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At least 649 records · Page 36Linked to original sources
PubMed2005

GM-CSF promoter chromatin remodelling and gene transcription display distinct signal and transcription factor requirements.

Granulocyte-macrophage colony stimulating factor (GM-CSF) plays a key role in myeloid cell function and is rapidly and transiently expressed in T cells in response to immune or inflammatory stimuli. Induction of GM-CSF gene expression is accompanied by changes in chromatin structure across the proximal promoter region of the gene. We show that the promoter remodelling and subsequent gene transcription occurs with distinct signal and transcription factor requirements. Activation of the protein kinase C (PKC) signalling pathway is sufficient to induce changes in chromatin structure across the promoter, but both the PKC and calcium signalling pathways are required for efficient gene transcription. Although NFAT transcription factors contribute to GM-CSF gene transcription, they are not required for promoter remodelling. However, the presence of the nuclear factor-kappaB transcription factor, c-Rel, in the nucleus is strongly correlated with and required for the events of chromatin remodelling.

Animals↗
PubMed1992

Transcription by RNA polymerase II: initiator-directed formation of transcription-competent complexes.

Studies of transcription by RNA polymerase II have revealed two promoter elements, the TATA motif and the initiator (Inr), capable of directing specific transcription initiation. Although binding to the TATA motif by one of the components of the transcription machinery has been shown to be the initial recognition step in transcription complex formation, many promoters that lack a traditional TATA motif have recently been described. In such TATA-less promoters, the Inr element is critical in positioning RNA polymerase II. Various Inr elements have been described and classified according to sequence homology. These Inr elements are recognized specifically by Inr-binding proteins. Interaction between these Inr-binding proteins and components of the basal transcription machinery provides a means through which a transcription competent complex can be formed.

Adenoviridae↗
PubMed1998

The tumor suppressor Smad4/DPC4 and transcriptional adaptor CBP/p300 are coactivators for smad3 in TGF-beta-induced transcriptional activation.

Smads regulate transcription of defined genes in response to TGF-beta receptor activation, although the mechanisms of Smad-mediated transcription are not well understood. We demonstrate that the TGF-beta-inducible Smad3 uses the tumor suppressor Smad4/DPC4 and CBP/p300 as transcriptional coactivators, which associate with Smad3 in response to TGF-beta. The association of CBP with Smad3 was localized to the carboxyl terminus of Smad3, which is required for transcriptional activation, and a defined segment in CBP. Furthermore, CBP/p300 stimulated both TGF-beta- and Smad-induced transcription in a Smad4/DPC4-dependent fashion. Smad3 transactivation and TGF-beta-induced transcription were inhibited by expressing E1A, which interferes with CBP functions. The coactivator functions and physical interactions of Smad4 and CBP/p300 with Smad3 allow a model for the induction of gene expression in response to TGF-beta.

Adenovirus E1A Proteins↗
PubMed1993

Rapid transcriptional autoregulation of a yeast metalloregulatory transcription factor is essential for high-level copper detoxification.

Copper detoxification in the yeast Candida glabrata is carried out in large part by a family of metallothionein (MT) genes: a unique MT-I gene, a tandemly amplified MT-IIa gene, and a single unlinked MT-IIb gene. In response to elevated environmental copper levels, members of this MT gene family are transcriptionally activated by a copper-dependent, sequence-specific DNA-binding transcription factor, AMT1. AMT1 shares several structural and functional features with the Saccharomyces cerevisiae copper metalloregulatory transcription factor ACE1, which is constitutively expressed and poised for rapid transcriptional responses to the toxic metal copper. In this paper, we demonstrate that AMT1 is subject to positive transcriptional autoregulation, which is exerted through binding of copper-activated AMT1 to a single copper responsive element in the AMT1 promoter. A nonautoregulatory amt1 mutant displayed a marked decrease in both copper tolerance and expression of the MT-II genes, which are critical for high-level copper detoxification in Candida glabrata. Kinetic analysis demonstrated the remarkably rapid AMT1 mRNA accumulation in the presence of copper, which is followed by increased expression of the metallothionein gene products. These results demonstrate that AMT1-positive autoregulation plays a critical role in metal detoxification and suggest that the rapid autoactivation of the AMT1 metalloregulatory transcription factor biosynthesis is essential for C. glabrata to quickly build up a cellular defense line to protect cells upon exposure to high environmental copper levels.

