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P Matthias

Publications and source records attributed to P Matthias.

At least 19 recordsLinked to original sources
PubMed2001

The RING finger protein Siah-1 regulates the level of the transcriptional coactivator OBF-1.

The transcriptional coactivator OBF-1, which interacts with Oct-1 and Oct-2 and the octamer site DNA, has been shown to be critical for development of a normal immune response and the formation of germinal centers in secondary lymphoid organs. Here we have identified the RING finger protein Siah-1 as a protein interacting specifically with OBF-1. This interaction is mediated by the C-terminal part of Siah-1 and by residues in the N-terminus of OBF-1, partly distinct from the residues required for formation of a complex with the Oct POU domains and the DNA. Interaction between Siah-1 and OBF-1 leads to downregulation of OBF-1 protein level but not mRNA, and to a corresponding reduction in octamer site-dependent transcription activation. Inhibition of the ubiquitin-proteasome pathway in B cells leads to elevated levels of OBF-1 protein. Furthermore, in immunized mice, OBF-1 protein amounts are dramatically increased in primary activated B cells, without concomitant increase in OBF-1 mRNA. These data suggest that Siah-1 is part of a novel regulatory loop controlling the level of OBF-1 protein in B cells.

Amino Acid Sequence↗
PubMed2001

Estrogen receptor (ER)-alpha, but not ER-beta, mediates regulation of the insulin-like growth factor I gene by antiestrogens.

The importance of insulin-like growth factor I (IGF-I) on maintenance of skeletal integrity has been widely recognized. Although osteoblasts secrete some IGF-I, the liver is the primary endocrine source for IGF-I. We have studied the regulation of the human IGF-I promoter in the hepatocyte cell line Hep3B, and we have shown that the IGF-I promoter, when co-transfected in Hep3B cells together with an estrogen receptor (ER)-alpha expression vector, was transcriptionally regulated by raloxifene or raloxifene-like molecules but not by 17beta-estradiol and 4(OH)-tamoxifen. The induction mediated by raloxifene is antagonized by 17beta-estradiol and mediated selectively by ER-alpha, but not by ER-beta. Transfer of IGF-I promoter sequences from -733 to -65 or from -375 to -65 to a minimal Fos promoter resulted in a comparable responsiveness to raloxifene. This region contains two CAAT/enhancer-binding protein sites and an activator protein 1 site, both of which have been shown to be involved in estrogen receptor-mediated transactivation. When the CAAT/enhancer-binding protein sites were mutated in a construct bearing the sequence from -375 to -65 in front of the minimal Fos promoter, raloxifene induction was reduced, whereas mutation of the other elements did not affect induction. In addition, using chimeric proteins, we delineated the domains of ER-alpha that confer to ER-alpha transactivation abilities on the IGF-I promoter that are not exhibited by ER-beta. These data shed new light on the mechanism of action of antiestrogens and might help explain, at least in part, the bone-protective effects observed for some antiestrogens in ovariectomized animals.

Base Sequence↗
PubMed2001

B cell development and immunoglobulin gene transcription in the absence of Oct-2 and OBF-1.

Oct-2 and OBF-1 (also called OCA-B or Bob-1) are B cell-specific transcription factors that bind to the conserved octamer site of immunoglobulin promoters, yet their role in immunoglobulin transcription has remained unclear. We generated mice in which the lymphoid compartment was reconstituted with cells that lack both Oct-2 and OBF-1. Even in the absence of these two transcription factors, B cells develop normally to the membrane immunoglobulin M-positive (IgM+) stage and immunoglobulin gene transcription is essentially unaffected. These observations imply that the ubiquitous factor Oct-1 plays a previously unrecognized role in the control of immunoglobulin gene transcription and suggest the existence of another, as yet unidentified, cofactor. In addition, both factors are essential for germinal center formation, although OBF-1 is more important than Oct-2 for IgG production after immunization.

Animals↗
PubMed2000

Cutting edge: lack of peripheral B cells and severe agammaglobulinemia in mice simultaneously lacking Bruton's tyrosine kinase and the B cell-specific transcriptional coactivator OBF-1.

