysaccharide and anti-IgM stimulation of primary B lymphocytes, leads to a persistent nuclear accumulation of RelB.

In this study we analyzed the effect of CD40 stimulation on the activity and nuclear appearance of Rel/nuclear factor kappaB (NF-kappaB) factors in primary murine B lymphocytes. We show that triggering of CD40 signaling pathway(s) by CD40 ligands expressed on L cells led to strong activation of an NF-kappaB-controlled beta-globin reporter gene in primary B lymphocytes from transgenic mice. Analyses of nuclear translocation of individual members of Rel proteins after CD40 induction of primary B cells showed a strong and long-lasting accumulation of RelB and, less pronounced, of c-Rel. LPS stimulation did not give rise to a persistent nuclear accumulation of RelB and c-Rel, whereas nuclear c-Rel, but not RelB, accumulated after B cell receptor stimulation. CD40 induced not only nuclear translocation but also de novo synthesis of RelB RNA and protein. S107 plasmacytoma cells, which express CD40 but are defective for the nuclear appearance of p50/p65-NF-kappaB, do not express RelB after CD40 stimulation. In S107 cells stably transfected with relB genes, stimulation of nuclear RelB translocation by CD40 was observed. These results indicate that stimulation of CD40 signaling pathways exerts a long-lasting stimulatory effect on both the transcription and nuclear translocation of RelB. Since LPS and anti-IgM were unable to activate RelB, CD40 appears to trigger a special program of gene expression involved in the proliferation and/or differentiation of B lymphocytes.

Animals↗
PubMed1996

Coordinate suppression of myeloma-specific genes and expression of fibroblast-specific genes in myeloma X fibroblast somatic cell hybrids.

In most instances, fusion of differentiated cell types with fibroblasts has resulted in the extinction of the differentiation-specific traits of the non-fibroblast parental cell. To explore the genetic basis of this phenomenon, we have studied a series of somatic cell hybrids between mouse myeloma and fibroblasts. All the hybrids were adherent having a fibroblast-like phenotype. Molecular analysis revealed that plasma cell specific genes like the productively rearranged Ig genes, the J chain gene and genes for the cell surface markers CD20 and PC1, were extinguished in the hybrids. In contrast, fibroblast specific genes like fibronectin, alpha 2(I) and III collagens, as well as the receptor for fibroblast growth factor (flg), were expressed. Extinction was not due to chromosomal loss or lack of the relevant genes. To learn about the mechanism(s) of this phenomenon we have looked for the presence of positive and negative transcription factors in our hybrids. Expression of the PU.1 transcription factor, a member of the Ets transcription factor family normally expressed in B cells and macrophages, was lost in the cell hybrids. Interestingly, we found that the B-cell-specific Oct-2 transcription factor was still expressed at somewhat variable levels in several of the hybrid cell lines. In contrast, expression of the recently identified octamer coactivator BOB.1/OBF.1 was extinguished in all cell hybrids. This supports a critical role of this transcriptional coactivator for B-cell-specific gene expression. In addition, the Id and HLH462 genes coding for proteins known to repress bHLH transcription factors by formation of heterodimers, were found to be expressed at increased levels in fibroblasts and in the hybrids, indicating that their increased levels might also contribute to the suppression of myeloma-specific genes. Our results show that in myeloma x fibroblast hybrids, the phenotype of the fibroblast is dominant. It is suggested that fibroblasts contain regulatory "master" genes that are responsible for activation of the fibroblast differentiation pathway and suppress differentiation programs of other cell types.

Animals↗
PubMed1996

A role for nuclear NF-kappaB in B-cell-specific demethylation of the Igkappa locus.

The immunoglobulin kappa gene is specifically demethylated during B-cell maturation in a process which utilizes discrete cis-acting modules such as the intronic kappa enhancer element and the matrix attachment region (MAR). While any MAR sequence is sufficient for this reaction, mutation analysis indicates that tissue specificity is mediated by kappaB binding sequences within the kappa intronic enhancer. The plasmacytoma cell line S107 lacks kappaB binding activity and fails to demethylate the kappa locus. However, B-cell specific demethylation is restored by the introduction of an active kappaB binding protein gene relB. This represents the first demonstration of a trans-acting factor involved in cell-type-specific demethylation, and suggests that the same protein-DNA recognition system used for transcription may also contribute to the earlier developmental events that bring about activation of the kappa locus.

B-Lymphocytes↗
PubMed1996

CRISP-3, a protein with homology to plant defense proteins, is expressed in mouse B cells under the control of Oct2.

