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PubMed2005

Components of the transcriptional Mediator complex are required for asymmetric cell division in C. elegans.

Asymmetric cell division is a fundamental process that produces cellular diversity during development. In C. elegans, the Wnt signaling pathway regulates the asymmetric divisions of a number of cells including the T blast cell. We found that the let-19 and dpy-22 mutants have defects in their T-cell lineage, and lineage analyses showed that the defects were caused by disruption in the asymmetry of the T-cell division. We found that let-19 and dpy-22 encode homologs of the human proteins MED13/TRAP240 and MED12/TRAP230, respectively, which are components of the Mediator complex. Mediator is a multi-component complex that can regulate transcription by transducing the signals between activators and RNA polymerase in vitro. We also showed that LET-19 and DPY-22 form a complex in vivo with other components of Mediator, SUR-2/MED23 and LET-425/MED6. In the let-19 and dpy-22 mutants, tlp-1, which is normally expressed asymmetrically between the T-cell daughters through the function of the Wnt pathway, was expressed symmetrically in both daughter cells. Furthermore, we found that the let-19 and dpy-22 mutants were defective in the fusion of the Pn.p cell, a process that is regulated by bar-1/beta-catenin. Ectopic cell fusion in bar-1 mutants was suppressed by the let-19 or dpy-22 mutations, while defective cell fusion in let-19 mutants was suppressed by lin-39/Hox mutations, suggesting that let-19 and dpy-22 repress the transcription of lin-39. These results suggest that LET-19 and DPY-22 in the Mediator complex repress the transcription of Wnt target genes.

Amino Acid Sequence↗

PubMed2005

The yeast Mediator complex and its regulation.

The Mediator complex acts as a bridge, conveying regulatory information from enhancers and other control elements to the basal RNA polymerase II transcription machinery. Mediator is required for the regulated transcription of nearly all RNA polymerase II-dependent genes in Saccharomyces cerevisiae, and post-translational modifications of specific Mediator subunits can affect global patterns of gene transcription.

Animals↗

PubMed2006

Med19(Rox3) regulates Intermodule interactions in the Saccharomyces cerevisiae mediator complex.

The Saccharomyces cerevisiae Mediator is a 25-subunit complex that facilitates both transcriptional activation and repression. Structural and functional studies have divided Mediator subunits into four distinct modules. The Head, Middle, and Tail modules form the core functional Mediator complex, whereas a fourth, the Cyc-C module, is variably associated with the core. By purifying Mediator from a strain lacking the Med19(Rox3) subunit, we have found that a complex missing only the Med19(Rox3) subunit can be isolated under mild conditions. Additionally, we have established that the entire Middle module is released when the Deltamed19(rox3) Mediator is purified under more stringent conditions. In contrast to most models of the modular structure of Mediator, we show that release of the Middle module in the Deltamed19(rox3) Mediator leaves a stable complex made up solely of Head and Tail subunits. Both the intact and Head-Tail Deltamed19(rox3) Mediator complexes have defects in enhanced basal transcription, enhanced TFIIH phosphorylation of the CTD, as well as binding of RNA Pol II and the CTD. The largely intact Deltamed19(rox3) complex facilitates activated transcription at levels similar to the wild type Mediator. In the absence of the Middle module, however, the Deltamed19(rox3) Mediator is unable to facilitate activated transcription. Although the Middle module is unnecessary for holding the Head and Tail modules together, it is required for the complex to function as a conduit between activators and the core transcription machinery.

Gene Expression Regulation, Fungal↗

PubMed2001

Novel critical role of a human Mediator complex for basal RNA polymerase II transcription.

Human Mediator complexes have been described as important bridging factors that enhance the effect of activators in purified systems and in chromatin. Here we report a novel basal function of a human Mediator complex. A monoclonal antibody was generated that depleted the majority of Mediator components from crude cell extracts. The removal of human Mediator abolished transcription by RNA polymerase II. This was observed on all genes tested, on TATA-containing and TATA-less promoters, both in the presence and absence of activators. To identify the relevant complex a combined biochemical and immunopurification protocol was applied. Two variants termed Mediator and basal Mediator were functionally and structurally distinguished. Basal Mediator function relies on additional constraints, which is reflected in the observation that it is essential in crude but not in purified systems. We conclude that basal Mediator is a novel general transcription factor of RNA polymerase II.

