An essential role for NF-kappa B in IL-18-induced IFN-gamma expression in KG-1 cells.
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IL-18 is a multifunctional cytokine playing various regulatory roles in the immune system including induced cytokine production. As a part of our ongoing studies on the molecular mechanisms of IL-18-induced IFN-gamma production, we have examined the transcriptional regulation of the IFN-gamma gene by IL-18 in a human myelomonocytic cell line, KG-1. On the basis of DNA/protein binding, we have determined an IL-18-inducible NF-kappa B binding site located at -786 to -776 of the IFN-gamma gene regulatory region (designated KBBsite). Transient transfection of promoter-reporter gene constructs revealed that the KBBsite is required for full IL-18-induced activation of the IFN-gamma gene transcription induced by IL-18. In addition, stable transformants of a dominant-negative form of the I kappa B alpha showed an inhibition of IL-18-dependent I kappa B alpha degradation, NF-kappa B activation, and expression of IFN-gamma. These results are the first to show the actual significance of the NF-kappa B pathway in the regulation of IFN-gamma gene expression by IL-18. (+info)
Differential effects of the widely expressed dMax splice variant of Max on E-box vs initiator element-mediated regulation by c-Myc.
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dMax, a naturally occurring splice variant of the Myc binding protein Max, lacks the DNA binding basic region and helix 1 of the Helix-Loop-Helix domain; dMax interacts with c-Myc in vitro and in vivo, and inhibits E-box Myc site driven transcription in transient transfection assays. Here we have investigated the expression, function and interactions of dMax. RT/PCR analyses detected dmax mRNA in multiple tissues of the developing, newborn and adult mouse. Functionally, dMax reduced the ability of c-Myc to cooperate with the progression factor A-Myb to promote S phase entry of quiescent smooth muscle cells. In contrast, dMax failed to ablate inhibition of initiator element (Inr)-mediated transcription by c-Myc in Jurkat T cells. In in vitro protein:protein association assays, dMax interacted with c-Myc, N-Myc, L-Myc, Mad1, Mxi1, Mad3 and Mad4, but not with itself or wild-type Max. These interactions required an intact leucine zipper. Inhibition of E-box-mediated transactivation by induction of dMax overexpression resulted in apoptosis of WEHI 231 B cells. Thus, dMax is a widely expressed, naturally occurring protein, with the capacity to bind most members of the Myc/Max superfamily; dMax has little effect on Inr-mediated repression by c-Myc, but can significantly decrease E-box-mediated events promoting proliferation and cell survival. (+info)
Cloning and expression of rat pancreatic beta-cell malonyl-CoA decarboxylase.
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To gain insight into the function and regulation of malonyl-CoA decarboxylase (MCD) we have cloned rat MCD cDNA from a differentiated insulin-secreting pancreatic beta-cell-line cDNA library. The full-length cDNA sequence shows 69% identity with the cDNA cloned previously from the goose uropygial gland, and predicts a 492 amino acid protein of 54.7 kDa. The open reading frame contains an N-terminal mitochondrial targeting sequence and the C-terminal part of the enzyme ends with a peroxisomal (Ser-Lys-Leu) targeting motif. Since the sequence does not reveal hydrophobic domains, MCD is most likely expressed in the mitochondrial matrix and inside the peroxisomes. A second methionine residue, located 3' of the mitochondrial presequence, might be the first amino acid of a putative cytosolic MCD, since the nucleotide sequence around it fits fairly well with a consensus Kozak site for translation initiation. However, primer extension detects the presence of only one transcript initiating upstream of the first ATG, indicating that the major, if not exclusive, transcript expressed in the pancreatic beta-cell encodes MCD with its mitochondrial presequence. The sequence also shows multiple possible sites of phosphorylation by casein kinase II and protein kinase C. mRNA tissue-distribution analysis indicates a transcript of 2.2 kb, and that the MCD gene is expressed over a wide range of rat tissues. The distribution of the enzyme shows a broad range of activities from very low in the brain to elevated in the liver and heart. The results provide the foundations for further studies of the role of MCD in lipid metabolism and metabolic signalling in various tissues. (+info)
Central role of transcription factor NF-IL6 for cytokine and iron-mediated regulation of murine inducible nitric oxide synthase expression.
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We have previously shown that iron regulates the transcription of inducible nitric oxide synthase (iNOS). To elucidate the underlying mechanisms we performed a series of transient transfections of murine fibroblast (NIH-3T3) and macrophage-like cells (J774.A1) with reporter plasmids containing the iNOS promoter and deletions thereof. By means of this and subsequent DNase I footprinting analysis we identified a regulatory region between -153 and -142 bp upstream of the transcriptional start site of the iNOS promoter that was sensitive to regulation by iron perturbation. Gel shift and supershift assays revealed that the responsible protein for this observation is NF-IL6, a member of the CCAAT/enhancer binding protein family of transcription factors. Binding of NF-IL6 to its consensus motif within the iNOS promoter was inducible by IFN-gamma and/or LPS, was reduced by iron, and was enhanced by the iron chelator desferrioxamine. Introduction of a double mutation into the NF-IL6 binding site (-153/-142) of an iNOS promoter construct resulted in a reduction of IFN-gamma/LPS inducibility by >90% and also impaired iron mediated regulation of the iNOS promoter. Our results provide evidence that this NF-IL6 binding site is of central importance for maintaining a high transcriptional rate of the iNOS gene after IFN-gamma/LPS stimulation, and that NF-IL6 may cooperate with hypoxia inducible factor-1 in the orchestration of iron-mediated regulation of iNOS. (+info)
Structural organization of the human Elk1 gene and its processed pseudogene Elk2.
