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B Lüscher

Publications and source records attributed to B Lüscher.

At least 55 records · Page 3Linked to original sources
PubMed1997

Neuronal subtype-specific expression directed by the GABA(A) receptor delta subunit gene promoter/upstream region in transgenic mice and in cultured cells.

The promoter of the GABA(A) receptor delta subunit gene was analyzed in transgenic mice and in cultured cells to study sequences involved in neuronal subtype-specific gene expression. A 6.4-kb genomic fragment faithfully directed neuron-specific transcription of a lacZ reporter gene in the central nervous system. The transgene expression pattern in the cerebral cortex, hippocampal formation, thalamus, and brainstem was consistent with the regional and neuronal subtype-specific expression of the endogenous delta subunit protein in both developing and mature brain. In the cerebellum, however, the transgene was ectopically expressed in Purkinje cells and silent in granule cells, where the endogenous delta subunit is abundantly expressed. These mice provide a useful tool for investigating activity-dependent regulation of GABA(A) receptor expression under physiological and pathological conditions. Transfection studies using primary cortical neurons and astroglial cells revealed that the delta subunit gene promoter was selectively active in neurons even when truncated to a 267-bp core fragment. In conclusion, the delta subunit gene promoter/upstream region contains the information for neuronal subtype-specific expression in the entire brain except in the cerebellum and is selectively active in primary cortical neurons in vitro.

Aging↗
PubMed1997

Activation of the GABA(A)-receptor delta-subunit gene promoter following pentylenetetrazole-induced seizures in transgenic mice.

An impairment of GABA(A)-receptor-mediated inhibitory neurotransmission has been implicated in the development of epileptic seizures. To determine whether seizures affect GABA(A)-receptor gene transcription in vivo, a transgenic mouse line carrying a lacZ-fusion gene driven by GABA(A)-receptor delta-subunit promoter and upstream sequences was subjected to pentylenetetrazol (PTZ)-induced seizures. After injection of a single convulsive dose of PTZ, the activity of the delta-subunit promoter, as monitored by beta-galactosidase immunohistochemistry, was increased selectively in neurons of layers II-IV of neocortex. In contrast, mice kindled by repeated administration of initially subconvulsive doses of PTZ did not show a change in transgene expression, even shortly after the last PTZ-induced seizure. These results show that transient changes in transcription of the GABA(A)-receptor delta-subunit gene occur after acute seizures, but not after kindling. The limited responsiveness of the GABA(A)-receptor delta-subunit promoter after repeated stimulation may reflect an inappropriate adaptation of cellular responses to recurrent excitation, thereby contributing to the development of seizure disorders.

Animals↗
PubMed1996

Interaction of the co-activator CBP with Myb proteins: effects on Myb-specific transactivation and on the cooperativity with NF-M.

The oncoprotein v-Myb is a potent inducer of myeloid leukemias, and its cellular homolog c-Myb plays a crucial role in the regulation of hematopoiesis. Both proteins function as transcriptional regulators. We demonstrate that this function is mediated at least in part by the nuclear co-activator CREB binding protein (CBP). This protein interacts directly with both c-Myb and v-Myb and potentiates Myb-specific transcription as measured on the mim-1 promoter. In contrast, dominant negative mutants of CBP lead to repression, as does E1A, an antagonist of CBP function. Phosphorylation of c-Myb does not appear to be required for interaction with CBP, thus indicating that the binding may be constitutive. Furthermore, the C/EBP family member NF-M, which cooperates with c-Myb in transactivating the mim-1 promoter through an adjacent DNA binding site, is co-activated by CBP in a Ras-dependent manner. Not only the individual activities of c-Myb and NF-M are stimulated by CBP, but also their synergistic transcriptional function, while it is negatively regulated by dominant negative forms of CBP. These data suggest that CBP is recruited by both Myb proteins and NF-M and potentiates their transcriptional activity. We suggest that CBP can bridge between c-Myb and NF-M, thus providing an explanation for the strong synergism between these two proteins.

Animals↗
PubMed1996

C/EBP, c-Myb, and PU.1 cooperate to regulate the neutrophil elastase promoter.

