Mouse Myelin Basic Protein Gene Research Articles

Expression of SV40 T antigen gene in the oligodendroglia induced primitive neuroectodermal tumor‐like tumors in the mouse brain

Primitive neuroectodermal tumors (PNET) are classified as the embryonal tumors developed in the brain, except for the cerebellum. Although many studies have been reported, the origin and pathogenesis of PNET are still unclear. In this study, we observed the development of undifferentiated tumors indistinguishable from PNET in the transgenic mice which expressed simian virus 40 T antigen (SV40-Tag) selectively in the oligodendroglia under the control of mouse myelin basic protein gene promoter. These PNET-like tumors reproducibly developed in the brain stem of the founder mice and the transgenic progeny derived from one founder mouse. Oligodendroglia-specific expression of SV40-Tag in these transgenic mice was observed by immunohistochemical analysis. Furthermore, expression of the oligodendroglia-specific marker genes was decreased in the tumors as well as in the transgenic brains. These findings suggested that tumors developed in transgenic mice were indistinguishable from PNET, and one of them showed oligodendroglia-like characteristics. Consequently, this transgenic line is a useful animal model to study the pathogenesis of undifferentiated tumor.

Oligodendrocyte precursor hypercellularity and abnormal retina development in mice overexpressing PDGF-B in myelinating tracts.

Platelet-derived growth factor (PDGF) influences the generation of neurons and glia during embryogenesis and in early postnatal life. In an attempt to determine the consequences of an overexpression of PDGF-B during the first weeks of life, we targeted transgenic expression of a human PDGF-B cDNA to myelinating tracts using the promoter region of the myelin basic protein (MBP) gene. Transgenic mRNA and protein were expressed in the brain and the expression profile of the human PDGF-B during early postnatal development closely paralleled that of the endogenous mouse MBP gene. The gross morphological appearance of transgenic brains was normal but at the cellular level several phenotypic alterations could be identified. In white matter tracts such as the corpus callosum and cerebellar medulla, there was a marked hypercellularity. The number of oligodendrocyte precursors was increased and astrocytes were more abundant. In adult mice carrying the MBP-PDGF-B transgene, however, myelination appeared normal and the amount of oligodendrocytes was similar to that of control littermates. In addition to the phenotypic alterations in the brain, investigation of eye structure revealed a striking disorganization of retinal architecture. The retina was folded with cells collected in papillar or follicular-like structures. Retinal whole mount preparations after India ink perfusion revealed capillary disorganization with large-caliber vessels supporting only a few fine branches. Our observations strengthen the notion that PDGF is an important effector molecule in postnatal CNS development.

Tumor necrosis factor modulates transcription of myelin basic protein gene through nuclear factor kappa B in a human oligodendroglioma cell line

Tumor necrosis factor-α (TNF-α) is a major mediator of inflammation and it is involved in many neurological disorders such as multiple sclerosis. Levels of TNF-α and lymphotoxin-α have been found elevated in plaques, bloods, and cerebral spinal fluids from multiple sclerosis patients. The expression of myelin basic protein (MBP), a major protein of the myelin sheath, is affected by cytokines secreted by activated immune cells. To determine the signal transduction pathway involving tumor necrosis factor’s action in myelination and demyelination, we have cloned and analyzed cis-elements on promoters of the human and mouse MBP genes. There are two putative nuclear factors kappa-B (NF-κB) cis-elements on the human and one on the mouse gene promoter. In an electrophoretic mobility shift assay, all three NF-κB cis-elements showed binding to a protein, which was recognized by an antibody against NF-κB P65 component. The specificity of the binding was demonstrated in a competitive assay using NF-κB consensus oligonucleotides. A two base pair site-directed mutation on the mouse NF-κB cis-element abolished its binding activity. We created a DNA construct by linking the mouse MBP gene promoter containing the NF-κB cis-element to luciferase gene. Transfection of this construct into a human oligodendroglioma cell line showed TNF-α increased the transgene expression. Furthermore the mutation of NF-κB site abolished TNF-α -induction of the transgene. The data demonstrate that NF-κB is the mediator between tumor necrosis factor’s action and MBP gene expression. Elucidating the molecular mechanisms underlying TNF-α regulation of MBP gene expression provides new scientific bases for the development of therapy against oligodendrocyte-specific and myelin-related disorders such as multiple sclerosis.