OBF-1 is a B cell-restricted transcriptional coactivator that is recruited to octamer-containing promoters by interacting with the POU domain of Oct-1 or Oct-2. We have shown earlier that mice lacking OBF-1 were dramatically impaired in their ability to mount humoral immune responses and did not develop germinal centers in the spleen; however, they had a largely normal B cell development in the bone marrow. In this study, we demonstrate that OBF-1-deficient mice also have an early defect in B cell development and show that OBF-1-/- immature B cells are greatly impaired at the transition from the bone marrow to the spleen. In addition, when the OBF-1 mutation is combined to a mutation in the gene encoding Bruton's tyrosine kinase, a striking phenotype is observed. These double-deficient animals lack peripheral B cells and have virtually no serum Igs, thus closely resembling human X chromosome-linked agammaglobulinemia.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2000

The lymphoid-specific cofactor OBF-1 is essential for the expression of a V(H) promoter/HS1,2 enhancer-linked transgene in late B cell development.

Mice deficient for the lymphoid-specific cofactor OBF-1 display reduced levels of IgG, IgA and IgE. To examine whether the lowered immunoglobulin expression is linked to reduced activity of IgH cis-regulatory elements, OBF-1(-/-) mice were crossed with mice expressing transgenes driven by a V(H) or beta-globin promoter linked to the HS1,2 enhancer. Here we show that OBF-1 is essential for the induced expression of a V(H) promoter-linked transgene, in contrast to a beta-globin promoter-dependent transgene, in LPS/IL-4 or CD40-stimulated splenic B cells. Furthermore, impaired transgene expression is observed in OBF-1(-/-) peritoneal B cells. This deficiency may be linked to OBF-1, as peritoneal cells from normal mice express OBF-1 protein constitutively. Our data link OBF-1 to IgH gene expression in late B lymphoid development.

Animals↗
PubMed2000

Molecular mechanisms of extinction: old findings and new ideas.

Fusion experiments between somatic cells have been used for a long time as a means to understand the regulation of gene expression. In hybrids between differentiated cells such as hepatocytes or lymphocytes and undifferentiated cells such as fibroblasts a phenomenon called extinction has been described. In such hybrids expression of cell-specific genes derived from the more differentiated parental cell is selectively turned off (extinguished), whereas genes expressed from both cells like housekeeping genes remain active after fusion. Study of the molecular basis of extinction of the liver-specifically expressed tyrosine aminotransferase gene and of the B-cell-specifically expressed immunoglobulin genes has revealed that in hybrids the transcriptional program of the differentiated cells is reset. This is accompanied by a loss of expression or activity of many of the regulatory molecules that were operating in the differentiated cells. In the light of new insights in eukaryotic gene regulation we speculate that molecular mechanisms such as chromatin remodelling, recruitment to heterochromatin or subnuclear localization could underly the extinction process.

Animals↗
PubMed2000

Correlation of anti-viral B cell responses and splenic morphology with expression of B cell-specific molecules.

This study attempted to evaluate and compare the role of various B cell-specific markers for anti-viral immune responses in mouse strains lacking molecules belonging to the B cell receptor (BCR) complex (IgM, Ig alpha and C(kappa)), the co-stimulatory molecules (CD19 and CD22), the protein kinases [Bruton's tyrosine kinase (Btk)] or the transcription factors (OBF-1). These mice were tested in two model infections [vesicular stomatitis virus (VSV) and lymphocytic choriomeningitis virus (LCMV)] using T cell-independent (TI) or T cell-dependent (TD) antigens. All mice controlled an LCMV infection indicating that cytotoxic T cell functions were within normal ranges. In contrast, OBF-1(-/-) mice were partially protected and mb-1(delta c/delta c) mice not at all protected against VSV infection, a virus that is controlled virtually exclusively by neutralizing antibodies. Susceptibility to VSV infection was correlated with structural defects in the spleen: absence of mature B cells and follicles with marginal zone macrophages and absence of germinal centers with follicular dendritic cells correlated with lack or substantial reduction of protective IgM and IgG responses respectively. The lack of kappa light chain did not affect the neutralizing response, indicating that it could easily be replaced by the lambda chain. Absence of the co-stimulatory molecules CD19 and CD22 or of the signaling molecule Btk had modulating effects, but did not increase susceptibility to VSV or LCMV. Our findings suggest that there are crucial molecules for B cell activation at the beginning (BCR complex) and the end (transcription) of the signaling cascade, whereas fine-tuning factors modulating the response in between exhibit considerable functional overlap.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Lymphoid-specific transcription mediated by the conserved octamer site: who is doing what?