Antibodies, Monoclonal↗

PubMed1999

The Med1 subunit of the yeast mediator complex is involved in both transcriptional activation and repression.

The mediator complex is essential for regulated transcription in vitro. In the yeast Saccharomyces cerevisiae, mediator comprises >15 subunits and interacts with the C-terminal domain of the largest subunit of RNA polymerase II, thus forming an RNA polymerase II holoenzyme. Here we describe the molecular cloning of the MED1 cDNA encoding the 70-kDa subunit of the mediator complex. Yeast cells lacking the MED1 gene are viable but show a complex phenotype including partial defects in both repression and induction of the GAL genes. Together with results on other mediator subunits, this implies that the mediator is involved in both transcriptional activation and repression. Similar to mutations in the SRB10 and SRB11 genes encoding cyclin C and the cyclin C-dependent kinase, a disruption of the MED1 gene can partially suppress loss of the Snf1 protein kinase. We further found that a lexA-Med1 fusion protein is a strong activator in srb11 cells, which suggests a functional link between Med1 and the Srb10/11 complex. Finally, we show that the Med2 protein is lost from the mediator on purification from Med1-deficient cells, indicating a physical interaction between Med1 and Med2.

Cloning, Molecular↗

PubMed2006

Differential roles of transcriptional mediator complex subunits Crsp34/Med27, Crsp150/Med14 and Trap100/Med24 during zebrafish retinal development.

The transcriptional mediator complex has emerged as an important component of transcriptional regulation, yet it is largely unknown whether its subunits have differential functions in development. We demonstrate that the zebrafish mutation m885 disrupts a subunit of the mediator complex, Crsp34/Med27. To explore the role of the mediator in the control of retinal differentiation, we employed two additional mutations disrupting the mediator subunits Trap100/Med24 and Crsp150/Med14. Our analysis shows that loss of Crsp34/Med27 decreases amacrine cell number, but increases the number of rod photoreceptor cells. In contrast, loss of Trap100/Med24 decreases rod photoreceptor cells. Loss of Crsp150/Med14, on the other hand, only slightly reduces dopaminergic amacrine cells, which are absent from both crsp34(m885) and trap100(lessen) mutant embryos. Our data provide evidence for differential requirements for Crsp34/Med27 in developmental processes. In addition, our data point to divergent functions of the mediator subunits Crsp34/Med27, Trap100/Med24, and Crsp150/Med14 and, thus, suggest that subunit composition of the mediator contributes to the control of differentiation in the vertebrate CNS.

Alleles↗

PubMed2004

Ras induces mediator complex exchange on C/EBP beta.

C/EBPbeta is an intrinsically repressed transcription factor that regulates genes involved in differentiation, proliferation, tumorigenesis, and apoptosis. C/EBPbeta acts as a repressor that is turned into an activator by the Ras oncoprotein through phosphorylation of a MAPK site. C/EBPbeta activation is accompanied by a conformational change. Active and repressive C/EBPbeta interacts with multisubunit Mediator complexes through the CRSP130/Sur2 subunit. The CRSP130/Sur2 subunit is common to two distinct types of Mediator complexes, characterized by CRSP70 and CDK8 proteins as transcriptionally active and inactive Mediator, respectively. Knockdown of CRSP130/Sur2 prevents Mediator binding and transactivation through C/EBPbeta. Oncogenic Ras signaling or activating mutations in C/EBPbeta selects the transcriptionally active Mediator complex that also associates with RNA polymerase II. These results show that a Ras-induced structural alteration of C/EBPbeta determines differential gene activation through selective interaction with distinct Mediator complexes.

Apoptosis↗

PubMed1988

Histopathological lung changes in immune complex mediated anaphylactic shock in humans elicited by dextran.