Base Sequence↗
PubMed2005

Genomic binding and transcriptional regulation by the Drosophila Myc and Mnt transcription factors.

Deregulated expression of members of the myc oncogene family has been linked to the genesis of a wide range of cancers, whereas their normal expression is associated with growth, proliferation, differentiation, and apoptosis. Myc proteins are transcription factors that function within a network of transcriptional activators (Myc) and repressors (Mxd/Mad and Mnt), all of which heterodimerize with the bHLHZ protein Mad and bind E-box sequences in DNA. These transcription factors recruit coactivator or corepressor complexes that in turn modify histones. Myc, Mxd/Max, and Mnt proteins have been thought to act on a specific subset of genes. However, expression array studies and, most recently, genomic binding studies suggest that these proteins exhibit widespread binding across the genome. Here we demonstrate by immunostaining of Drosophila polytene chromosome that Drosophila Myc (dMyc) is associated with multiple euchromatic chromosomal regions. Furthermore, many dMyc-binding regions overlap with regions containing active RNA polymerase II, although dMyc can also be found in regions lacking active polymerase. We also demonstrate that the pattern of dMyc expression in nuclei overlaps with histone markers of active chromatin but not pericentric heterochromatin. dMyc binding is not detected on the X chromosome rDNA cluster (bobbed locus). This is consistent with recent evidence that in Drosophila cells dMyc regulates rRNA transcription indirectly, in contrast to mammalian cells where direct binding of c-Myc to rDNA has been observed. We further show that the dMyc antagonist dMnt inhibits rRNA transcription in the wing disc. Our results support the view that the Myc/Max/Mad network influences transcription on a global scale.

Animals↗
PubMed2004

The Etched1 gene of Zea mays (L.) encodes a zinc ribbon protein that belongs to the transcriptionally active chromosome (TAC) of plastids and is similar to the transcription factor TFIIS.

Etched1 (et1) is a pleiotropic, recessive mutation of maize that causes fissured and cracked mature kernels and virescent seedlings. Microscopic examinations of the et1 phenotype revealed an aberrant plastid development in mutant kernels and mutant leaves. Here, we report on the cloning of the et1 gene by transposon tagging, the localization of the gene product in chloroplasts, and its putative function in the plastid transcriptional apparatus. Several alleles of Mutator (Mu)-induced et1 mutants, the et1-reference (et1-R) mutant, and Et1 wild-type were cloned and analyzed at the molecular level. Northern analyses with wild-type plants revealed that Et1 transcripts are present in kernels, leaves, and other types of tissue, and no Et1 expression could be detected in the et1 mutants analyzed. The ET1 protein is imported by chloroplasts and has been immunologically detected in transcriptionally active chromosome (TAC) fractions derived from chloroplasts. Accordingly, the relative transcriptional activity of TAC fractions was significantly reduced in chloroplasts of et1-R plants. ET1 is the first zinc ribbon (ZR) protein shown to be targeted to plastids. With regard to its localization and its striking structural similarity to the eukaryotic transcription elongation factor TFIIS, it is feasible that ET1 functions in plastid transcription elongation by reactivation of arrested RNA polymerases.

Amino Acid Sequence↗
PubMed1994

The modulation of apolipoprotein E gene expression by 3,3'-5-triiodothyronine in HepG2 cells occurs at transcriptional and post-transcriptional levels.