The conserved octamer site found in the promoters of Immunoglobulin (Ig) genes has been identified early on as a key element for B cell-specific Ig promoter activity and has also been associated with expression of other B cell-specific genes. Several transcription factors--ubiquitous or cell-restricted--interact specifically with the octamer motif in B cells: Oct-1 and Oct-2 are POU domain transcription factors that bind directly to the octamer site and thereby can recruit the coactivator OBF-1 to the promoters of various genes. Yet, in spite of intense work, the precise role played by each of these factors for transcription activation through the octamer motif is unclear. In particular, genetic inactivation in mice of the lymphoid cell-specific transcription factor Oct-2 or of the B cell-specific coactivator OBF-1 led to surprising findings that are being discussed.

Animals↗
PubMed1998

The large subunit of basal transcription factor SNAPc is a Myb domain protein that interacts with Oct-1.

The human RNA polymerase II and III snRNA promoters have similar enhancers, the distal sequence elements (DSEs), and similar basal promoter elements, the proximal sequence elements (PSEs). The DSE, which contains an octamer motif, binds broadly expressed activator Oct-1. The PSE binds a multiprotein complex referred to as SNAPc or PTF. On DNAs containing both an octamer site and a PSE, Oct-1 and SNAPc bind cooperatively. SNAPc consists of at least four stably associated subunits, SNAP43, SNAP45, SNAP50, and SNAP190. None of the three small subunits, which have all been cloned, can bind to the PSE on their own. Here we report the isolation of cDNAs corresponding to the largest subunit of SNAPc, SNAP190. SNAP190 contains an unusual Myb DNA binding domain consisting of four complete repeats (Ra to Rd) and a half repeat (Rh). A truncated protein consisting of the last two SNAP190 Myb repeats, Rc and Rd, can bind to the PSE, suggesting that the SNAP190 Myb domain contributes to recognition of the PSE by the SNAP complex. SNAP190 is required for snRNA gene transcription by both RNA polymerases II and III and interacts with SNAP45. In addition, SNAP190 interacts with Oct-1. Together, these results suggest that the largest subunit of the SNAP complex is involved in direct recognition of the PSE and is a target for the Oct-1 activator. They also provide an example of a basal transcription factor containing a Myb DNA binding domain.

Amino Acid Sequence↗
PubMed1998

Coactivator OBF-1 makes selective contacts with both the POU-specific domain and the POU homeodomain and acts as a molecular clamp on DNA.

The lymphoid-specific transcriptional coactivator OBF-1 (also known as OCA-B or Bob-1) is recruited to octamer site-containing promoters by interacting with Oct-1 or Oct-2 and thereby enhances the transactivation potential of these two Oct factors. For this interaction the POU domain is sufficient. By contrast, OBF-1 does not interact with the POU domains of other POU proteins, such as Oct-4, Oct-6, or Pit-1, even though these factors bind efficiently to the octamer motif. Here we examined the structural requirements for selective interaction between the POU domain and OBF-1. Previous data have shown that formation of a ternary complex among OBF-1, the POU domain, and the DNA is critically dependent on residues within the octamer site. By methylation interference analysis we identified bases that react differently in the presence of OBF-1 compared to the POU domain alone, and using phosphothioate backbone-modified probes in electrophoretic mobility shift assays, we identified several positions influencing ternary complex formation. We then used Oct-1/Pit-1 POU domain chimeras to analyze the selectivity of the interaction between OBF-1 and the POU domain. This analysis indicated that both the POU specific domain (POUS) and the POU homeodomain (POUH) contribute to complex formation. Amino acids that are different in the Pit-1 and Oct-1 POU domains and are considered to be solvent accessible based on the Oct-1 POU domain/DNA cocrystal structure were replaced with alanine residues and analyzed for their influence on complex formation. Thereby, we identified residues L6 and E7 in the POUS and residues K155 and I159 in the POUH to be critical in vitro and in vivo for selective interaction with OBF-1. Furthermore, in an in vivo assay we could show that OBF-1 is able to functionally recruit two artificially separated halves of the POU domain to the promoter DNA, thereby leading to transactivation. These data allow us to propose a model of the interaction between OBF-1 and the POU domain, whereby OBF-1 acts as a molecular clamp holding together the two moieties of the POU domain and the DNA.

Amino Acid Sequence↗
PubMed1997

Effects of varying marijuana potency on deposition of tar and delta9-THC in the lung during smoking.