The Oct2 transcription factor is expressed throughout the B-lymphoid lineage and plays an essential role during the terminal phase of B-cell differentiation. Several genes specifically expressed in B lymphocytes have been identified that contain a functional octamer motif in their regulatory elements. However, expression of only a single gene, the murine CD36 gene, has been shown to date to be dependent on Oct2. Here, we present the identification and characterization of a further gene, coding for cysteine-rich secreted protein 3 (CRISP-3), whose expression in B cells is regulated by Oct2. We show that CRISP-3 is expressed in the B-lymphoid lineage specifically at the pre-B-cell stage. By using different experimental strategies, including nuclear run-on experiments, we demonstrate that this gene is transcriptionally activated by Oct2. Furthermore, analysis of CRISP-3 expression in primary B cells derived from either wild-type or Oct2-deficient mice demonstrates the dependence on Oct2. Two variant octamer motifs were identified in the upstream promoter region of the crisp-3 gene, and Oct2 interacts with both of them in vitro. Cotransfection experiments with expression vectors for Oct1 and Oct2 together with a reporter driven by the crisp-3 promoter showed that transcriptional activation of this promoter can only be achieved with Oct2. The C-terminal transactivation domain of Oct2 is required for this activation. Finally, introducing specific mutations in the two variant octamer motifs revealed that both of them are important for full transcriptional activation by Oct2.

3T3 Cells↗
PubMed1995

Functional characterization of the murine homolog of the B cell-specific coactivator BOB.1/OBF.1.

B cell-specific transcriptional promoter activity mediated by the octamer motif requires the Oct1 or Oct2 protein and additional B cell-restricted cofactors. One such cofactor, BOB.1/OBF.1, was recently isolated from human B cells. Here, we describe the isolation and detailed characterization of the murine homolog. Full-length cDNAs and genomic clones were isolated, and the gene structure was determined. Comparison of the deduced amino acids shows 88% sequence identity between mouse and human BOB.1/OBF.1. The NH2-terminal 126 amino acids of BOB.1/OBF.1 are both essential and sufficient for interaction with the POU domains of either Oct1 or Oct2. This protein-protein interaction does not require the simultaneous binding of Oct proteins to DNA, and high resolution footprinting of the Oct-DNA interaction reveals that binding of BOB.1/OBF.1 to Oct1 or Oct2 does not alter the interaction with DNA. BOB.1/OBF.1 can efficiently activate octamer-dependent promoters in fibroblasts; however, it fails to stimulate octamer-dependent enhancer activity. Fusion of subdomains of BOB.1/OBF.1 with the GAL4 DNA binding domain reveals that both NH2- and COOH-terminal domains of BOB.1/OBF.1 contribute to full transactivation function, the COOH-terminal domain is more efficient in this transactivation assay. Consistent with the failure of full-length BOB.1/OBF.1 to stimulate octamer-dependent enhancer elements in non B cells, the GAL4 fusions likewise only stimulate from a promoter-proximal position.

Amino Acid Sequence↗
PubMed1995

Lymphoid- and myeloid-specific activity of the PU.1 promoter is determined by the combinatorial action of octamer and ets transcription factors.

The putative oncogene PU.1/Spi-1 is a member of the ets-family of transcription factors normally expressed in a subset of hematopoietic cell types. Here we have characterized the role of the PU.1 promoter region for the cell-type specific expression. The proximal 120 bp are sufficient to mediate a high level of activity specifically in B cells and macrophages. Three important motifs could be identified within this region. Two of them, an ets binding site (EBS) and a variant octamer motif were most important for cell-type-specific promoter activity in B cells and macrophages. An additional Sp1 motif stimulates basal activity of this promoter element. The relative contribution to overall activity of octamer motif and EBS differs in B cells and macrophages. In B cells, both octamer motif and EBS combine to mediate high level activity, whereas in macrophages the EBS predominantly confers promoter activity. Both the Oct1 and Oct2 transcription factors, presumably in combination with a B-cell-restricted coactivator, are responsible for the activity of the variant octamer motif in B cells. Interestingly, the PU.1 transcription factor can functionally interact with the EBS in its own promoter, suggesting a positive feedback regulation.

3T3 Cells↗
PubMed1995

Identification of CD36 as the first gene dependent on the B-cell differentiation factor Oct-2.

The Oct-2 transcription factor is expressed predominantly in B lymphocytes and has been shown previously to be important for the terminal phase of B-cell differentiation in mice. A number of genes specifically expressed in B cells contain Oct-2-binding sites in their regulatory regions. However, the analysis of expression levels of these genes in Oct-2-deficient B cells revealed that they were unaffected. Hence, there were no genes known that critically depend on Oct-2 for their expression. To understand the molecular basis for the Oct-2 effect on B-cell development, we searched for Oct-2 target genes by subtractive cDNA cloning. We show here that expression of the murine CD36 gene in B cells and macrophages requires a functional Oct-2 protein. Nuclear run-on experiments demonstrate that this gene is regulated transcriptionally by Oct-2. Moreover, CD36 levels correlated with the levels of Oct-2 expression in several mouse B-cell and macrophage cell lines. Finally, compared to wild-type and heterozygous mice, CD36 mRNA levels were markedly reduced in spleens and B-cell-enriched splenocyte fractions from oct-2-/- mice. The data identify CD36 as the first target gene critically dependent on Oct-2 for its expression. Because CD36 expression is also dependent on Oct-2 in vivo, it is a candidate gene through which Oct-2 could affect B-cell differentiation.