Targeted expression of Cre recombinase to myelinating cells of the central nervous system in transgenic mice.

genesisVolume 26, Issue 2 p. 127-129 ArticleFree Access Targeted expression of Cre recombinase to myelinating cells of the central nervous system in transgenic mice Michiko Niwa-Kawakita, Michiko Niwa-Kawakita INSERM U434, Fondation Jean Dausset-CEPH, Paris, FranceSearch for more papers by this authorVincent Abramowski, Vincent Abramowski INSERM U434, Fondation Jean Dausset-CEPH, Paris, FranceSearch for more papers by this authorMichel Kalamarides, Michel Kalamarides INSERM U434, Fondation Jean Dausset-CEPH, Paris, FranceSearch for more papers by this authorGilles Thomas, Gilles Thomas INSERM U434, Fondation Jean Dausset-CEPH, Paris, FranceSearch for more papers by this authorMarco Giovannini, Corresponding Author Marco Giovannini marco.giovannini@cephb.fr INSERM U434, Fondation Jean Dausset-CEPH, Paris, FranceINSERM U434, Fondation Jean Dausset-CEPH, 27 rue Juliette Dodu, 75010 Paris, FranceSearch for more papers by this author Michiko Niwa-Kawakita, Michiko Niwa-Kawakita INSERM U434, Fondation Jean Dausset-CEPH, Paris, FranceSearch for more papers by this authorVincent Abramowski, Vincent Abramowski INSERM U434, Fondation Jean Dausset-CEPH, Paris, FranceSearch for more papers by this authorMichel Kalamarides, Michel Kalamarides INSERM U434, Fondation Jean Dausset-CEPH, Paris, FranceSearch for more papers by this authorGilles Thomas, Gilles Thomas INSERM U434, Fondation Jean Dausset-CEPH, Paris, FranceSearch for more papers by this authorMarco Giovannini, Corresponding Author Marco Giovannini marco.giovannini@cephb.fr INSERM U434, Fondation Jean Dausset-CEPH, Paris, FranceINSERM U434, Fondation Jean Dausset-CEPH, 27 rue Juliette Dodu, 75010 Paris, FranceSearch for more papers by this author First published: 23 February 2000 https://doi.org/10.1002/(SICI)1526-968X(200002)26:2<127::AID-GENE8>3.0.CO;2-HCitations: 11AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume26, Issue2Special Issue: Tissue Specific Expression of Cre Recombinase in MiceFebruary 2000Pages 127-129 ReferencesRelatedInformation

Cell-type specific factors bind to regulatory elements located downstream of the TATA-box element in the mouse myelin basic protein (MBP) gene promoter

Cell-type specific transcription of the myelin basic protein (MBP) gene in primary oligodendrocytes (OL) is regulated by cis-acting regulatory elements located at both upstream and downstream of the TATA-box region of the MBP promoter. To identify cell-type specific factors that bind to the downstream regulatory elements, we utilised DNase I footprinting analysis and gel retardation assays with nuclear extracts from myelin-forming OL as well as a non-myelin forming cell line, C6 glioma (C6) cells. Several regions of DNA were protected from DNAse I digestion by nuclear extracts of both cell types. However, two regions, from −17 to +17 and from +47 to +58 were protected specifically in OL, while three regions, from +17 to +22, from +43 to +49 and from +58 to +64 were protected only with C6 nuclear extracts. Inspection of the protected regions for homology with known transcription factor binding sites revealed that sequences at from +47 to +58 and from +56 to +68 showed extensive homology to the negative regulatory element (NRE1), of the mouse renin gene and to the interferon (IFN) consensus sequence of major histocompatibility complex class I genes (MHC I-ICS), respectively. Gel retardation assays using a MHC I-ICS oligonucleotide and transient transfection assays using MBP-CAT constructs were used to study the effect of IFNs on MBP promoter activity in OL and C6 cells. In OL, IFN-α/β caused little induction of CAT activity, but IFN-γ resulted in a 2–3.5-fold decrease in CAT activity. In contrast, in C6 cells both IFN-α/β and IFN-γ induced a 1.5–2.5-fold increase in CAT activity. The cooperative effects of factors binding to NREs and ICS may be responsible for the cell-type specific regulation of MBP gene transcription.