To determine whether smoking more, compared to less, potent marijuana (MJ) cigarettes to a desired level of intoxication ("high") reduces pulmonary exposure to noxious smoke components, in 10 habitual smokers of MJ, we measured respiratory delivery and deposition of tar and delta9-tetrahydrocannabinol (THC), carboxyhemoglobin (COHb) boost, smoking topography, including cumulative puff volume (CPV) and breathholding time, change in heart rate (deltaHR) and "high" during ad lib smoking of 0, 1.77, and 3.95% MJ cigarettes on 3 separate days. At each session, subjects had access to only a single MJ cigarette. On average, smoking topography and COHb boost did not differ across the different strengths of MJ, while THC delivery, as well as HR, were significantly greater (p < 0.01) and tar deposition significantly less (p < 0.03) for 3.95% than 1.77% MJ. Although individual adaptations in smoking topography for 3.95% compared to 1.77% MJ were highly variable, three subjects with the lowest 3.95% MJ:1.77% MJ ratios for CPV also displayed the lowest 3.95% MJ:1.77% MJ ratios for tar deposition. In vitro studies using a standardized smoking technique revealed a mean 25% lower tar yield from 3.95% than 1.77% MJ (p < 0.05), but no difference between 1.77% and 0% marijuana. Under the conditions of this study, we conclude that tar delivery is reduced relative to THC content in a minority of subjects, and this reduction appears to be due to a reduced intake of smoke (decreased CPV) and/or a reduced tar yield from the stronger MJ preparation.

Adult↗
PubMed1997

The B cell-specific coactivator OBF-1 (OCA-B, Bob-1) is inducible in T cells and its expression is dispensable for IL-2 gene induction.

The transcriptional coactivator variously known as OBF-1, OCA-B or Bob-1 has previously been shown to be expressed in a highly B cell-specific manner. Here we show that expression of the OBF-1 gene is also observed in several T cell lines as well as in primary T cells following activation. This suggests that this coactivator may also be involved in transcriptional control in T lymphocytes. In addition we show that several potential T cell-specific target genes which have octamer sites in their regulatory regions, such as the genes coding for interleukin-2, -3 and -4, are still properly regulated in T cells isolated from OBF-1-/- mice.

Animals↗
PubMed1997

Extinction of immunoglobulin gene expression in B cells upon fusion with HeLa cells is preceded by rapid nuclear depletion of essential transcription factors and is accompanied by widespread inactivation of genes expressed in a B cell-specific manner.

When immunoglobulin (Ig) expressing B cells are fused with non-B cells, Ig expression is rapidly suppressed at the level of transcription, a phenomenon termed extinction. Here we demonstrate that fusion of HeLa cells with either diploid or tetraploid B cells (Daudi) results in widespread extinction of several other B cell-encoded genes that are expressed in a B cell-specific manner. In contrast, expression of B cell-expressed genes that are not dependent on cell-specific controls is unaffected. We show that the molecular mechanism(s) underlying Ig gene extinction can be explained, at least in part, by a lack of transcription factors that are essential for Ig gene transcription. These transcription factors are either not produced due to block of transcription of their respective genes (Oct-2, OBF-1, PU.1), or are rendered inactive posttranslationally (NF-kappa B, E47). By isolating Daudi x HeLa heterokaryons a few hours after fusion, we have studied the initial fate of two B cell-specific transcription factors involved in Ig gene transcription, Oct-2 and NF-kappa B. This report provides the first demonstration that upon fusion with HeLa cells, the nuclear contents of B cell-expressed transcription factors are depleted within a few hours with kinetics that are as fast or faster than that of Ig gene extinction. Thus, the extinguishing mechanism is effective very early after fusion. We suggest that extinction of Ig genes is part of a global mechanism that suppresses the differentiation program foreign to the HeLa phenotype.

B-Lymphocytes↗
PubMed1996

B-cell-specific coactivator OBF-1/OCA-B/Bob1 required for immune response and germinal centre formation.