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In the ets gene family of transcription factors, ELK1 belongs to the subfamily of Ternary Complex Factors (TCFs) which bind to the Serum Response Element (SRE) in conjunction with a dimer of Serum Response Factors (SRFs). The primary structure of the human Elk1 gene was determined by genomic cloning. The gene structure of Elk1 spans 15.2 kb and consists of seven exons and six introns. The coding sequence resides on exons 3, 4, 5, 6 and 7. Sequencing of cDNA clones isolated from human hippocampus library revealed that the second exon was often skipped by an alternative splicing event. All introns commenced with nucleotides GT at the 5' boundary and ended with nucleotides AG at the 3' boundary, in agreement with the proposed consensus sequence for intron spliced donor and acceptance sites. Sequence inspection of the 5'-flanking region revealed the absence of a 'TATA' box and the presence of putative cis-acting regulatory elements such as Sp1, GATA-1, CCAAT, and c-Myb. Moreover, the sequence analysis of Elk2 locus on 14q32.3 confirmed that Elk2 gene corresponds to a processed pseudogene of Elk1 which has been reported between alpha 1 gene (IGHA1) and pseudo gamma gene (IGHGP) of immunoglobulin heavy chain. Furthermore, the results of Southern analysis using DNAs from human-mouse hybrid cell lines carrying a part of 14q32 region revealed that there is another locus hybridizing to Elk1 cDNA on 14q32.2 --> qter region in addition to Elk2 locus between IGHA1 and IGHGP loci. (+info)
Transcriptional targeting of retroviral vectors to the erythroblastic progeny of transduced hematopoietic stem cells.
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Targeted expression to specific tissues or cell lineages is a necessary feature of a gene therapy vector for many clinical applications, such as correction of hemoglobinopathies or thalassemias by transplantation of genetically modified hematopoietic stem cells. We developed retroviral vectors in which the constitutive viral enhancer in the U3 region of the 3' LTR is replaced by an autoregulatory enhancer of the erythroid-specific GATA-1 transcription factor gene. The replaced enhancer is propagated to the 5' LTR upon integration into the target cell genome. The modified vectors were used to transduce human hematopoietic cell lines, cord blood-derived CD34(+) stem/progenitor cells, and murine bone marrow repopulating stem cells. The expression of appropriate reporter genes (triangle upLNGFR, EGFP) was analyzed in the differentiated progeny of transduced stem cells in vitro, in liquid culture as well as in clonogenic assay, and in vivo, after bone marrow transplantation in lethally irradiated mice. The GATA-1 autoregulatory enhancer effectively restricts the expression of the LTR-driven proviral transcription unit to the erythroblastic progeny of both human progenitors and mouse-repopulating stem cells. Packaging of viral particles, integration into the target genome, and stability of the integrated provirus are not affected by the LTR modification. Enhancer replacement is therefore an effective strategy to target expression of a retroviral transgene to a specific progeny of transduced hematopoietic stem cells. (+info)
Missense and silent tau gene mutations cause frontotemporal dementia with parkinsonism-chromosome 17 type, by affecting multiple alternative RNA splicing regulatory elements.
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Frontotemporal dementia with parkinsonism, chromosome 17 type (FTDP-17) is caused by mutations in the tau gene, and the signature lesions of FTDP-17 are filamentous tau inclusions. Tau mutations may be pathogenic either by altering protein function or gene regulation. Here we show that missense, silent, and intronic tau mutations can increase or decrease splicing of tau exon 10 (E10) by acting on 3 different cis-acting regulatory elements. These elements include an exon splicing enhancer that can either be strengthened (mutation N279(K)) or destroyed (mutation Delta280(K)), resulting in either constitutive E10 inclusion or the exclusion of E10 from tau transcripts. E10 contains a second regulatory element that is an exon splicing silencer, the function of which is abolished by a silent FTDP-17 mutation (L284(L)), resulting in excess E10 inclusion. A third element inhibiting E10 splicing is contained in the intronic sequences directly flanking the 5' splice site of E10 and intronic FTDP-17 mutations in this element enhance E10 inclusion. Thus, tau mutations cause FTDP-17 by multiple pathological mechanisms, which may explain the phenotypic heterogeneity observed in FTDP-17, as exemplified by an unusual family described here with tau pathology as well as amyloid and neuritic plaques. (+info)
Activation and repression of transcription by auxin-response factors.
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Auxin-response factors (ARFs) bind with specificity to TGTCTC auxin-response elements (AuxREs), which are found in promoters of primary/early auxin-response genes. Nine different ARFs have been analyzed for their capacity to activate or repress transcription in transient expression assays employing auxin-responsive GUS reporter genes. One ARF appears to act as a repressor. Four ARFs function as activators and contain glutamine-rich activation domains. To achieve transcriptional activation on TGTCTC AuxREs in transient expression assays, ARFs require a conserved dimerization domain found in both ARF and Aux/IAA proteins, but they do not absolutely require their DNA-binding domains. Our results suggest that ARFs can activate or repress transcription by binding to AuxREs directly and that selected ARFs, when overexpressed, may potentiate activation further by associating with an endogenous transcription factor(s) (e.g., an ARF) that is bound to AuxREs. Transfection experiments suggest that TGTCTC AuxREs are occupied regardless of the auxin status in cells and that these occupied AuxREs are activated when exogenous auxin is applied to cells or when ARF activators are overexpressed. The results provide new insight into mechanisms involved with auxin regulation of primary/early-response genes. (+info)