A minimal human MBP promoter-lacZ transgene is appropriately regulated in developing brain and after optic enucleation, but not in shiverer mutant mice.

Previous studies, both in vitro and in vivo, suggest that small portions of the mouse myelin basic protein (MBP) promoter are sufficient to activate regulated expression of MBP. To confirm our previous in vitro studies, we prepared transgenic mice with short regions of the human MBP promoter fused to the lacZ reporter gene. We found that 750 nucleotides of the proximal human MBP promoter is sufficient to activate oligodendrocyte-specific, developmentally regulated expression of lacZ in three independent lines. This promoter, however, does not activate expression of lacZ in Schwann cells in peripheral nerve or in adult mouse brain. The relative levels of beta-galactosidase specific activity, mRNA, and transcription parallel those of MBP mRNA during myelinogenesis. Thus, we exploited this transgene as a quantitative tool to evaluate the response to stimuli known to affect myelination. Transgene expression is reduced 75 % after optic enucleation, as previously reported for levels of MBP mRNA, indicating that axons signal to this portion of the proximal MBP promoter to fully activate MBP expression during myelinogenesis. Instead, in adult shiverer mice, another setting in which MBP transcription is modulated, transgene expression is not increased, in contrast to the increased transcriptional activation of MBP previously reported in these mice. These data suggest that the regulatory region that mediates transcriptional activation of the MBP gene is modular, since discrete subregions are required for activation in Schwann cells, during myelinogenesis in oligodendrocytes, during maintenance myelination in adult brain, and in the dysmyelinating mutant shiverer mouse.

Analysis of Glial-Specific Gene Expression Usingin VitroTranscription Assays

Thein vitrotranscription system using extracts from animal tissues is one of the most powerful and convenient ways to investigate transcriptional regulation of tissue-specific genes when appropriate cultured cells are not available. In the present study, we established a protocol for preparing transcription-competent nuclear extracts from mouse brain. Transcription in the extract could mimicin vivotissue specificity qualitatively, suggesting that this technique is beneficial for studying transcriptional regulation of nervous system-specific genes. By using these methods, we analyzedcis-elements of the glial cell-specific mouse myelin basic protein gene.

Identification of a Sequence-specific Single-stranded DNA Binding Protein That Suppresses Transcription of the Mouse Myelin Basic Protein Gene

The myelin basic protein (MBP) gene is expressed only in oligodendrocytes and Schwann cells, and expression follows a tightly regulated developmental time course. Cell type- and developmental stage-specific expression of the MBP gene appears to be regulated by a series of cis-acting elements located upstream of the transcription start site. The proximal element of the MBP regulatory region (MB1), located between nucleotides -14 and -50, is one of several elements participating in the programmed expression of MBP. In this report, we describe the molecular cloning and characterization of myelin gene expression factor-2 (Myef-2), a protein isolated from mouse brain that binds specifically to single-stranded DNA derived from the MB1 element and represses transcription of the MBP gene in transient transfection assay. Myef-2 mRNA is developmentally regulated in mouse brain; its peak expression occurs at postnatal day 7, prior to the onset of MBP expression. The developmental pattern of Myef-2 mRNA expression coincides with that previously described for SCIP, a POU domain transcription factor that also represses myelin basic protein expression. The myef-2 gene maps to mouse chromosome 2. The relevance of these findings for regulation of MBP gene expression and oligodendrocyte differentiation is discussed.

