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At least 1,243 records · Page 69Linked to original sources
PubMed2004

Regulation of transcription of the steroidogenic acute regulatory protein (StAR) gene: temporal and spatial changes in transcription factor binding and histone modification.

We examined the binding of transcription factors and histone modifications associated with induction of expression of the steroidogenic acute regulatory protein (StAR) gene in MA-10 cells using a quantitative chromatin immunoprecipitation (ChIP) assay. GATA-4, SF-1/Ad4BP, and cyclic AMP response element binding protein binding protein (CBP) bind rapidly to the StAR proximal promoter, but in different patterns following 8-Br-cAMP stimulation. Concomitantly, histone modifications occur in a spatial and temporal sequence including increased association of acetylated histone H3 with the proximal promoter region, increased association of dimethylated lysine 4 histone H3 with exonic sequences, a modification that marks actively transcribed regions, and reduced association of a marker linked to gene silencing (lysine 9 dimethylated histone H3). Our findings demonstrate that transcription factors and coactivators are rapidly associated with the StAR proximal promoter, that the patterns of binding differ which has implications for postulated direct interactions among these factors, and that multiple histone modifications are demonstrable in a spatially- and temporally-specific pattern along the StAR gene. These observations suggest that a combinatorial code of transcription factors including reciprocal changes in histone modifications associated with active transcription and gene silencing control StAR gene expression.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2005

Function of a eukaryotic transcription activator during the transcription cycle.

Site-specific photocrosslinkers positioned within the central transcription-activating region of yeast Gcn4 were used to identify, in an unbiased way, three polypeptides in direct physical proximity to the activator during the process of transcription activation. Crosslinking was specific and did not change during different steps of the transcription cycle. The crosslinking targets were identified as Tra1, Gal11, and Taf12, subunits of four complexes (SAGA, NuA4, Mediator, and TFIID) known to play a role in gene regulation. Using this crosslinking assay, an activating region mutant, and extracts depleted of individual complexes containing the crosslinking targets, we found that contact with Tra1/SAGA is critical for activation, Gal11 contact has a modest effect on activation, and contact with TFIID and NuA4 is of little or no importance for activation under our conditions. Thus, a single activating region contacts multiple factors, and each contact makes differential contributions to transcriptional activation.

Amino Acid Sequence↗
PubMed2004

Ligand-regulated association of ErbB-4 to the transcriptional co-activator YAP65 controls transcription at the nuclear level.

It has been proposed that ligand-dependent Regulated Intramembrane Proteolysis (RIP) of ErbB-4 receptors generates 80 kDa Intra-Cellular Domains (E4.ICDs) that relocate to the nuclear compartments where they implement the signaling abilities of the ErbB-4 receptors. The E4.ICD may directly regulate gene transcription or, in an alternative scenario, the tyrosine kinase activity of E4.ICDs may target proteins involved in transcriptional regulation upon its relocation into the nucleus. We have identified the transcriptional coactivator YAP65, here referred as YAP (Yes Associated Protein), as binding partner of ErbB-4 in a two hybrid screening in yeast. Interaction between YAP and ErbB-4 occurs via the WW domain of YAP and the PPPPY at positions 1297-1301 and the PPPAY at positions 1052-1056 of the amino acid sequence of the Cyt-1 isoform of ErbB-4. Stechiometry of binding is regulated by the ligand-dependent phosphorylation of Tyr 1056 in the PPPAYTPM module that function as "biochemical switch" to decrease the association of YAP to ErbB-4. In principle, this novel interaction highlights new mechanisms of signaling propagation from the ErbB-4 receptors, offering supporting evidences that the E4.ICDs forms released following ligand-receptor engagement may recruit YAP and relocate to the nucleus to implement or regulate transcription.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Real-time quantitative reverse transcription-polymerase chain reaction for the detection of AML1-MTG8 fusion transcripts in t(8;21)-positive acute myelogenous leukemia.

Quantification of AML1-MTG8 fusion transcripts was performed by using real-time reverse transcription-polymerase chain reaction (RT-PCR) and the clinical value of this method was evaluated in t(8;21)-positive acute myelogenous leukemia (AML). A t(8;21)-positive cell line, Kasumi-1, was used for constructing standard curves and the corrected AML1-MTG8 mRNA expression level relative to the expression of the GAPDH housekeeping gene was calculated. Bone marrow samples from 14 patients with t(8;21)-positive AML were sequentially examined. The corrected AML1-MTG8 expression level at diagnosis varied in the range from 0.4 to 2.7 (median, 1.5) among the patients. When samples at 1, 3 and 6 months were examined after diagnosis, the corrected AML1-MTG8 expression level was found to decrease sequentially in all but one. AML1-MTG8 fusion transcripts were also detected in four of eight samples from patients in remission for more than 1 year. In conclusion, real-time RT-PCR can provide a rapid and accurate quantification of AML1-MTG8 fusion transcripts. This system could be useful to reveal the prognostic relevance of minimal residual disease in t(8;21)-positive AML.

Adolescent↗
PubMed2003

Positive and negative regulators for neuronal BC1 RNA transcription by RNA polymerase III are possible members of the RNA polymerase II transcription system.

