Biomedical subjects

M Wegner

Publications and source records attributed to M Wegner.

At least 37 records · Page 2Linked to original sources

PubMed1999

Activation of AP-1 and nuclear factor-kappaB transcription factors is involved in hydrogen peroxide-induced apoptotic cell death of oligodendrocytes.

H2O2-induced onset and execution of programmed cell death in mature rat brain oligodendrocytes in culture is accompanied by the induction and nuclear translocation of the transcription factors AP-1 and nuclear factor-kappaB (NF-kappaB), both of which have been discussed as regulators of cell death and survival. Supershift analysis of nuclear extracts indicated that the AP-1 complex consists of c-Jun, c-Fos, JunD, and possibly JunB proteins, and that the NF-kappaB complex contains p50, p65, and c-Rel proteins. The first signs of DNA fragmentation were seen already during the first hour after the treatment. DNA fragmentation could be prevented by the antioxidants pyrrolidine dithiocarbamate and vitamin E, by the nuclease inhibitor aurintricarboxylic acid, and by preincubation with the iron chelator deferoxamine (DFO). Additionally, DFO protected oligodendrocytes from H2O2-induced cytotoxic effects as assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and suppressed the formation of free radicals. DFO alone led to a slight increase and in combination with H2O2 synergistically induced DNA-binding activities of AP-1 and NF-kappaB in oligodendrocytes. Our data suggest that although low levels of H2O2 directly activate AP-1 and NF-kappaB and might contribute to signal transduction pathways promoting cell survival, the formation and action of hydroxyl radicals promote cell death mechanisms that can be attenuated by the iron chelator DFO.

Animals↗

PubMed1999

A molecular analysis of the yemenite deaf-blind hypopigmentation syndrome: SOX10 dysfunction causes different neurocristopathies.

The Yemenite deaf-blind hypopigmentation syndrome was first observed in a Yemenite sister and brother showing cutaneous hypopigmented and hyperpigmented spots and patches, microcornea, coloboma and severe hearing loss. A second case, observed in a girl with similar skin symptoms and hearing loss but without microcornea or coloboma, was reported as a mild form of this syndrome. Here we show that a SOX10 missense mutation is responsible for the mild form, resulting in a loss of DNA binding of this transcription factor. In contrast, no SOX10 alteration could be found in the other, severe case of the Yemenite deaf-blind hypopigmentation syndrome. Based on genetic, clinical, molecular and functional data, we suggest that these two cases represent two different syndromes. Moreover, as mutations of the SOX10 transcription factor were previously described in Waardenburg-Hirschsprung disease, these results show that SOX10 mutations cause various types of neurocristopathy.

Amino Acid Sequence↗

PubMed1999

The J domain of papovaviral large tumor antigen is required for synergistic interaction with the POU-domain protein Tst-1/Oct6/SCIP.

Large T antigens from polyomaviruses are multifunctional proteins with roles in transcriptional regulation, viral DNA replication, and cellular transformation. They have been shown to enhance the activity of various cellular transcription factors. In the case of the POU protein Tst-1/Oct6/SCIP, this enhancement involves a direct physical interaction between the POU domain of the transcription factor and the amino-terminal region of large T antigen. Here we have analyzed the structural requirements for synergistic interaction between the two proteins in greater detail. Tst-1/Oct6/SCIP and the related POU protein Brn-1 were both capable of direct physical interaction with large T antigen. Nevertheless, only Tst-1/Oct6/SCIP functioned synergistically with large T antigen. This differential behavior was due to differences in the amino-terminal regions of the proteins, as evident from chimeras between Tst-1/Oct6/SCIP and Brn-1. Synergy was specifically observed for constructs containing the amino-terminal region of Tst-1/Oct6/SCIP. Large T antigen, on the other hand, functioned synergistically with Tst-1/Oct6/SCIP only when the integrity of its J-domain-containing amino terminus was maintained. Mutations that disrupted the J domain concomitantly abolished the ability to enhance the function of Tst-1/Oct6/SCIP. The J domain of T antigen was also responsible for the physical interaction with Tst-1/Oct6/SCIP and could be replaced in this property by other J domains. Intriguingly, a heterologous J domain from a human DnaJ protein partially substituted for the amino terminus of T antigen even with regard to the synergistic enhancement of Tst-1/Oct6/SCIP function. Given the general role of J domains, we propose chaperone activity as the underlying mechanism for synergy between Tst-1/Oct6/SCIP and large T antigens.