The regulation of the synthesis and secretion of apolipoprotein E (apoE) is incompletely understood. This study examines the mechanisms responsible for regulating apoE gene expression in HepG2 cells by thyroid hormone (3,3'-5-triiodothyronine). The secretion rate of apoE was by thyroid hormone increased (1.5-1.8-fold) in pulse/chase experiments. Thyroid hormone doubled apoE mRNA concentration as determined by Northern-blot analysis. Inhibition of protein synthesis by cycloheximide increased the thyroid-hormone-induced stimulation of apoE mRNA. This suggests that the synthesis of new protein is not required for thyroid hormone to stimulate apoE mRNA. Actinomycin D was used to inhibit new transcription; there was a more rapid degradation of mature apoE mRNA in thyroid hormone-treated HepG2 cells than in control cells, suggesting that thyroid hormone acts post-transcriptionally to regulate apoE gene expression. Cycloheximide blocked the action of thyroid hormone, suggesting that thyroid hormone regulates the turnover of apoE mRNA via the synthesis of de novo protein. Nuclear run-on transcription assays demonstrated that thyroid hormone stimulated apoE gene transcription threefold in 24 h. These findings indicate that the expression of the apoE gene is controlled at both transcriptional and post-transcriptional loci by the thyroid hormone.

Apolipoproteins E↗
PubMed2006

Characterization of human deoxyribonuclease I gene (DNASE1) promoters reveals the utilization of two transcription-starting exons and the involvement of Sp1 in its transcriptional regulation.

Levels of deoxyribonuclease I (DNase I) activity in vivo have been shown to be altered by physiological and/or pathological processes. However, no information is available on the regulation of DNase I gene (DNASE1) expression in vivo or in vitro. We first mapped the transcription start sites of DNASE1 in human pancreas and in the DNase I-producing human pancreatic cancer cell line QGP-1, and revealed a novel site approximately 12 kb upstream of exon 1, which was previously believed to be the single transcription-starting exon. This initiation site marks an alternative starting exon, designated 1a. Exons 1 and 1a were used simultaneously as transcription-starting exons in pancreas and QGP-1 cells. Promoter assay, EMSA and chromatin immunoprecipitation analysis with QGP-1 cells showed the promoter region of exon 1a in which the Sp1 transcription factor is specifically involved in promoter activity. This is the first to be identified as a transcription factor responsible for gene expression of vertebrate DNase I genes. Furthermore, RT-PCR analysis indicated alternative splicing of human DNASE1 pre-mRNA in pancreas and QGP-1 cells. Only two transcripts among eight alternative splicing products identified can be translated to produce intact DNase I protein. These results suggest that human DNASE1 expression is regulated through the use of alternative promoter and alternative splicing.

Alternative Splicing↗
PubMed1991

Control of transcription of the Bacillus subtilis spoIIIG gene, which codes for the forespore-specific transcription factor sigma G.

The Bacillus subtilis spoIIIG gene codes for a sigma factor termed sigma G which directs transcription of genes expressed only in the forespore compartment of the sporulating cell. Use of spoIIIG-lacZ transcriptional fusions showed that spoIIIG is cotranscribed with the spoIIG operon beginning at t0.5-1 of sporulation. However, this large mRNA produced little if any sigma G, and transferring the spoIIIG gene without the spoIIG promoter into the amyE locus resulted in a Spo+ phenotype. Significant translation of spoIIIG began at t2.5-3 with use of an mRNA whose 5' end is just upstream of the spoIIIG coding sequence. Synthesis of this spoIIIG-specific mRNA was not abolished by a deletion in spoIIIG itself. Similar results were obtained when a spoIIIG-lacZ translational fusion lacking the spoIIG promoter was integrated at the amyE locus. These data suggest that synthesis of sigma G is dependent neither on transcription from the spoIIG promoter nor on sigma G itself but can be due to another transcription factor. This transcription factor may be sigma F, the product of the spoIIAC locus, since a spoIIAC mutation blocked spoIIIG expression, and sequences upstream of the 5' end of the spoIIIG-specific mRNA agree well with the recognition sequence for sigma F. RNA polymerase containing sigma F (E sigma F) initiated transcription in vitro on a spoIIIG template at the 5' end found in vivo, as did E sigma G. However, E sigma F showed a greater than 20-fold preference for spoIIIG over a known sigma G-dependent gene compared with the activity of E sigma G.