Severe dextran-induced anaphylactic reaction (DIAR) is being recognized as a form of immediate IgG mediated immune complex reaction. Support for this pathogenesis is found in the correlation between the titer of dextran-reactive antibodies of IgG class and the severity of the reaction. Autopsy records were reviewed in 27 certified cases of fatal DIAR. The most frequent macroscopic findings were dilatation of the right side of the heart and acute pulmonary stasis. Autopsy lung specimens were collected from 17 of these patients. In 15 of the 17 lung specimens pulmonary microemboli were found. The microemboli had the appearance of hyaline eosinophilic globules, and the lung vasculature also contained leukocytes, platelets and disintegrated erythrocytes. These findings show similarity to the findings in a monkey model of known IgG mediated anaphylaxis, and give further support to the proposed pathogenesis of severe DIAR.

Adult↗

PubMed2001

The Swi5 activator recruits the Mediator complex to the HO promoter without RNA polymerase II.

Regulation of HO gene expression in the yeast Saccharomyces cerevisiae is intricately orchestrated by an assortment of gene-specific DNA-binding and non-DNA binding regulators. Binding of the early G1 transcription factor Swi5 to the distal URS1 element of the HO promoter initiates a cascade of events through recruitment of the Swi/Snf and SAGA complexes. In late G1, binding of transcription factor SBF to promoter proximal sequences results in the timely expression of HO. In this work we describe an important additional layer of complexity to the current model by identifying a connection between Swi5 and the Mediator/RNA polymerase II holoenzyme complex. We show that Swi5 recruits Mediator to HO by specific interaction with the Gal11 module of the Mediator complex. Importantly, binding of both the Gal11 and Srb4 mediator components to the upstream region of HO is independent of the SBF factor. Swi/Snf is required for Mediator binding, and genetic suppression experiments suggest that Swi/Snf and Mediator act in the same genetic pathway of HO activation. Experiments examining the kinetics of binding show that Mediator binds to HO promoter elements 1.5 kb upstream of the transcription start site in early G1, but this binding occurs without RNA Pol II. RNA Pol II does not bind to HO until late G1, when HO is actively transcribed, and binding occurs exclusively to the TATA region.

Cell Cycle Proteins↗

PubMed1996

Cu(2+)-mediated complex formation between polyacrylic acid (PAA) and bovine serum albumin.

Cu(2+)-mediated complex formation between polyacrylic acid (PAA) and negatively charged bovine serum albumin (BSA) was studied in neutral water in the presence of Cu2+. Depending on the concentration of Cu2+, the reaction between PAA-Cu2+ complexes and BSA appeared to follow one of two possible paths. At low Cu2+ concentrations (nCu/nAA < 0.15), a further increase in BSA concentration led to the breakdown of the complex as in mechanism I: [formula: see text] At higher Cu2+ concentrations (nCu/nAA > 0.15), a further increase in BSA concentration led to the formation of non-stoichiometric polycomplexes (mechanism II): [formula: see text] The immunogenic properties of ternary mixtures of BSA-Cu(2+)-PAA were investigated and the relationship between immunogenicity and complex formation in solution was analyzed. The addition of Cu2+ to solutions of PAA with BSA gave rise to a considerable increase in BSA-specific immunogenicity. Data obtained from the analysis of the immunogenicity of BSA-Cu(2+)-PAA mixtures formed using different ratios of the components suggested that (1) the highest immunogenic activity is exhibited by stable ternary complexs, and (2) immunoactive polyelectrolyte complexes have a non-stoichiometric composition. We thus propose a novel method, based on Cu2+ mediated complex formation, to enhance protein-specific antibody responses.

Acrylic Resins↗

PubMed1985

Adverse effect of exercise on immune complex-mediated glomerulonephritis.

To assess the effect of strenuous daily exercise on immune complex-mediated glomerulonephritis (GN), rabbits were randomly assigned to one of three experimental groups: Group I (n = 12): treadmill exercise for 28 days plus twice weekly intravenous injections of saline. Group II (n = 10): treadmill exercise for 28 days plus twice weekly intravenous bovine serum albumin (BSA) injection. Group III (n = 9): intravenous doses of BSA, as in group II, but no exercise. Blood and urine samples were collected from each animal periodically during the 28-day experimental period. On the 29th day of the study all animals were sacrificed and tissue taken for renal histopathologic studies. We found that in group II (exercise + GN) abnormal albuminuria was more frequent (p less than 0.001), blood urea nitrogen (BUN) levels rose significantly with time (p less than 0.02) and hematuria (blood in renal tubules) was more common (p less than 0.05), compared to group III (GN only). The differences between groups II and III could not be explained by the effect of exercise alone since group I (exercise only) developed no abnormal albuminuria, BUN levels or hematuria during the course of the study. These findings suggest that strenuous exercise superimposed on active immune complex-mediated GN results in worsening of the abnormal glomerular function.