Antigens, Viral, Tumor↗

PubMed1998

Functional analysis of Sox10 mutations found in human Waardenburg-Hirschsprung patients.

The Sry-related protein Sox10 is selectively expressed in neural crest cells during early stages of development and in glial cells of the peripheral and central nervous systems during late development and in the adult. Mutation of the Sox10 gene leads to neural crest defects in the Dominant megacolon mouse mutant and to combined Waardenburg-Hirschsprung syndrome in humans. Here, we have studied the four Sox10 mutations found to date in Waardenburg-Hirschsprung patients both in the context of the rat and the human cDNA. Unlike the rat Sox10 protein, which failed to show transcriptional activity on its own, human Sox10 displayed a weak, but reproducible, activity as a transcriptional activator. All mutant Sox10 proteins, including the one that only lacked the 106 last amino acids were deficient in this capacity, indicating that the carboxyl terminus of human Sox10 carries a transactivation domain. Whereas all four mutants failed to transactivate, only two failed to synergistically enhance the activity of other transcription factors. Synergy required both the ability to bind to DNA and a region in the amino-terminal part of Sox10. Those mutants that failed to synergize were unable to bind to DNA. Analysis of the naturally occurring Sox10 mutations not only helps to dissect Sox10 structure, but also allows limited predictions on the severity of the disease.

Animals↗

PubMed1998

Expression of the SOX10 gene during human development.

SOX10, a new member of the SOX gene family, is a transcription factor defective in the Dom (Dominant megacolon) mouse and in the human Shah-Waardenburg syndrome. To help unravel its physiological role during human development, we studied SOX10 gene expression in embryonic, fetal, and adult human tissues by Northern blot and in situ hybridization. As in mice, the human SOX10 gene was essentially expressed in the neural crest derivatives that contribute to the formation of the peripheral nervous system, and in the adult central nervous system. Nevertheless, it was more widely expressed in humans than in rodents. The spatial and temporal pattern of SOX10 expression supports an important function in neural crest development.

Blotting, Northern↗

PubMed1998

Cooperative function of POU proteins and SOX proteins in glial cells.

Glial cells of the oligodendrocyte lineage express several highly related POU proteins including Tst-1/Oct6/SCIP and Brn-1. Tst-1/Oct6/SCIP, but not Brn-1 efficiently cooperated with Sox10, the only SRY box protein so far identified in oligodendrocytes. Here we show that, in addition to Sox10, cells of the oligodendrocyte lineage contain significant amounts of the related SRY box proteins Sox4 and Sox11. During development, Sox11 was strongly expressed in the central nervous system. It was first detected in neural precursors throughout the neuroepithelium. During later stages of neural development, Sox11 was additionally expressed in areas of the brain in which neurons undergo differentiation. In agreement with its expression in neural precursors, Sox11 levels in cells of the oligodendrocyte lineage were high in precursors and down-regulated during terminal differentiation. Outside the nervous system, expression of Sox11 was also detected in the developing limbs, face, and kidneys. Structure function analysis revealed that Sox11 has a strong intrinsic transactivation capacity which is mediated by a transactivation domain in its carboxyl-terminal part. In addition, Sox11 efficiently synergized with Brn-1. Synergy was dependent on binding of both proteins to adjacent DNA elements, and required the presence of the respective transactivation domain in each protein. Our data suggest the existence of a specific code in which POU proteins require specific Sox proteins to exhibit cooperative effects in glial cells.

Amino Acid Sequence↗

PubMed1998

Muscarinic acetylcholine receptors activate expression of the EGR gene family of transcription factors.

In order to search for genes that are activated by muscarinic acetylcholine receptors (mAChRs), we used an mRNA differential display approach in HEK293 cells expressing m1AChR. The zinc-finger transcription factor genes Egr-1, Egr-2, and Egr-3 were identified. Northern blot analyses confirmed that mRNA levels of Egr-1, Egr-2, and Egr-3 increased readily after m1AChR stimulation and that a maximum was attained within 50 min. At that time, Egr-4 mRNA was also detectable. Western blots and electromobility shift assays demonstrated synthesis of EGR-1 and EGR-3, as well as binding to DNA recognition sites in response to m1AChR activation. Activation of m1AChR increased transcription from EGR-dependent promoters, including the acetylcholinesterase gene promoter. Activity-dependent regulation of Egr-1 mRNA expression and EGR-1 protein synthesis was also observed in cells expressing m2, m3, or m4AChR subtypes. Increased EGR-1 synthesis was mimicked by phorbol myristate acetate, but not by forskolin, and receptor-stimulated EGR-1 synthesis was partially inhibited by phorbol myristate acetate down-regulation. Together, our results demonstrate that muscarinic receptor signaling activates the EGR transcription factor family and that PKC may be involved in intracellular signaling. The data suggest that transcription of EGR-dependent target genes, including the AChE gene, can be under the control of extracellular and intracellular signals coupled to muscarinic receptors.