Bacillus subtilis↗
PubMed2004

Transcriptional activation by Bacillus subtilis ResD: tandem binding to target elements and phosphorylation-dependent and -independent transcriptional activation.

The expression of genes involved in nitrate respiration in Bacillus subtilis is regulated by the ResD-ResE two-component signal transduction system. The membrane-bound ResE sensor kinase perceives a redox-related signal(s) and phosphorylates the cognate response regulator ResD, which enables interaction of ResD with ResD-dependent promoters to activate transcription. Hydroxyl radical footprinting analysis revealed that ResD tandemly binds to the -41 to -83 region of hmp and the -46 to -92 region of nasD. In vitro runoff transcription experiments showed that ResD is necessary and sufficient to activate transcription of the ResDE regulon. Although phosphorylation of ResD by ResE kinase greatly stimulated transcription, unphosphorylated ResD, as well as ResD with a phosphorylation site (Asp57) mutation, was able to activate transcription at a low level. The D57A mutant was shown to retain the activity in vivo to induce transcription of the ResDE regulon in response to oxygen limitation, suggesting that ResD itself, in addition to its activation through phosphorylation-mediated conformation change, senses oxygen limitation via an unknown mechanism leading to anaerobic gene activation.

Bacillus subtilis↗
PubMed2006

Roles of human parainfluenza virus type 3 bases 13 to 78 in replication and transcription: identification of an additional replication promoter element and evidence for internal transcription initiation.

The genomic promoter of human parainfluenza virus type 3 (HPIV3) contains multiple cis-elements controlling transcription and replication. Previous work showed that regions 1 to 12 and 79 to 96 were critical in promoting replication of an HPIV3 minireplicon, while the intergenic sequence and N gene start signal (IS/Ngs, bases 49 to 61) were important for transcription. Because these data were collected primarily using point mutations, not every base from position 1 to 96 was analyzed, and some important control elements may have been missed. To clarify the role of bases 13 to 78 in transcription and replication, a series of mutations were made which collectively scanned this entire region. Mutation of bases 13 to 28 resulted in markedly decreased HPIV3 minireplicon replication, indicating these bases constitute an additional cis-element involved in the synthesis of the HPIV3 antigenomic RNA. The position dependence of the IS/Ngs was also examined. Analysis of mutants in which the IS/Ngs was shifted 5' or 3' showed that this segment could be moved without significantly disrupting transcription initiation. Additional mutants which contained two successive IS/Ngs segments were created to test whether the polymerase accessed the gene start signal by proceeding along the template 3' to 5' or by binding internally at the gene start signal. Based on analysis of the double gene start mutants, we propose a model of internal transcription initiation in which the polymerase enters the template at approximately the location of the natural N gene start but then scans the template bidirectionally to find a gene start signal and initiate transcription.

Genome, Viral↗
PubMed1994

Transcriptional activation of the herpes simplex virus type 1 UL38 promoter conferred by the cis-acting downstream activation sequence is mediated by a cellular transcription factor.

The herpes simplex virus (HSV) type 1 strict late (gamma) UL38 promoter contains three cis-acting transcriptional elements: a TATA box, a specific initiator element, and the downstream activation sequence (DAS). DAS is located between positions +20 and +33 within the 5' untranslated leader region and strongly influences transcript levels during productive infection. In this communication, we further characterize DAS and investigate its mechanism of action. DAS function has a strict spacing requirement, and DAS contains an essential 6-bp core element. A similarly positioned element from the gamma gC gene (UL44) has partial DAS function within the UL38 promoter context, and the promoter controlling expression of the gamma US11 transcript contains an identically located element with functional and sequence similarity to UL38 DAS. These data suggest that downstream elements are a common feature of many HSV gamma promoters. Results with recombinant viruses containing modifications of the TATA box or initiator element of the UL38 promoter suggest that DAS functions to increase transcription initiation and not the efficiency of transcription elongation. In vitro transcription assays using uninfected HeLa nuclear extracts show that, as in productive infection with recombinant viruses, the deletion of DAS from the UL38 promoter dramatically decreases RNA expression. Finally, electrophoretic mobility shift assays and UV cross-linking experiments show that DAS DNA forms a specific, stable complex with a cellular protein (the DAS-binding factor) of approximately 35 kDa. These data strongly suggest that the interaction of cellular DAS-binding factor with DAS is required for efficient expression of UL38 and other HSV late genes.