Animals↗

PubMed2001

Mediator complexes and transcription.

Over the past decade, various components of the transcription machinery have been identified as potential targets for activators. Recently, metazoan versions of yeast Mediator have been isolated and found to act as key coactivators to many transcription factors. Recent work has defined the composition, function and biology of metazoan mediator complexes, which has led us to propose a new nomenclature for the variously named versions of the mediator complex.

Animals↗

PubMed1984

Autoimmunity and inner ear disorders: an immune-complex mediated sensorineural hearing loss.

An immune-complex mediated form of autoimmune disease is described with the inner ear as one of its target organs. Around 40 dB bilateral sensorineural hearing loss improved on short-term corticosteroid therapy to normal levels. This case report is presented against the background of a general survey on autoimmunity and inner ear diseases. Cellular and humoral immune reactions may be etiologic moments in an audiovestibular dysfunction of autoimmune character.

Adolescent↗

PubMed2006

Arp2/3 complex-mediated actin polymerisation occurs on specific pre-existing networks in cells and requires spatial restriction to sustain functional lamellipod extension.

The classical Arp2/3-mediated dendritic network defines the cytoskeleton at the leading edge of crawling cells, and it is generally assumed that Arp2/3-mediated actin polymerization generates the force necessary to extend lamellipods. Our previous work suggested that successful lamellipod extension required not only free barbed ends for actin polymerization but also a proper ultrastructural organization of the cytoskeleton. To further explore the structural role of the Arp2/3 complex-mediated networks in lamellipod morphology and function, we performed a detailed analysis of the ultrastructure of the Arp2/3-mediated networks, using the WA domains of Scar and WASp to generate mislocalised Arp2/3 networks in vivo, and to reconstruct de novo Arp2/3-mediated actin nucleation and polymerization on extracted cytoskeletons. We present here evidence that spatially unrestricted Arp2/3-mediated networks are intrinsically three-dimensional and multilayered by nature and, as such, cannot sustain significant polarized extension. Furthermore, such networks polymerize only at preferred locations in extracted cells, corresponding to pre-existing Arp2/3 networks, suggesting that the specific molecular organization of the actin cytoskeleton, in terms of structure and/or biochemical composition, dictates the location of Arp2/3 complex-mediated actin polymerization. We propose that successful lamellipod extension depends not only on localized actin polymerization mediated through local signalling, but also on spatial restriction of the Arp2/3 complex-mediated nucleation of actin polymerization, both in terms of location within the cell and ultrastructural organization of the resulting network.

Actin-Related Protein 2-3 Complex↗

PubMed2003

Association of the Mediator complex with enhancers of active genes.

The multiprotein Mediator complex has been shown to interact with gene-specific regulatory proteins and RNA polymerase II in vitro. Here, we use chromatin immunoprecipitation to analyze the recruitment of Mediator to GAL genes of yeast in vivo. We find that Mediator associates exclusively with transcriptionally active and not inactive GAL genes. This association maps to the upstream activating sequence, rather than the core promoter, and is independent of RNA polymerase II, general transcription factors, and core promoter sequences. These findings support the idea of Mediator as a primary conduit of regulatory information from enhancers to promoters in eukaryotic cells.

Enhancer Elements, Genetic↗

PubMed1999

Ceftizoxime-induced hemolysis due to immune complexes: case report and determination of the epitope responsible for immune complex-mediated hemolysis.

