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Biomedical subjects

M Busslinger

Publications and source records attributed to M Busslinger.

At least 55 records · Page 3Linked to original sources
PubMed1996

C-terminal activating and inhibitory domains determine the transactivation potential of BSAP (Pax-5), Pax-2 and Pax-8.

Pax-5 encodes the transcription factor BSAP which plays an essential role in early B cell development and midbrain patterning. In this study we have analysed the structural requirements for transcriptional activation by BSAP. In vitro mutagenesis and transient transfection experiments indicate that the C-terminal serine/threonine/proline-rich region of BSAP contains a potent transactivation domain of 55 amino acids which is active from promoter and enhancer positions. This transactivation domain was found to be inactivated by a naturally occurring frameshift mutation in one PAX-5 allele of the acute lymphoblastic leukemia cell line REH. The function of the transactivation domain is negatively regulated by adjacent sequences from the extreme C-terminus. The activating and inhibitory domains function together as an independent regulatory module in different cell types as shown by fusion to the GAL4 DNA binding domain. The same arrangement of positively and negatively acting sequences has been conserved in the mammalian Pax-2 and Pax-8, the zebrafish Pax-b as well as the sea urchin Pax-258 proteins. These data demonstrate that the transcriptional competence of a subfamily of Pax proteins is determined by a C-terminal regulatory module composed of activating and inhibitory sequences.

Alleles↗
PubMed1996

The estrogen-dependent c-JunER protein causes a reversible loss of mammary epithelial cell polarity involving a destabilization of adherens junctions.

Members of the epidermal growth factor (EGF) receptor family are known to be specifically involved in mammary carcinogenesis. As a nuclear target of activated receptors, we examined c-Jun in mammary epithelial cells. For this, we used a c-JunER fusion protein which was tightly controlled by estrogen. Activation of the JunER by hormone resulted in the transcriptional regulation of a variety of AP-1 target genes. Hormone-activated JunER induced the loss of epithelial polarity, a disruption of intercellular junctions and normal barrier function and the formation of irregular multilayers. These changes were completely reversible upon hormone withdrawal. Loss of epithelial polarity involved redistribution of both apical and basolateral proteins to the entire plasma membrane. The redistribution of E-cadherin and beta-catenin was accompanied by a destabilization of complexes formed between these two proteins, leading to an enrichment of beta-catenin in the detergent-soluble fraction. Uninduced cells were able to form three-dimensional tubular structures in collagen I gels which were disrupted upon JunER activation, leading to irregular cell aggregates. The JunER-induced disruption of tubular structures was dependent on active signaling by growth factors. Moreover, the effects of JunER could be mimicked in normal cells by the addition of acidic fibroblast growth factor (aFGF). These data suggest that a possible function of c-Jun in epithelial cells is to modulate epithelial polarity and regulate tissue organization, processes which may be equally important for both normal breast development and as initiating steps in carcinogenesis.

Animals↗
PubMed1995

Low affinity binding of interleukin-1 beta and intracellular signaling via NF-kappa B identify Fit-1 as a distant member of the interleukin-1 receptor family.

The fit-1 gene gives rise to two different mRNA isoforms, which code for soluble (Fit-1S) and membrane-bound (Fit-1M) proteins related to the type I interleukin (IL)-1 receptor. To investigate IL-1 binding, we have synthesized and purified histidine-tagged polypeptides corresponding to Fit-1S and the extracellular domain of the type I IL-1 receptor using a vaccinia expression system. Fit-1S is shown to interact with IL-1 beta, but not with IL-1 alpha. However, Fit-1S binds IL-1 beta only with low affinity in contrast to the IL-1 receptor, suggesting that IL-1 beta is not a physiological ligand of Fit-1S. Moreover, expression of the membrane-bound protein Fit-1M in transiently transfected Jurkat cells did not result in activation of the transcription factor NF-kappa B following IL-1 beta treatment. However, a chimeric protein consisting of the extracellular domain of the type I IL-1 receptor and of the transmembrane and intracellular regions of Fit-1M stimulated NF-kappa B-dependent transcription as efficiently as the full-length type I IL-1 receptor. These data indicate that Fit-1M is a signaling molecule belonging to the IL-1 receptor family.

Base Sequence↗
PubMed1995

Deregulated expression of PAX5 in medulloblastoma.