Animals↗
PubMed2001

Activation of human immunodeficiency virus transcription in T cells revisited: NF-kappaB p65 stimulates transcriptional elongation.

Human immunodeficiency virus type 1 (HIV-1) is able to establish a persistent latent infection during which the integrated provirus remains transcriptionally silent. Viral transcription is stimulated by NF-kappaB, which is activated following the exposure of infected T cells to antigens or mitogens. Although it is commonly assumed that NF-kappaB stimulates transcriptional initiation alone, we have found using RNase protection assays that, in addition to stimulating initiation, it can also stimulate elongation from the HIV-1 long terminal repeat. When either Jurkat or CCRF/CEM cells were activated by the mitogens phorbol myristate acetate and phytohemagglutinin, elongation, as measured by the proportion of full-length transcripts, increased two- to fourfold, even in the absence of Tat. Transfection of T cells with plasmids carrying the different subunits of NF-kappaB demonstrated that the activation of transcriptional elongation is mediated specifically by the p65 subunit. It seems likely that initiation is activated because of NF-kappaB's ability to disrupt chromatin structures through the recruitment of histone acetyltransferases. To test whether p65 could stimulate elongation under conditions where it did not affect histone acetylation, cells were treated with the histone deacetylase inhibitor trichostatin A. Remarkably, addition of p65 to the trichostatin A-treated cell lines resulted in a dramatic increase in transcription elongation, reaching levels equivalent to those observed in the presence of Tat. We suggest that the activation of elongation by NF-kappaB p65 involves a distinct biochemical mechanism, probably the activation of carboxyl-terminal domain kinases at the promoter.

Acetylation↗
PubMed2004

Adenovirus protein VII condenses DNA, represses transcription, and associates with transcriptional activator E1A.

Adenovirus protein VII is the major protein component of the viral nucleoprotein core. It is highly basic, and an estimated 1070 copies associate with each viral genome, forming a tightly condensed DNA-protein complex. We have investigated DNA condensation, transcriptional repression, and specific protein binding by protein VII. Xenopus oocytes were microinjected with mRNA encoding HA-tagged protein VII and prepared for visualization of lampbrush chromosomes. Immunostaining revealed that protein VII associated in a uniform manner across entire chromosomes. Furthermore, the chromosomes were significantly condensed and transcriptionally silenced, as judged by the dramatic disappearance of transcription loops characteristic of lampbrush chromosomes. During infection, the protein VII-DNA complex may be the initial substrate for transcriptional activation by cellular factors and the viral E1A protein. To investigate this possibility, mRNAs encoding E1A and protein VII were comicroinjected into Xenopus oocytes. Interestingly, whereas E1A did not associate with chromosomes in the absence of protein VII, expression of both proteins together resulted in significant association of E1A with lampbrush chromosomes. Binding studies with proteins produced in bacteria or human cells or by in vitro translation showed that E1A and protein VII can interact in vitro. Structure-function analysis revealed that an N-terminal region of E1A is responsible for binding to protein VII. These studies define the in vivo functions of protein VII in DNA binding, condensation, and transcriptional repression and indicate a role in E1A-mediated transcriptional activation of viral genes.

Adenovirus E1A Proteins↗
PubMed1986

Control of transcription initiation in vitro requires binding of a transcription factor to the distal promoter of the ovalbumin gene.