Regulation of mouse myelin basic protein gene transcription by a sequence-specific single-stranded DNA-binding protein in vitro

Cell- and stage-specific transcription of the myelin basic protein (MBP)-encoding gene ( Mbp) in brain is regulated by arrays of cis-acting regulatory elements positioned at the 5'-flanking region of the gene. The proximal element between nucleotides - 14 to - 50, termed MBI, has previously been shown to have an important role in the cell-type-specific transcription of Mbp in cells derived from the central nervous system (CNS). Here, we utilized band-shift and in vitro transcription assays to examine the ability of MEF-2, an expression factor encoded by a cDNA isolated from mouse brain, in binding to the MB1 element and regulating transcription of the Mbp promoter. Results from the bandshift assays indicated that the bacterially produced MEF-2 recognizes specific nt in the MB1 motif, and its binding to these nt reduces the overall transcriptional activity of Mbp in a cell-free extract.

The 3′-Untranslated Region of Mouse Myelin Basic Protein Gene Increases the Amount of mRNA in Immortalized Mouse Oligodendrocytes

The 3′-untranslated region (UTR) of the myelin basic protein (MBP) mRNA has been found previously to enhance the translational efficiency of the coding region by two-fold in cell-free translational systems. In this study, we transfected eukaryotic expression vectors containing the reporter cDNA for chloramphenicol acetyltransferase (CAT) with or without the mouse MBP cDNA 3′-UTR into cultured cells. CAT activity in the mouse oligodendrocyte cell line, N20.1, transfected with a CAT cDNA containing the MBP 3′-UTR [CAT-MBP 3′-UTR], was twice as high as that of the CAT cDNA without the 3′-UTR; CAT activities for the two constructs were the same in the mouse fibroblast cell line, NIH 3T3. Using reverse transcriptase PCR quantitative analysis, the expression of mRNA was determined. The level of the [CAT-MBP 3′-UTR] mRNA was about ten times higher than CAT mRNA in N20.1 cells but they were the same in NIH 3T3 cells. We conclude that the 3′-UTR of MBP gene increases gene expression at both the mRNA and protein levels in oligodrocyte cell lines, probably through a post-transcriptional mechanism such as message stabilization.

Selective expression of foreign genes in glioma cells: use of the mouse myelin basic protein gene promoter to direct toxic gene expression.

We have previously demonstrated that retrovirus-mediated genes were transferred to mouse glioma cells in a meningeal gliomatosis model (Yamada et al.: Japanese Journal of Cancer Research 83:1244-1247, 1992). This retrovirus vector contains the Escherichia coli. beta-galactosidase (beta-gal) gene as a marker for integration of the lacZ gene, which is controlled by the SV40 early promoter. We investigated whether lacZ genes could be specifically controlled in mouse glioma cells by glial-specific promoters, including the 2.5 kb 5' flanking region of the mouse glial fibrillary acidic protein (GFAP) gene, the 1.3 kb 5' flanking region of the myelin basic protein (MBP) gene, and the 1.5 kb 5' flanking region of the myelin proteolipid protein (PLP) gene. Psi-2 packaging cells were transfected with each retrovirus vector (GFAP promoter-, MBP promoter-, and PLP promoter-lacZ) and the infectious virus particles were recovered from the supernatants. Blue staining for beta-gal was detected in various fibroblast, myeloma, and glioma cell lines transduced with the retrovirus BAG vector. On the other hand, blue staining was only detected in glioma cells after transduction with the lacZ gene-bearing retrovirus controlled by glial-specific promoters. The strongest promoter activity was detected after transduction with the retrovirus in which the MBP promoter controlled the lacZ gene. Mouse glioma cells transduced with retrovirus containing the MBP promoter directing the herpes simplex virus type 1 thymidine kinase (HTK) gene were extremely sensitive to ganciclovir, while the parental cells and cells transduced with retrovirus containing the lacZ gene were not sensitive to ganciclovir.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and developmental regulation of Golli-mbp, a 105-kilobase gene that encompasses the myelin basic protein gene and is expressed in cells in the oligodendrocyte lineage in the brain.