Medulloblatoma is a pediatric brain tumor originating in the human cerebellum. A collection of 23 medulloblastomas was analyzed for expression of the developmental control genes of the PAX and EN gene families by RNase protection and in situ hybridization. Of all nine PAX genes investigated, only PAX5 and PAX6 were consistently expressed in most medulloblastomas (70 and 78% of all cases, respectively), as were the genes EN1 (57%) and EN2 (78%). EN1, EN2, and PAX6 genes were also expressed in normal cerebellar tissue, and their expression in medulloblastoma is consistent with the hypothesis that this tumor originates in the external granular layer of the developing cerebellum. PAX5 transcripts were, however, not detected in the neonatal cerebellum, indicating that this gene is deregulated in medulloblastoma. In the desmoplastic variant of medulloblastoma, PAX5 expression was restricted to the reticulin-producing proliferating tumor areas containing undifferentiated cells; PAX5 was not expressed in the reticulin-free nonproliferating islands undergoing neuronal differentiation. These data suggest that deregulated expression of PAX5 correlates positively with cell proliferation and inversely with neuronal differentiation in desmoplastic medulloblastoma.

Adolescent↗
PubMed1995

Immediate effects of reversible HTLV-1 tax function: T-cell activation and apoptosis.

The tax protein of Human T-cell leukemia virus type 1 (HTLV-1) is important for the transforming properties of this virus in vitro and is considered to be responsible for the early stages of leukemogenesis in infected hosts. To address the early consequences of HTLV-1 tax function, we have constructed fusion proteins containing tax sequence either aminoterminal (taxER) or carboxy-terminal (ERtax) of the hormone binding domain of the human estrogen receptor (ER). Addition of estrogen or the antagonist hydroxytamoxifen to Jurkat T-cells expressing these constructs led to the trans-activation or responsive promoters and upregulation of cell surface markers CD28, CD69 and CD5 but not CD25 (IL2R-alpha subunit) or B7 (ligand for CD28). Additional stimulation of the T-cell receptor CD3 complex, led to the upregulation of CD25. B7 was upregulated by concomittent activation of ERtax and CD3 or CD28 pathways. These events were in part reversible upon withdrawal of hormone and inactivation of ERtax. Severe inhibition of proliferation, and apoptosis was observed with cells which had been subjected to short term (3 days) activation of the tax fusion proteins and the CD3 complex. Induction of ERtax activity for longer than 3 days promoted cell death independently of CD3 stimulation. Co-stimulation through the CD28 cell surface molecule did not suppress induction of apoptosis.

Apoptosis↗
PubMed1995

The gene coding for the B cell surface protein CD19 is localized on human chromosome 16p11.

The CD19 gene codes for one of the earliest markers of the human B cell lineage and is a target for the B lymphoid-specific transcription factor BSAP (Pax-5). The transmembrane protein CD19 has been implicated in controlling proliferation of mature B lymphocytes by modulating signal transduction through the antigen receptor. In this study, we have employed Southern blot and fluorescence in situ hybridization analyses to localize the CD19 gene to human chromosome 16p11.

Animals↗
PubMed1995

The role of BSAP (Pax-5) in B-cell development.

The hierarchy of transcriptional control in B-cell development has recently been analyzed by targeted gene inactivation in the mouse. In this manner, the paired box containing gene Pax-5, encoding the B cell specific transcription factor BSAP, has been shown to play a key role in early B lymphopoiesis. Other experimental strategies have implicated BSAP in the control of cell proliferation, isotype switching and transcription of the immunoglobulin heavy-chain gene at late stages of B-cell differentiation.

Animals↗
PubMed1995

DNA-binding and transactivation properties of Pax-6: three amino acids in the paired domain are responsible for the different sequence recognition of Pax-6 and BSAP (Pax-5).

Pax-6 is known to be a key regulator of vertebrate eye development. We have now isolated cDNA for an invertebrate Pax-6 protein from sea urchin embryos. Transcripts of this gene first appear during development at the gastrula stage and are later expressed at high levels in the tube foot of the adult sea urchin. The sea urchin Pax-6 protein is highly homologous throughout the whole protein to its vertebrate counterpart with the paired domain and homeodomain being virtually identical. Consequently, we found that the DNA-binding and transactivation properties of the sea urchin and mouse Pax-6 proteins are very similar, if not identical. A potent activation domain capable of stimulating transcription from proximal promoter and distal enhancer positions was localized within the C-terminal sequences of both the sea urchin and mouse Pax-6 proteins. The homeodomain of Pax-6 was shown to cooperatively dimerize on DNA sequences consisting of an inverted repeat of the TAAT motif with a preferred spacing of 3 nucleotides. The consensus recognition sequence of the Pax-6 paired domain deviates primarily only at one position from that of BSAP (Pax-5), and yet the two proteins exhibit largely different binding specificities for individual, naturally occurring sites. By creating Pax-6-BSAP fusion proteins, we were able to identify a short amino acid stretch in the N-terminal part of the paired domain which is responsible for these differences in DNA-binding specificity. Mutation of three Pax-6-specific residues in this region (at positions 42, 44, and 47 of the paired domain) to the corresponding amino acids of BSAP resulted in a complete switch of the DNA-binding specificity from Pax-6 to BSAP. These three amino acids were furthermore shown to discriminate between the Pax-6- and BSAP-specific nucleotide at the divergent position of the two consensus recognition sequences.