The B-lymphocyte-specific transcriptional factor called Oct binding factor (OBF)-1, OCA-B or Bob1 (refs 1-3) is thought to be involved in the transcription of immunoglobulin genes through recruitment to the highly conserved octamer site of immunoglobulin promoters, mediated by either Oct-1 or Oct-2. To define the in vivo role of OBF-1 we have used gene targeting in embryonic stem cells to generate mice lacking the coactivator OBF-1. Such OBF-1-/- mice are born normally, are fertile and seem healthy, and surprisingly, rearrangement and transcription of immunoglobulin genes are largely unaffected. However, mice deficient in OBF-1 have reduced numbers of mature B cells and a severe reduction in the number of recirculating B cells, but otherwise show normal B-cell differentiation. Serum IgA and particularly IgG levels are greatly reduced. If mutant mice are immunized with either a thymus-independent or a thymus-dependent antigen, their immune responses are dramatically weakened. Strikingly, germinal centres completely fail to develop after immunization with thymus-dependent antigen. Our results demonstrate that in vivo OBF-1 is not required for initial transcription of immunoglobulin genes or for B cell development, but instead is essential for the response of B cells to antigens, and is required for the formation of germinal centres.

Animals↗
PubMed1996

The ability of the inhibitory domain of the POU family transcription factor Oct-2 to interfere with promoter activation by different classes of activation domains is dependent upon the nature of the basal promoter elements.

The Oct-2 transcription factor contains an inhibitory domain which is able to repress transcription following DNA binding. Here we show that within the neuronally expressed Oct-2.5 form, the inhibitory domain can strongly inhibit activation by transcription factor activation domains which are either composed predominantly of acidic residues or contain the HOB motif, whereas it has a weaker effect or no effect on proline-rich activation domains and on a glutamine-rich domain. In contrast, the isolated inhibitory domain of Oct-2 can efficiently repress all types of activation domains. This effect is observed however, only on TATA box-containing promoters and not on promoters containing an initiator motif. This widespread inhibition of different activation domains and its dependence on the nature of the basal promoter elements indicate that the inhibitory domain is likely to act by contacting a common downstream target of activation domains within the basal transcriptional complex bound at the TATA box rather than quenching specific activation domains by direct interaction. These effects are discussed in terms of the functional role of the inhibitory domain within Oct-2.5 and the mechanism by which it acts.

Animals↗
PubMed1996

Mapping of the transcriptional repression domain of the lymphoid-specific transcription factor oct-2A.

The lymphoid-specific transcription factor Oct-2a is implicated in B cell-specific transcriptional activity via the octamer motif. Structure/function analysis of various Oct-2a effector regions in the context of the GAL4 DNA-binding domain revealed that Oct-2a contains two functionally different activation domains at the N and the C termini. The transcriptional activity of both domains is strongly potentiated by interactions with distinct B cell-specific coactivators. Recently, we have identified a repression domain located within the N terminus of Oct-2a (amino acids 2-99). When this domain was transferred to a potent activator, transcription was strongly inhibited. In this study we present a deletion analysis of the N-terminal region of Oct-2a to determine the minimal repression domain. We identified a stretch of 23 amino acids, rich in serine and threonine residues, which was responsible for most of the repression activity. We show that repression is strongly dependent on the type of enhancer present in the reporter plasmid as well as on the cell line tested. The possibility that Oct-2a can act as an activator and/or a repressor may have important consequences for the function of Oct-2a in B cell differentiation and other developmental processes.

Amino Acid Sequence↗
PubMed1996

Gene structure and characterization of the murine homologue of the B cell-specific transcriptional coactivator OBF-1.

The B cell-specific activity of immunoglobulin (Ig) gene promoters is to a large extent mediated by the conserved octamer motif ATTTGCAT. This requires the DNA binding octamer factors Oct-1 and/or Oct-2, as well as an additional B cell-restricted non-DNA binding cofactor. We recently cloned such a coactivator specific for Oct-1 or Oct-2 from human B cells and called it OBF-1. Here we report the isolation and characterization of the murine homologue. Full-length cDNA clones as well as genomic clones were isolated and the gene structure was determined. The deduced protein sequence shows that the mouse protein has an identical length, is likewise proline rich and shows 89% overall identity to the human protein. The OBF-1 gene is expressed in a very highly B cell-specific manner and is transcribed in cells representative of all stages of B cell differentiation, including the earliest ones. We show that OBF-1 interacts in the absence of DNA with the POU domain of Oct-1 or Oct-2 and also with the general transcription factors TBP and TFIIB. Furthermore, we demonstrate that although OBF-1 efficiently activates promoter octamer sites, it does not activate enhancer octamer sites.

Amino Acid Sequence↗