We have identified a novel transcription unit of 105 kilobases (called the Golli-mbp gene) that encompasses the mouse myelin basic protein (MBP) gene. Three unique exons within this gene are alternatively spliced into MBP exons and introns to produce a family of MBP gene-related mRNAs that are under individual developmental regulation. These mRNAs are temporally expressed within cells of the oligodendrocyte lineage at progressive stages of differentiation. Thus, the MBP gene is a part of a more complex gene structure, the products of which may play a role in oligodendrocyte differentiation prior to myelination. One Golli-mbp mRNA that encodes a protein antigenically related to MBP is also expressed in the spleen and other non-neural tissues.

Cell and tissue-specific expression of a heterologous gene under control of the myelin basic protein gene promoter in transgenic mice

Myelin basic protein (MBP) is the second most abundant protein in CNS myelin. We have used transgenic mice to investigate the ability of the 5′ flanking sequence of the mouse MBP gene to regulate the cell-type-specific- and temporal expression of a heterologous gene under its control. Transgenic mice were produced with a construct containing the bacterial chloramphenicol acetyltransferase (CAT) gene downstream of the MBP 5′ flanking sequence and CAT expression was monitored both enzymatically and histochemically. The results indicate that 1323 bp of 5′ flanking is sufficient to direct CAT expression specifically to the tissue and cell-type, in which MBP is normally synthesized. Additionally, this length of the sequence also retains the ability to temporally regulate CAT levels in a manner analogous to endogenous MBP levels.

Molecular basis for oligodendrocyte degeneration in jimpy mouse

The mouse proteolipid protein (PLP) gene was cloned into the λ bacteriophage Charon 4A. The organization and the nucleotide sequence of the exons of the mouse PLP gene were quite similar to those of their human counterparts, consisting of seven exons. The transcription of the PLP gene started from multiple sites. There was a unique sequence tandemly repeated four times, sharing homology with the herpes simplex virus DR2 sequence, upstream from the transcribed region.Expression of the myelin basic protein (MBP) is also restricted to the oligodendrocytes in the central nervous system as is the PLP expression. Homology search against the mouse MBP gene revealed that several boxes in the 5′-flanking region of PLP show a high degree of homology with the sequence present in the MBP 5′-flanking region, possibly of importance in the concomitant expression of both genes in the central nervous system.PLP-mRNA in jimpy mutant mice does not contain exon 5 and its content is greatly reduced. We analyzed the jimpy PLP-mRNA and showed that the transcription initiated from the same sites as those in normal mice. Cloning and sequencing of the 5′-flanking region of the jimpy PLP gene revealed that there were no mutations in the promoter region of the jimpy PLP gene. Therefore, it is likely that a mutation, presumably existing within the jimpy PLP gene, caused the skipping of exon 5 and directly affected the mRNA level.

Effects of chronic administration of antidepressants on 3H-flunitrazepam binding in rat brain

The mouse proteolipid protein (PLP) gene was cloned into the λ bacteriophage Charon 4A. The organization and the nucleotide sequence of the exons of the mouse PLP gene were quite similar to those of their human counterparts, consisting of seven exons. The transcription of the PLP gene started from multiple sites. There was a unique sequence tandemly repeated four times, sharing homology with the herpes simplex virus DR2 sequence, upstream from the transcribed region.Expression of the myelin basic protein (MBP) is also restricted to the oligodendrocytes in the central nervous system as is the PLP expression. Homology search against the mouse MBP gene revealed that several boxes in the 5′-flanking region of PLP show a high degree of homology with the sequence present in the MBP 5′-flanking region, possibly of importance in the concomitant expression of both genes in the central nervous system.PLP-mRNA in jimpy mutant mice does not contain exon 5 and its content is greatly reduced. We analyzed the jimpy PLP-mRNA and showed that the transcription initiated from the same sites as those in normal mice. Cloning and sequencing of the 5′-flanking region of the jimpy PLP gene revealed that there were no mutations in the promoter region of the jimpy PLP gene. Therefore, it is likely that a mutation, presumably existing within the jimpy PLP gene, caused the skipping of exon 5 and directly affected the mRNA level.