Amino Acid Sequence↗
PubMed1995

Transcriptional activation of the fra-1 gene by AP-1 is mediated by regulatory sequences in the first intron.

Constitutive expression of c-Fos, FosB, Fra-1, or c-Jun in rat fibroblasts leads to up-regulation of the immediate-early gene fra-1. Using the posttranslational FosER induction system, we demonstrate that this AP-1-dependent stimulation of fra-1 expression is rapid, depends on a functional DNA-binding domain of FosER, and is a general phenomenon observed in different cell types. In vitro mutagenesis and functional analysis of the rat fra-1 gene in stably transfected Rat-1A-FosER fibroblasts indicated that basal and AP-1-regulated expression of the fra-1 gene depends on regulatory sequences in the first intron which comprise a consensus AP-1 site and two AP-1-like elements. We have also investigated the transactivating and transforming properties of the Fra-1 protein to address the significance of fra-1 up-regulation. The entire Fra-1 protein fused to the DNA-binding domain of Ga14 is shown to lack any transactivation function, and yet it possesses oncogenic potential, as overexpression of Fra-1 in established rat fibroblasts results in anchorage-independent growth in vitro and tumor development in athymic mice, fra-1 is therefore not only induced by members of the Fos family, but its gene product may also contribute to cellular transformation by these proteins. Together, these data identify fra-1 as a unique member of the fos gene family which is under positive control by AP-1 activity.

Animals↗
PubMed1994

Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP.

The Pax5 gene, coding for the transcription factor BSAP, was mutated in the mouse germline by targeted disruption. Homozygous mutant mice were born alive, became growth retarded, and usually died within three weeks. About 5% of mutants survived to adulthood and were fertile, but severely runted. Morphogenesis of the posterior midbrain was affected as early as embryonic day 16.5, leading to a reduction of the inferior colliculus near the midline and to altered foliation of the anterior cerebellum. Moreover, all mutants failed to produce small pre-B, B, and plasma cells owing to a complete arrest of B cell development at an early precursor stage. These data define a key role for Pax5 in early B lymphopoiesis and midbrain patterning.

Animals↗
PubMed1994

Molecular cloning and characterization of a human PAX-7 cDNA expressed in normal and neoplastic myocytes.

The myogenic basic helix-loop-helix proteins are essential components of the regulatory network controlling vertebrate myogenesis. However, determined myoblasts appear in the limb buds which do not initially express any member of this transcription factor family. In a search for potential novel regulators of myogenesis, a human PAX-7 cDNA was isolated from primary myoblasts. Analysis of the DNA-binding properties of the Pax-7 paired domain revealed that it binds DNA in a sequence-specific manner indistinguishable from that of the paralogous Pax-3 protein. Each of the two proteins also binds to palindromic homeodomain-binding sites by cooperative dimerization. Both Pax-3 and Pax-7, which are known to partially overlap in their expression during development, can also efficiently form heterodimers on these sites and stimulate reporter gene transcription in transient transfection experiments which, in the case of Pax-7, is dependent on the transactivation function encoded by the C-terminal sequences. Thus, the formation of heterodimers might have important consequences for target gene recognition and regulation during development. PAX-7 was found to be weakly expressed in normal human myoblasts, while PAX-3 could not be detected in these cells at all. However, transcripts for either PAX-3 and/or PAX-7 were expressed at elevated levels in tumorigenic rhabdomyosarcoma cell lines. Hence, overexpression of these PAX genes may be involved in the genesis of myogenic tumors.

Amino Acid Sequence↗
PubMed1994

The transcription factor BSAP (NF-HB) is essential for immunoglobulin germ-line epsilon transcription.

Treatment of murine splenic B lymphocytes and certain B-lineage cell lines with mitogen (LPS) and the lymphokine IL-4 has been shown to induce expression of germ-line epsilon transcripts (l epsilon transcripts) and class switching to the C epsilon gene. Three protein complexes, one of which (complex 3) is constitutively expressed, have been shown to bind to a 179-base pair LPS/IL-4-responsive l epsilon promoter (Rothman, P., S. C. Li, B. Gorham, L. Glimcher, F. W. Alt, and M. Boothby. 1991. Mol. Cell. Biol. 11:5551). Complex 3 is indispensable for this inducible promoter activity. In this report, we have used electrophoretic mobility shift assays (EMSA) to demonstrate that the early B cell-specific transcription factor (BSAP) is involved in the formation of complex 3. In addition, BSAP is implicated functionally in l epsilon transcription because a BSAP binding site either from a sea urchin histone promoter (H2A-2.2) or from 5' of murine immunoglobulin S gamma 2a can substitute for the epsilon-associated site (epsilon(foot), as assayed by transient transfection assays of the l epsilon:CAT reporter constructs into the M12.4.1 B cell line. Like the sea urchin histone BSAP site, the complex 3 binding site (epsilon(foot)) functions as an upstream promoter element when assayed in the OVEC vector. These results indicate that BSAP is an essential protein required for LPS/IL-4 induction of the l epsilon promoter. In addition, experiments showing that a BSAP binding site from 5' of S gamma 2a also functions as an upstream promoter element in OVEC suggest a potential role for BSAP in regulation of the IgG2a isotype.