The murine neutrophil elastase (NE) gene is expressed specifically in immature myeloid cells. A 91-bp NE promoter region contains three cis elements which are conserved evolutionarily and are essential for activation of the promoter in differentiating 32D cl3 myeloid cells. These elements bound c-Myb (at -49), C/EBPalpha (at -57), and PU.1 (at -82). In NIH 3T3 cells, the NE promoter was activated by c-Myb, C/EBPalpha, and PU.1, via their respective binding sites. Cooperative activation was seen by any combination of c-Myb, C/EBPalpha, and PU.1, including all three together, again via their DNA-binding sites. In CV-1 cells, but not in NIH 3T3 cells, cooperation between Myb and C/EBPalpha depended on the integrity of the PU.1-binding site. In addition to C/EBPalpha, C/EBPdelta strongly activated the NE promoter, alone or with c-Myb, but C/EBPbeta was less active. Either of C/EBPalpha's two transactivation domains cooperatively activated the promoter with c-Myb, in both NIH 3T3 and 32D c13 cells. Synergistic binding to DNA in a gel shift assay between C/EBPalpha, c-Myb, and PU.1 could not be demonstrated. Also, separation of the C/EBP- and c-Myb-binding sites by 5 or 10 bp did not prevent cooperativity. These results suggest that a coactivator protein mediates cooperative activation of the NE promoter by a C/EBP and c-Myb. These factors, together with PU.1, direct restricted expression of the NE promoter to immature myeloid cells.

3T3 Cells↗
PubMed1995

Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors.

Vigilance, anxiety, epileptic activity, and muscle tone can be modulated by drugs acting at the benzodiazepine (BZ) site of gamma-aminobutyric acid type A (GABAA) receptors. In vivo, BZ sites are potential targets for endogenous ligands regulating the corresponding central nervous system states. To assess the physiological relevance of BZ sites, mice were generated containing GABAA receptors devoid of BZ sites. Following targeted disruption of the gamma 2 subunit gene, 94% of the BZ sites were absent in brain of neonatal mice, while the number of GABA sites was only slightly reduced. Except for the gamma 2 subunit, the level of expression and the regional and cellular distribution of the major GABAA receptor subunits were unaltered. The single channel main conductance level and the Hill coefficient were reduced to values consistent with recombinant GABAA receptors composed of alpha and beta subunits. The GABA response was potentiated by pentobarbital but not by flunitrazepam. Diazepam was inactive behaviorally. Thus, the gamma 2 subunit is dispensable for the assembly of functional GABAA receptors but is required for normal channel conductance and the formation of BZ sites in vivo. BZ sites are not essential for embryonic development, as suggested by the normal body weight and histology of newborn mice. Postnatally, however, the reduced GABAA receptor function is associated with retarded growth, sensorimotor dysfunction, and drastically reduced life-span. The lack of postnatal GABAA receptor regulation by endogenous ligands of BZ sites might contribute to this phenotype.

Animals↗
PubMed1995

Differential effects by Mad and Max on transformation by cellular and viral oncoproteins.

c-Myc is an essential component of the regulatory mechanisms controlling cell growth. Max is the obligatory partner of c-Myc for all its biological functions analysed to date. Recently two Max interacting proteins, Mad and Mxi1, have been identified. It has been suggested that these two proteins modulate c-Myc function, in the simplest model by competing with c-Myc for the interaction with Max. We have analysed different aspects of Mad function in comparison to Max. Native Mad/Max heterodimers bound specifically to a c-Myc/Max consensus DNA binding site. Furthermore Mad inhibited efficiently c-Myc, mutant p53, adenovirus E1a, or human papilloma virus type 16 transformation of rat embryo cells in cooperation with activated Ha-Ras. Myc transformed clones showed an increased cell cycle time and a reduced immortalization frequency after cotransfection with either mad or max. In contrast to Mad, Max did not inhibit E1a/Ha-Ras cotransformation but repressed c-Myc/Ha-Ras transformation efficiently. Mad delta N, an N-terminal deletion mutant of Mad, was as efficient in repressing c-Myc/Ha-Ras cotransformation as full length Mad but showed little inhibitory activity when assayed on E1a/Ha-Ras. Unlike wt Mad, Mad delta N had little effect on cell growth. Our data suggest that Mad affects cell growth at least in part by a c-Myc independent mechanism.