Sequences involved in brain-specific in vitro transcription from the core promoter of the mouse myelin basic protein gene

In the previous study, we have shown that a short DNA stretch from -35 to -17 in the core promoter of the mouse myelin basic protein (MBP) gene is mainly responsible for brain-specificity in in vitro transcription. In this study, we found from intensive mutation analysis that the TATA-box sequence at -34 is not critical, but sequences downstream from the TATA-box especially around -20 were important for brain-specificity. The existence of a tissue-specific factor for the MBP core promoter different from the TATA-box-binding factor TFIID is implicated.

Demonstration of a transcription element in vitro between the capping site and translation initiation site of the mouse myelin basic protein gene

A transcription element was identified, by in vitro analyses, just downstream from the capping site of the mouse myelin basic protein (MBP) gene. Deletion of this element caused a dramatic drop of transcription efficiency in mouse brain, rat liver and HeLa cell nuclear extracts, regardless of the form of DNA being closed circular or linear form. DNase I footprint analysis demonstrated the presence of a ubiquitous trans-acting factor for this region. This element functioned even when it is located in the normal direction downstream from the adenovirus major late promoter. Mutation analysis suggested that an essential part of the downstream element was located between +25 and +45.

Core promoter of the mouse myelin basic protein gene governs brain-specific transcription in vitro.

The core promoter of the mouse myelin basic protein (MBP) gene from -36 to +12 was preferentially transcribed in brain nuclear extracts. Both the TATA at -34 and downstream elements to +12 were required for efficient, accurate and brain-specific transcription. From brain and liver nuclear extracts, we have partially purified the general transcription factor TFIID. The partially purified fractions contained TATA element binding factors of the MBP promoter as well as adenovirus major late promoter (MLP). The tissue-derived TFIID was functionally exchangeable for the HeLa TFIID, and directed transcription from the MLP. Surprisingly, the brain TFIID activated transcription from the MBP core promoter while the liver TFIID did to a much lesser extent. Exchange of the TATA-containing short DNA stretch to the MBP core promoter for a corresponding region of the mouse albumin promoter or MLP abolished the brain specificity. We found that several tissue-specific promoters other than MBP, such as mouse neurofilament and human alpha-1-antitrypsin promoters were also transcribed much more efficiently by the brain and liver TFIID, respectively. We suggest that different tissues contain functionally non-equivalent TFIID or TFIID-like activities.

Rapid preimplantation detection of mutant (shiverer) and normal alleles of the mouse myelin basic protein gene allowing selective implantation and birth of live young.

As a model for the detection of human genetic disease in preimplantation embryos, we describe a method in which trophectoderm biopsy samples from viable mouse blastocysts are simultaneously analyzed for the presence of a normal or mutant allele of the myelin basic protein gene by the polymerase chain reaction. The biopsied embryos are kept in culture during analysis of biopsied material and later reintroduced to a foster mother. Prenatal diagnosis can be completed in less than 7 hr. The identity of either amplification product was proved conclusively by direct sequence analysis of amplified products. Ninety-six percent of recovered blastocysts survived biopsy, as judged by re-formation of a blastocyst cavity in culture. Fifty-nine percent of the biopsied embryos established pregnancy by day 6.5, compared to 88% of unmanipulated controls. This approach can be applied to preimplantation diagnosis of human genetic diseases by using extraembryonic cells from blastocysts obtained after in vitro fertilization or uterine lavage. It will make possible the elimination of a mutant allele from a family in a single generation.

Expression of a Novel Transcript of the Myelin Basic Protein Gene

A cDNA (M41) corresponding to a mouse myelin basic protein (MBP) mRNA with a longer 5'-untranslated region than predicted from earlier studies of MBP gene structure has been isolated and characterized. The additional 5'-untranslated region is encoded by two previously unidentified exons upstream of the major transcription start site of the gene. Using a DNA probe specific for M41-MBP mRNAs, Northern blot analysis indicated that expression of this transcript follows a developmental course in mouse brain similar to that of the majority of MBP mRNAs, but that the level of expression varies between brain and spinal cored. Expression of MBP mRNAs similar to the mouse M41-MBP also was identified in rat brain. The results suggest that the structure of the MBP gene is more complex than originally thought, containing at least two more exons. There appears to be at least one more MBP gene promoter that directs the synthesis of a subset of MBP mRNAs with a unique 5'-untranslated region.