Animals↗
PubMed1994

Alternative promoter usage of the Fos-responsive gene Fit-1 generates mRNA isoforms coding for either secreted or membrane-bound proteins related to the IL-1 receptor.

Fit-1 has been identified previously as a Fos-responsive gene of rat fibroblasts. Here we show that Fit-1 is directly regulated by the estrogen-inducible transcription factor Fos-ER and that it belongs to the family of delayed early genes. Two different mRNA isoforms are expressed from the Fit-1 gene. The Fit-1M mRNA isolated from spleen codes for a membrane-bound protein which is most closely related in its extracellular, transmembrane and intracellular domains to the type I interleukin-1 (IL-1) receptor. The Fit-1S mRNA of fibroblasts directs, instead, the synthesis of a secreted protein consisting of only the extracellular domain. Analysis of the exon-intron structure of the Fit-1 gene indicated that the Fit-1S and Fit-1M mRNAs are transcribed from two different promoters and that the sequence differences at their 3' ends result from alternative 3' processing. Northern blot analysis with specific 5' and 3' probes directly demonstrated tight coupling between alternative promoter usage and 3' processing of the Fit-1 transcripts. The orthologous gene of the mouse (known as T1 or ST2) is expressed during ontogeny first in the fetal liver of the embryo and then in lung and hematopoietic tissues of the adult. The mRNA coding for the membrane-bound protein is more abundantly expressed in all of these tissues, while the transcript for the secreted form predominates in fibroblasts and mammary epithelial cells. Differential regulation of two distinct promoters is thus used to determine the ratio between secreted and membrane-bound forms of Fit-1 (T1/ST2) which may modulate signaling in response to IL-1.

Alternative Splicing↗
PubMed1993

A selective transcriptional induction system for mammalian cells based on Gal4-estrogen receptor fusion proteins.

Most mammalian cells neither express any Gal4-like activity nor endogenous estrogen receptor, thus rendering estrogen an inert signal for them. For these two reasons we have developed a selective induction system based on the estrogen-regulable transcription factor Gal-ER. Gal-ER consists of the DNA-binding domain of the yeast Gal4 protein fused to the hormone-binding domain of the human estrogen receptor and hence should exclusively regulate a transfected gene under the control of a Gal4-responsive promoter in mammalian cells. Two major improvements of this induction system were made. First, a synthetic Gal4-responsive promoter was constructed which consisted of four Gal4-binding sites, an inverted CCAAT element, a TATA box, and the adenovirus major late initiation region. This promoter showed extremely low basal activity in the absence and high inducibility in the presence of ligand-activated Gal-ER. Second, the transcription factor Gal-ER was rendered more potent and less susceptible to cell type-specific variation by fusing the strong activating domain of the herpesvirus protein VP16 onto its C terminus. In response to estrogen, Gal-ER-VP16 induced the Gal4-responsive promoter at least 100-fold in transiently transfected NIH 3T3 and P19 cells. Rat fibroblast cell lines expressing integrated Gal-ER and Gal4-responsive fos genes were transformed in a strictly estrogen-dependent manner. The exogenous fos gene was rapidly induced to maximal levels within 1-2 hr of estrogen addition. Elevated Fos activity in turn stimulated transcription of the endogenous fra-1 gene. These data demonstrate the utility of the Gal-ER induction system as a powerful genetic switch for regulating heterologous genes and, in particular, for identifying Fos targets in mammalian cells.

Animals↗
PubMed1993

Chromosomal localization of seven PAX genes and cloning of a novel family member, PAX-9.

In the human paired box-containing (PAX) gene family, only two members, PAX-3 and PAX-6, which are associated with Waardenburg's syndrome and aniridia, respectively have been mapped to human chromosomes. We have now isolated cosmids for six additional human PAX genes (PAX-1,-2,-5,-7,-8,-9) and a polymerase chain reaction fragment for PAX-4. PAX-9 is a novel family member which is closely related in its paired domain to PAX-1. The chromosomal location of all cloned PAX genes was determined by analysis of somatic cell hybrids and (except PAX-4) by fluorescence in situ hybridization to metaphase chromosomes. PAX-1 and PAX-7 map to chromosomal regions containing previously assigned disease loci.

Amino Acid Sequence↗