BACKGROUND: Several occurrences of immune complex-mediated, cephalosporin-induced intravascular hemolysis have been reported. This report describes the first case of hemolytic anemia caused by an immune-complex mechanism associated with ceftizoxime and delineates the epitope responsible for hemolysis. CASE REPORT: The patient's serum was tested for antibody that reacted with five penicillins and 30 cephems (all types of cephalosporins) by using protocols to detect drug-adsorption and immune-complex mechanisms. The patient's antibody that formed immune complexes with ceftizoxime reacted with 10 of 30 cephems. These 10 drugs were classified as oxime-type cephalosporins, which have a common structural formula consisting of [(Z)-2-(2-amino-4-thiazolyl)-2-methoxyiminoacetoamido] at the C7 position on 7-aminocephalosporinic acid with or without substitution at the C3 position. CONCLUSION: The patient's antibody recognized a common structure in 10 oxime-type cephalosporins, and immune complexes formed by the antibody specifically or nonspecifically bound to red cell membranes. Therefore, when intermittent antibiotic therapy is required, as in this case, care should be taken in antibiotic selection to avoid drug-induced hemolytic anemia. In addition, when this type of hemolysis is observed, tests for antibody that reacts by adsorption and immune-complex mechanisms should be performed against penicillins and cephems to select antibiotics not showing a cross-reaction.

Adsorption↗

PubMed1985

The nonspecific clearance function of the reticuloendothelial system in patients with immune complex mediated diseases before and after therapeutic plasmapheresis.

The nonspecific clearance function of the reticuloendothelial system (RES) in six patients with immune complex mediated systemic vasculitis was determined by the evaluation of the disappearance rate of technetium 99m labelled microaggregated human serum albumin colloid (MHAC) injected IV before and after therapeutic plasma exchange. Three patients with systemic lupus erythematosus (SLE) and one patient with immune complex vasculitis (ICV) exhibited a significant clinical improvement after plasmapheresis which was paralleled by an accelerated MHAC elimination rate following plasma exchange therapy. One patient with ICV and unresponsive to plasma exchange showed delayed MHAC elimination. In one patient with myasthenia gravis (MG), the elimination rate was not altered by plasmapheresis. The data obtained indicate that nonspecific clearance of the RES may be one effect of plasma exchange therapy in patients with immune complex mediated diseases.

Adolescent↗

PubMed2003

FcgammaRI up-regulation induced by local adenoviral-mediated interferon-gamma production aggravates chondrocyte death during immune complex-mediated arthritis.

Using various FcgammaR-deficient mice, we have obtained suggestive evidence that FcgammaRI on macrophages is responsible for severe cartilage destruction during arthritis mediated by immune complexes (ICs). This role of FcgammaRI is pronounced in the presence of activated Th1 cells and a likely Th1 cell-derived cytokine mediating up-regulation of FcgammaRI expression is interferon (IFN)-gamma. We now investigated whether local overexpression of IFN-gamma using an adenoviral vector is able to elevate cartilage destruction during experimental immune complex-mediated arthritis (ICA) and to what extent this process is FcgammaRI-mediated. IFN-gamma overexpression during ICA had no significant effect on the total cell mass infiltrating the knee joint. However, a higher percentage of macrophages expressing markers for a proinflammatory phenotype was found and these macrophages were situated in close proximity of the cartilage surface. Interestingly, cartilage destruction as studied by matrix metalloproteinase (MMP)-mediated proteoglycan damage (VDIPEN expression), chondrocyte death, and erosion was significantly increased. This effect of IFN-gamma was only found in the presence of ICs, as IFN-gamma overexpression during zymosan-induced arthritis, which is not IC-dependent, did not lead to severe cartilage destruction. These results imply a crucial role for ICs and the IgG-binding receptors in the aggravation of cartilage damage by IFN-gamma. Local overexpression of IFN-gamma induced increased FcgammaRI mRNA levels in synovium. To study whether this up-regulation of FcgammaRI mediates aggravation of cartilage destruction, ICA was raised in FcgammaRI(-/-) and their wild-type controls. IFN-gamma resulted in elevated VDIPEN expression, which was still present in FcgammaRI(-/-). Of great interest, chondrocyte death remained low in FcgammaRI(-/-). These results indicate that IFN-gamma overexpression deteriorates cartilage destruction in the presence of ICs and that FcgammaRI is crucial in the development of chondrocyte death.

Adenoviridae↗