Cell Line↗

PubMed1998

Structural requirements for DNA binding of GCM proteins.

Members of the GCM family of transcription factors contain a DNA binding domain unrelated to any other known DNA binding domain and bind to a DNA sequence motif not recognized by any other known transcription factor. Here we show that positions 2, 3, 6 and 7 of the 5'-ATGCGGGT-3' motif are particularly important for DNA binding and that methylation of several G residues on the upper strand, but not on the lower strand, interfered with binding of GCM proteins. No differences were detected between the DNA binding of Drosophila GCM and mammalian mGCMa. Alanine scan mutagenesis of the DNA binding domain of mGCMa identified the three conserved amino acids K74, C76 and C125 as being essential for DNA binding. Conserved cysteine residues were also found to be important for maintaining the overall integrity of the DNA binding domain and for mediating redox sensitivity of DNA binding. These cysteine residues are arranged in a symmetrical structure that bears no resemblance to other cysteine-containing structures, such as zinc fingers. In agreement with this, DNA binding of mGCMa was not dependent on zinc ions. Our results give insights into the exact nature of the GCM binding sites expected in target genes and point to a role for redox regulation in the function of GCM proteins.

Amino Acid Sequence↗

PubMed1998

Mutation of the Sry-related Sox10 gene in Dominant megacolon, a mouse model for human Hirschsprung disease.

The spontaneous mouse mutant Dominant megacolon (Dom) is a valuable model for the study of human congenital megacolon (Hirschsprung disease). Here we report that the defect in the Dom mouse is caused by mutation of the gene encoding the Sry-related transcription factor Sox10. This assignment is based on (i) colocalization of the Sox10 gene with the Dom mutation on chromosome 15; (ii) altered Sox10 expression in the gut and in neural-crest derived structures of cranial ganglia of Dom mice; (iii) presence of a frameshift in the Sox10 coding region, and (iv) functional inactivation of the resulting truncated protein. These results identify the transcriptional regulator Sox10 as an essential factor in mouse neural crest development and as a further candidate gene for human Hirschsprung disease, especially in cases where it is associated with features of Waardenburg syndrome.

Amino Acid Sequence↗

PubMed1998

Sox10, a novel transcriptional modulator in glial cells.

Sox proteins are characterized by possession of a DNA-binding domain with similarity to the high-mobility group domain of the sex determining factor SRY. Here, we report on Sox10, a novel protein with predominant expression in glial cells of the nervous system. During development Sox10 first appeared in the forming neural crest and continued to be expressed as these cells contributed to the forming PNS and finally differentiated into Schwann cells. In the CNS, Sox10 transcripts were originally confined to glial precursors and later detected in oligodendrocytes of the adult brain. Functional studies failed to reveal autonomous transcriptional activity for Sox10. Instead, Sox10 functioned synergistically with the POU domain protein Tst-1/Oct6/SCIP with which it is coexpressed during certain stages of Schwann cell development. Synergy depended on binding to adjacent sites in target promoters, was mediated by the N-terminal regions of both proteins, and could not be observed between Sox10 and several other POU domain proteins. Interestingly, Sox10 also modulated the function of Pax3 and Krox-20, two other transcription factors involved in Schwann cell development. We propose a role for Sox10 in conferring cell specificity to the function of other transcription factors in developing and mature glia.

Age Factors↗

PubMed1998

Activation of SV40 DNA replication in vivo by amplification-promoting sequences of the mouse ribosomal gene cluster.

Mouse genomic DNA sequences from the region upstream of the ribosomal genes reported to contain amplification-promoting activity (APS1 and APS2) were tested for their ability to support SV40 replication in vivo. Both APSs can functionally replace the auxiliary sequence 2 on the late side of the SV40 origin, which facilitates initiation of replication. This stimulatory activity on the SV40 origin was orientation independent and was exerted over a distance of at least 0.9 kb. However, the APS2 element can not substitute for the auxiliary sequence 1 on the early side of the origin. Although both APS elements contain various prospective transcription factor binding sites, they do not trans-activate transcription from a minimal promoter. In this respect they differ from the enhancer element of the auxiliary sequence 2 of the SV40 origin. These results suggest that the rDNA sequences tested contain genetic information facilitating replication initiation on a viral core origin.