Animals↗
PubMed1995

High affinity DNA binding of native full length c-Myb and differential proteolytic sensitivity of its N- and C-terminal domains.

c-Myb is the prototype of a family of transcription factors characterised by a unique DNA binding domain. Previous analyses have concentrated on truncated versions of c-Myb as it has been very difficult to produce full length c-Myb. To overcome these difficulties we expressed full length c-Myb in HeLa cells using a recombinant vaccinia virus. Partially purified native full length c-Myb bound efficiently and specifically to DNA with a dissociation constant similar to that obtained with bacterially expressed DNA binding domains. No evidence was found for a negative effect of the leucine zipper on DNA binding. Furthermore the DNA binding domain was protease resistant in contrast to the transactivation and negative regulatory domains. Phosphorylation had no apparent effect on this differential protease sensitivity. The increased sensitivity of the C-terminal domain suggests a more open conformation, which may be relevant in the integration of signals and/or in protein-protein interactions.

Amino Acid Sequence↗
PubMed1995

Heterogeneity of GABAA-receptors: cell-specific expression, pharmacology, and regulation.

Vigilance, anxiety, memory, epileptogenic activity and muscle tension can be regulated by a modulation of GABAA-receptor function. A multitude of different GABAA-receptors exist in the brain due to the combinational assembly of various subunits encoded by at least 15 genes. The clarification of the physiological and pharmacological significance of GABAA-receptor subtypes, in combination with their cellular localization, will make it possible to identify the neuronal circuits regulating the respective CNS states and to provide strategies for the development of subtype-specific drugs for selective therapies.

Animals↗
PubMed1995

Tissue-specific expression of a FMR1/beta-galactosidase fusion gene in transgenic mice.

Fragile X syndrome is one of the most common genetic causes of mental retardation, yet the mechanisms controlling expression of the fragile X mental retardation gene FMR1 are poorly understood. To identify sequences regulating FMR1 transcription, transgenic mouse lines were established using a fusion gene consisting of an E.coli beta-galactosidase reporter gene (lacZ) linked to a 2.8 kb fragment spanning the 5'-region of FMR1. Five transgenic mouse lines showed lacZ expression in brain, in particular in neurons of the hippocampus and the granular layer of the cerebellum. Expression of the reporter gene was also detected in Leydig cells and spermatogonia in the testis, in many epithelia of adult mice, and in the two other steroidogenic cell types, adrenal cortex cells and ovarian follicle cells. Embryonic tissues which showed strong activity of the reporter gene included the telencephalon, the genital ridge, and the notochord. This expression pattern closely resembles the endogenous one, indicating that the 5' FMR1 gene promoter region used in this study contains most cis-acting elements regulating FMR1 transcription.

Animals↗
PubMed1995

Casein kinase II phosphorylation site mutations in c-Myb affect DNA binding and transcriptional cooperativity with NF-M.

Phosphorylation of c-Myb has been implicated in the regulation of the binding of c-Myb to DNA. We show that murine c-Myb is phosphorylated at Ser-11 and -12 in vivo and that these sites can be phosphorylated in vitro by casein kinase II (CKII), analogous to chicken c-Myb. An efficient method to study DNA binding properties of full-length c-Myb and Myb mutants under nondenaturing conditions was developed. It was found that a Myb mutant in which Ser-11 and -12 were replaced with Ala (Myb Ala-11/12), wild-type c-Myb, and Myb Asp-11/12 bound to the A site of the mim-1 promoter with decreasing affinities. In agreement with this finding, Myb Ala-11/12 transactivated better than wild-type c-Myb and Myb Asp-11/12 on the mim-1 promoter or a synthetic Myb-responsive promoter. Similar observations were made for the myeloid-specific neutrophil elastase promoter. The presence of NF-M or an NF-M-like activity abolished partially the differences seen with the Ser-11/12 mutants, suggesting that the reduced DNA binding due to negative charge at positions 11 and 12 can be compensated for by NF-M. Since no direct interaction of c-Myb and NF-M was observed, we propose that the cooperativity is mediated by a third factor. Our data offer two possibilities for how casein kinase II phosphorylation can influence c-Myb function: first, by reducing c-Myb DNA binding and thereby influencing transactivation, and second, by enhancing the apparent cooperativity between c-Myb and NF-M or an NF-M-like activity.