Animals↗
PubMed1995

High mobility group protein 2 functionally interacts with the POU domains of octamer transcription factors.

The octamer transcription factors Oct1 and Oct2 are involved in the transcriptional regulation of both lymphoid-specific and ubiquitously expressed genes. Their activity depends critically on their interaction with distinct cellular cofactors. Therefore, we have isolated cDNAs encoding proteins that physically interact with Oct2. Here we describe the analysis of one such clone, representing the murine homologue of high mobility group (HMG) protein 2. We have mapped the interaction domains for both proteins and have shown that HMG2 and Oct2 interact via their HMG domains and POU homeodomains, respectively. This interaction is not restricted to Oct2, as other members of the octamer transcription factor family like Oct1 and Oct6 also interact with HMG2. The interaction with HMG2 results in a marked increase in the sequence-specific DNA binding activity of the Oct proteins. Interestingly, the HMG2 protein is not present in the protein-DNA complex detected by an electrophoretic mobility shift assay. The Oct and HMG2 proteins also interact in vivo. A chimeric protein, in which the strong transactivation domain of VP16 was fused directly to the HMG domains of HMG2, stimulated the activity of an octamer-dependent reporter construct upon cotransfection. Furthermore, the expression of antisense RNA for HMG2 specifically reduces octamer-dependent transcription. These results suggest that one of the functions of HMG2 is to support the octamer transcription factors in their role as transcriptional activators.

Amino Acid Sequence↗
PubMed1995

False-positive sonographic hip examinations in newborns with congenital varus deformity of the proximal femur.

In cases of congenital varus deformity of the proximal femur, the screening of hip dysplasia can lead to misinterpretations. The false-positive ultrasound result is caused by the superiorly displaced greater trochanter in hips with coxa vara, which narrows the scan window. In this paper three typical patients are presented to demonstrate that the use of additional non-standard views cannot exclude hip dysplasia in all cases. Hence, such patients with femoral abnormalities and doubtful sonographic findings should be further evaluated by arthrography or magnetic resonance imaging to rule out additional hip dysplasia.

False Positive Reactions↗
PubMed1995

Molecular principles of Oct2-mediated gene activation in B cells.

The octamer motif is a crucial regulatory element for immunoglobulin promoter and enhancer function. We have investigated the molecular mechanisms that underlie octamer-mediated gene activation in B cells. This B cell-specific transcriptional regulation is subject to a novel type of regulatory mechanism. We could demonstrate that octamer-dependent transcription is not only regulated by specific DNA-binding transcription factors, but in addition requires the activity of B cell-restricted cofactors. Both octamer-dependent promoter and enhancer activation depend on such a combination of transcription factor and cofactors. However, the exact requirements differ for these two situations. Promoter activity can be achieved with either one of two distinct transcription factors, Oct1 and/or Oct2, together with the cofactor OCA-B1. In contrast, only Oct2 in conjunction with an additional cofactor, OCA-B2, can confer enhancer activity.

Animals↗
PubMed1994

Functional differences between the Oct2 transactivation domains determine the transactivation potential of individual Oct2 isoforms.

The lymphocyte specific transcription factor Oct2 is involved in mediating the B-cell specific transcriptional activity of the octamer motif. Mutational analyses in the context of the complete Oct2 protein had indicated that Oct2 contains two transactivation domains. These two domains appeared to be redundant for activation from a promoter proximal position, whereas stimulation from a remote enhancer position specifically required the C-terminal transactivation domain and an additional B-cell restricted activity. We have generated fusion proteins between the DNA binding domain of the yeast Gal4 transcription factor and individual Oct2 protein domains to analyze their transactivation potential separately. We show that both N- and C-terminal domains can stimulate transcription from a promoter proximal position independently. However, only the C-terminal transactivation domain activates from a distance and it can only do so in B-cells. The C-terminal transactivation domain represents a composite transactivation domain. Whereas removal of just 9 aminoacids from the extreme C-terminus lead to a complete inactivation of this domain deletions from the other side resulted in a gradual loss of activity. We also characterized the transactivation potential of different N-terminal regions of Oct2 generated by alternative splicing. We show that the N-terminus of one of the isoforms, Oct2.3, contains a negative regulatory domain (NRD), which can inactivate the neighbouring glutamine-rich transactivation in cis. The presence of this NRD affects the overall phosphorylation state of the Oct2 protein. This result suggests that the mechanism of inactivation might involve differential protein phosphorylation.

Amino Acids↗