Animals↗

PubMed1998

SOX10 mutations in patients with Waardenburg-Hirschsprung disease.

Waardenburg syndrome (WS; deafness with pigmentary abnormalities) and Hirschsprung's disease (HSCR; aganglionic megacolon) are congenital disorders caused by defective function of the embryonic neural crest. WS and HSCR are associated in patients with Waardenburg-Shah syndrome (WS4), whose symptoms are reminiscent of the white coat-spotting and aganglionic megacolon displayed by the mouse mutants Dom (Dominant megacolon), piebald-lethal (sl) and lethal spotting (ls). The sl and ls phenotypes are caused by mutations in the genes encoding the Endothelin-B receptor (Ednrb) and Endothelin 3 (Edn3), respectively. The identification of Sox10 as the gene mutated in Dom mice (B.H. et al., manuscript submitted) prompted us to analyse the role of its human homologue SOX10 in neural crest defects. Here we show that patients from four families with WS4 have mutations in SOX10, whereas no mutation could be detected in patients with HSCR alone. These mutations are likely to result in haploinsufficiency of the SOX10 product. Our findings further define the locus heterogeneity of Waardenburg-Hirschsprung syndromes, and point to an essential role of SOX10 in the development of two neural crest-derived human cell lineages.

Amino Acid Sequence↗

PubMed1998

Absence of polyomavirus JC in glial brain tumors and glioma-derived cell lines.

The human neurotropic papovavirus JC, a close relative of simian virus 40, has been associated with the formation of brain tumors in humans because of its ability to induce such tumors in other primates under experimental conditions. Here we have analyzed 30 brain tumors classified as either oligodendroglioma or astrocytoma and 22 cell lines derived from human gliomas for the presence of JC viral sequences using polymerase chain reaction with two different sets of primers. None of the tumors or cell lines contained JC viral sequences. Similarly, we failed to detect expression of JC T antigen in any of 26 human glioma lines analyzed in this study. We conclude that JC virus is not a major cause of human brain tumors.

Brain Neoplasms↗

PubMed1997

Redundancy of class III POU proteins in the oligodendrocyte lineage.

Class III POU proteins are prominent regulators of neural development. Tst-1/Oct6/SCIP, for instance, is essential for terminal differentiation of myelinating Schwann cells in the peripheral nervous system. Although Tst-1/Oct6/SCIP is also expressed in the myelin forming oligodendrocytes of the central nervous system, targeted deletion of Tst-1/Oct6/SCIP failed to reveal a gross alteration of myelination in the central nervous system. To better understand this apparent discrepancy, we examined the expression of POU proteins in both cultured primary oligodendrocytes and in the oligodendrocyte-like CG-4 cell line. These cells expressed Tst-1/Oct6/SCIP, Brn-1, and Brn-2 in significant amounts, indicating that Brn-1 and Brn-2 might have the capacity to compensate loss of Tst-1/Oct6/SCIP. We show that Tst-1/Oct6/SCIP, Brn-1, and Brn-2 were all down-regulated during the early phases of oligodendrocyte development both on RNA and protein level. All three POU proteins exhibited similar DNA binding characteristics. When promoters consisting of a single POU protein-binding site adjacent to a TATA box were used as reporters in transient transfections, Brn-1 proved to be a weaker transcriptional activator than Tst-1/Oct6/SCIP. In agreement with this, we found the transactivation domain of Brn-1, which we mapped between amino acids 119 and 237, significantly weaker than the transactivation domain of Tst-1/Oct6/SCIP. Taken together, our data imply a partial, but not complete redundancy between POU proteins in oligodendrocytes.

Amino Acid Sequence↗

PubMed1997

The regulator of early gliogenesis glial cells missing is a transcription factor with a novel type of DNA-binding domain.