Animals↗
PubMed1994

BSF1, a novel brain-specific DNA-binding protein recognizing a tandemly repeated purine DNA element in the GABAA receptor delta subunit gene.

The rat GABAA receptor delta subunit gene has been isolated and its promoter mapped to facilitate studies of neuron-specific gene transcription. The gene is transcribed from a single promoter with several clustered transcriptional initiation sites. The major start site maps to a prototype initiator sequence located in the center of a TATA-less CpG-rich island. Analysis of the 5'-flanking region of the GABAA receptor delta subunit gene revealed a novel tandemly repeated purine sequence element. Furthermore, gel retardation assays identified several tissue-specific and ubiquitous proteins that bind to this sequence. One of these factors, brain-specific factor (BSF) 1, was only observed in extracts from brain and was distinct from BETA and PAL, two DNA-binding proteins with similar purine-rich recognition sequences. The distribution of BSF1 in brain closely matched the expression pattern previously determined for the delta subunit mRNA, and its binding activity appeared simultaneously with GABAA receptor delta subunit mRNA during differentiation of cerebellar granule cells in vitro. Our data therefore suggest that BSF1 is a novel brain-specific and neuronal cell type-enriched factor possibly involved in differential transcription of the GABAA receptor delta subunit gene.

Amino Acid Sequence↗
PubMed1994

Biosynthesis of casein kinase II in lymphoid cell lines.

We have analyzed the biosynthesis of casein kinase II. In exponentially growing tissue culture cells, the beta subunit was synthesized in excess of the catalytic subunit (alpha). A substantial fraction of newly synthesized beta was degraded within the first hour. The remaining fraction of beta was incorporated into holoenzyme. In contrast, little degradation of newly synthesized alpha subunit was observed and most was quickly and efficiently incorporated into holoenzyme. The assembly of beta with alpha was paralleled by an increase in apparent molecular mass of beta due to phosphorylation. The subcellular distribution of newly synthesized [35S]Met-labelled casein kinase II and of enzyme labelled and chased in the presence of excess unlabelled methionine was very similar and compatible with a nuclear localization. The degradation of the excess beta subunit occurred through a non-lysosomal proteolytic system with a very low ATP requirement.

Animals↗
PubMed1994

Regulation of transcription factors c-Myc, Max, and c-Myb by casein kinase II.

A number of transcription factors have been shown to be phosphorylated by casein kinase II (CKII). We have identified CKII phosphorylation sites in c-Myc, Max, and c-Myb which are phosphorylated in the cell. Whereas little evidence to any functional significance of the CKII sites in c-Myc has been obtained, phosphorylation of its heterodimeric partner Max alters DNA binding properties. CKII phosphorylation of Ser-2 and -11 in Max resulted in enhanced DNA binding kinetics of both Max/Max homo- and Myc/Max heterodimers without altering steady state binding. Replacing these serine by alanine residues and comparing the wild type with the mutant Max proteins in transactivation assays did not reveal any significant differences. For c-Myb mutational analysis of the CKII phosphorylation sites showed altered steady state DNA binding. Replacing Ser-11/12 by alanine residues resulted in increased DNA binding compared to wt c-Myb or Myb Asp-11/12 as demonstrated by up to 10-fold differences in the dissociation constants. In transactivation assays, the Ala mutant showed consistently an increased activity both on a synthetic and on the mim-1 promoter. A potential CKII phosphorylation site in c-Fos was not phosphorylated in vitro. Analysis with peptides demonstrated that a proline residue at position +1 relative to the acceptor serine was inhibitory.

Amino Acid Sequence↗