We used a cell-free HeLa cell transcription system to identify and characterize transcription factors and the promoter elements that they recognize in RNA polymerase II-transcribed genes. Deletion of the region (-71 to -83) containing the GTCAAA direct repeat resulted in a marked decrease of specific transcription of the ovalbumin gene; transcription could be competed with DNA fragments containing this sequence. Furthermore, DNase I footprinting identified a protein-binding site including this direct repeat with crude extracts and one of the partially purified protein fractions required for transcription. We propose that a soluble factor activates transcription through binding to the direct repeat of GTCAAA sequence upstream from the ovalbumin gene.

Base Sequence↗
PubMed1988

Factors responsible for the higher transcriptional activity of extracts of adenovirus-infected cells fractionate with the TATA box transcription factor.

Extracts of adenovirus-infected HeLa cells have 5- to 10-fold-higher activity for transcription from the major late promoter in vitro than do extracts of mock-infected or E1A mutant-infected cells (K. Leong and A. J. Berk, Proc. Natl. Acad. Sci. USA 83:5844-5848, 1986). In this study, we analyzed extracts from mock-infected cells and from cells infected with an E1A mutant, pm975, which expresses principally the large E1A protein responsible for the stimulation of transcription. These extracts were fractionated by phosphocellulose chromatography, a procedure which separates factors required for transcription from this promoter (J. D. Dignam, B. S. Shastry, and R. G. Roeder, Methods Enzymol. 101:582-589, 1983), allowing the quantitative assay of individual factors (M. Samuels, A. Fire, and P. A. Sharp, J. Biol. Chem. 257:14419-14427, 1982). Fractions eluted with 0.04, 0.35, and 0.6 M KCl, which contained RNA polymerase II, the upstream factor MLTF, and three general polymerase II transcription factors, had similar activities when prepared from virus-infected or from mock-infected cells. The sequence-specific DNA-binding activity of MLTF was also similar in the virus-infected- and mock-infected-cell extracts. In contrast, the 1.0 M KCl fraction prepared from virus-infected cells consistently exhibited activity severalfold higher than that of the equivalent fraction prepared in parallel from mock-infected cells. E1A protein eluted principally (greater than 80%) in the 0.35 M KCl fraction. Results of others (M. Sawadogo and R. G. Roeder, Cell 43:165-175, 1985) have shown that the 1.0 M KCl fraction, containing 2 to 5% of the unfractionated protein extract, contains a factor which binds specifically to the major late promoter TATA box. These results, together with a recent genetic analysis of the E1B promoter which demonstrated that the TATA box was required for its efficient transcriptional activation (transactivation) by E1A (L. Wu, D. S. E. Rosser, M. Schmidt, and A. J. Berk, Nature (London) 326:512-515, 1987), are consistent with the model that E1A protein indirectly activates the TATA box transcription factor. Consistent with this model was the finding that mutants of the major late promoter containing only the TATA box and cap site region were transcribed at higher rates with extracts from virus-infected cells than with extracts from mock-infected cells. Other models consistent with the results are also discussed.

Adenovirus Early Proteins↗
PubMed1997

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

Alleles↗
PubMed1997

Optimization of nuclear transcript detection by FISH and combination with fluorescence immunocytochemical detection of transcription factors.

Detection of specific nuclear transcripts by fluorescence in situ hybridization (FISH) has constituted a major breakthrough in the study of the organization of transcription in the cell nucleus. Using the model of heat shock genes, we present an optimized procedure for nuclear transcripts that provides high efficiency for RNA detection and good preservation of cell morphology and nuclear texture. Using this procedure, we designed an original high-efficiency methodology combining FISH and fluorescence immunocytochemistry (FICC), which is used here for the simultaneous detection of heat-shock protein (hsp) nuclear transcripts and the specific heat-shock transcription factor 1 (HSF1). We show that the nuclear accumulation sites of HSF1 in heat-shocked cells do not correspond to the sites of transcription of the hsp70 gene.

Cell Nucleus↗