Absence or presence of glial cells missing (GCM) in cells of the developing nervous system of Drosophila decides over their future fate as neurons or glia with only those cells turning into glia that express GCM. To understand how GCM exerts its function we performed a detailed structure-function analysis. Using fusions between the DNA binding domain of the yeast GAL4 protein and GCM, we detected a transactivation function within the C-terminal part of GCM. In addition to this transactivation domain we mapped a sequence-specific DNA-binding domain within the N-terminal part of the GCM protein in close proximity to a bipartite nuclear localization signal. Binding site selection assays determined the motif 5'-AT(G/A)CGGGT-3' as the preferred binding site for GCM. Both the lack of homology to known proteins and the novel DNA binding specificity indicate that GCM contained a new type of DNA-binding domain. In transiently transfected cells, GCM also activated transcription from promoters consisting of the newly identified GCM-binding site and a TATA box. Thus, GCM is a novel type of transcription factor involved in early gliogenesis.

Amino Acid Sequence↗

PubMed1997

Expression of Krox proteins during differentiation of the O-2A progenitor cell line CG-4.

Krox proteins are important regulators of development and terminal differentiation. Using the rat glial progenitor cell line CG-4 as a model system for oligodendrocyte differentiation, we show that on the RNA level Krox-24 is the predominant member of the Krox family in these cells. Similar results were also obtained on the protein level as the major Krox protein from CG-4 cell extracts reacted specifically with an antibody against Krox-24. Whereas Krox-24 RNA and protein were abundant in undifferentiated CG-4 cells, a dramatic decrease in expression was detected after a 3-5-day period of differentiation during which we observed a reciprocal increase in the levels of myelin basic protein expression. Importantly, regulation of Krox-24 expression was very similar in CG-4 cells and primary oligodendrocyte cultures. When expression of Krox-24 in differentiating CG-4 cells was followed on a closer time scale, we observed a sharp and transient increase in Krox-24 RNA, protein, and DNA binding activity immediately after the onset of differentiation followed by an equally rapid decrease. This expression pattern implicates Krox-24 both in maintenance of the undifferentiated state and in the immediate early phase of differentiation of CG-4 cells and possibly oligodendrocytes.

3T3 Cells↗

PubMed1997

A cell-free replication system for human polyomavirus JC DNA.

The human polyomavirus JC virus (JCV) establishes persistent infections in most individuals and is the etiologic agent of progressive multifocal leukoencephalopathy. In this report, we describe the establishment of a soluble cell-free system that is capable of replicating exogenous plasmid DNA containing the JCV origin of replication. Replication in this system is completely dependent on the addition of JCV large T antigen (TAg). To prepare JCV TAg for replication analysis, a recombinant baculovirus containing the JCV TAg-coding sequence was generated. TAg expressed in insect cells was purified by metal chelate chromatography. JCV TAg supported initiation of JCV DNA replication in the presence of DNA polymerase alpha-primase, replication protein A, and topoisomerase I in a dose-dependent manner and was also capable of supporting DNA replication in crude human cell extracts. Point mutation of TAg-binding site I strongly diminished TAg binding and concomitantly reduced JCV DNA replication in vivo and in vitro by approximately 50%. Point mutation of TAg-binding site II or deletion of the early palindrome completely abolished replication of JCV origin-containing plasmid DNA in vivo and in vitro, marking these sequences as essential components of the JCV core origin. A comparison of several TAgs showed that simian virus 40 TAg, but not mouse polyomavirus (PyV) TAg, supported replication of a plasmid containing a JCV origin. These findings provide evidence that replication in the cell-free system faithfully mimics JCV DNA replication in vivo. Therefore, it may be a useful tool for future analysis of interactions between JCV and its host cell.

Animals↗

PubMed1996

Expression of the gene for the POU domain transcription factor Tst-1/Oct6 is regulated by an estrogen-dependent enhancer.

Expression of the POU domain protein Tst-1/Oct6 during development of glia and neurons is subject to a tight multifactorial control. Here we show that 17beta-estradiol increases the level of endogenous Tst-1/Oct6 in glial cells. This effect was mediated at the level of gene expression by an enhancer present in the 5' flanking region of the mouse gene for Tst-1/Oct6, approximately 5 kb upstream of the transcriptional start site. The enhancer contained as the functional element a sequence motif that closely resembled a classical estrogen response element. It consisted of an imperfect palindrome with a spacing of 3 bp, and was bound in vitro by activated estrogen receptor. Furthermore, this element was able to confer estrogen responsiveness when introduced into a heterologous promoter. In the Tst-1/Oct6 gene enhancer, a TPA response element was found in close proximity to the estrogen receptor binding site. As a consequence, TPA and estrogen activated transcription of the Tst-1/Oct6 gene in a synergistic manner.

Animals↗