Neuronal cell-specific BC1 RNA is a unique RNA polymerase III (Pol III) transcript. The transcription is controlled by an activator E2 site and by BCRE, a repressor element, in response to neuronal activity. BC1 RNA is localized to dendritic domains as ribonucleoprotein particles, and it has been suggested to play a functional role in translational regulation of dendritic mRNAs. In the present study, using a luciferase assay in NG108-15 cells, we found that the positive and negative regulators for BC1 RNA transcription can also function in the Pol II transcription system. Our results suggest that the neuronal activity-dependent expression of BC1 RNA by Pol III and a subset of neuronal mRNAs by Pol II may be simultaneously controlled by the E2 site and BCRE, as well as their binding proteins.

Animals↗
PubMed1997

The transcriptional activation pattern of lipopolysaccharide binding protein (LBP) involving transcription factors AP-1 and C/EBP beta.

Lipopolysaccharide (LPS) Binding Protein (LBP) is an acute phase protein with the ability to recognize bacterial LPS and transport it to the CD14 molecule or into HDL particles. It is synthesized in hepatocytes and secreted into the blood stream. LBP levels significantly rise during the acute phase response and levels of LBP may be important for an appropriate host reaction to bacterial challenge and for developing the sepsis syndrome. In order to elucidate the mechanisms of LBP regulation we investigated its transcription pattern and performed promoter studies under experimental conditions mimicking an acute phase scenario. In human hepatoma cell lines stimulation with IL-1 beta, IL-6, TNF-alpha and dexamethasone leads to strong transcriptional activation of the LBP gene in a dose- and time-dependent manner. IL-6 alone induces LBP significantly, whereas IL-1 beta mainly increases the IL-6 effect when applied in combination. Our results furthermore show that AP-1 and C/EBP beta are transcription factors involved in the activation of the LBP gene, as revealed by Luciferase reporter gene analysis and electromobility shift assays. Elucidating the mechanism of transcriptional activation of LBP potentially may help in understanding host-pathogen response patterns and mechanisms involved in the acute phase reaction and in the pathophysiology of sepsis.

Acute-Phase Proteins↗
PubMed2001

Cholecystokinin gene transcription: promoter elements, transcription factors and signaling pathways.

Cholecystokinin (CCK) is a neuropeptide expressed in the small intestine and in the central and peripheral nervous system. CCK gene expression is both spatially and temporally regulated. In neurons CCK production is increased by growth factors, cyclic adenosine 3', 5'-monophosphate (cAMP), dopamine, estrogen, and injury situations, while intestinal CCK expression is mainly regulated by food intake. The function of the proximal CCK promoter has been examined by transfection of human CCK-CAT reporter constructs in cultured cells, DNase I footprinting and gel shift assays. These studies have led to the identification of regulatory elements and transcription factors important for basal and stimulated gene expression and depicted the signaling pathways involved in growth factor and cAMP induced CCK transcription. The review outlines the current knowledge of the regulation of CCK transcription and describes the role of putative transcription factors in tissue-specific CCK gene expression.

Animals↗
PubMed1997

Transcriptional analysis of mga, a regulatory gene in Streptococcus pyogenes: identification of monocistronic and bicistronic transcripts that phase vary.

Transcription of several surface virulence proteins of Streptococcus pyogenes is regulated by Mga, a protein that shows homology to response regulators of two-component signal-transducing systems. Two of these surface virulence proteins, M protein and C5a peptidase, undergo phase variation. To determine whether Mga itself undergoes phase variation and might allow the phasing switch to coordinate the activity of these genes, expression of the mga gene was analyzed. We show for the first time that there are two mga-specific transcripts: a 3.8-kb bicistronic message that includes both mga and emm12 genes and a monocistronic 1.6-kb mga message. Both transcripts phase vary and are present in higher amounts in M+ variants than in M- variants. Incubation of RNA with rifampicin indicates that the smaller 1.6-kb message is not a processed product. Two promoters were mapped upstream of mga: P1 at position 666 (-395) and P2 at position 978 (-83). In strain CS46 (delta mga), transcription initiation from the P1 promoter does not occur, and multiple start sites are found around the P2 promoter. Complementation experiments indicate that sequences upstream of the P2 promoter are required for activation of emm12 and scpA by Mga in trans.

Amino Acid Sequence↗
PubMed2001

A common functional C-T substitution polymorphism in the promoter region of the human catalase gene influences transcription factor binding, reporter gene transcription and is correlated to blood catalase levels.

Oxidative stress is implicated in disease and aging. In order to obtain molecular genetic tools that can be used to determine the potential impact of oxidative stress we examined the human catalase gene promoter for possible variation. Genomic DNA isolated from 10 individuals was screened for polymorphisms in the 5'-flanking region by direct sequence analysis of PCR products (nt -307 to -46 from the transcription start site). A common C/T polymorphism -262 base pairs from the transcription start site was detected. Computer analysis indicated that the two variants bound different transcription factors. Indeed, gel retardation analysis revealed different protein binding patterns to the two variants. Expression studies with reporter constructs showed significantly higher transcriptional activity of the T variant in HepG2 and K562 cells (1.5-fold,p <.05 Wilcoxon test). Thus a higher expression in human liver and blood cells is possible. In order to test this hypothesis, catalase levels in red blood cells were determined in 29 donors. The corresponding genotype was determined with a restriction enzyme-based assay. It was found that catalase levels were significantly higher in donors carrying the T allele in comparison to donors homozygous for the C allele (p <.03). In conclusion, we report here the first common (allele frequency in a Swedish population, 28%) genetic variant in a fundamental oxidative stress protection gene with a defined phenotype.

Alleles↗
PubMedDate not supplied

DNA methylation and chromatin structure affect transcriptional and post-transcriptional transgene silencing in Arabidopsis.

In plants, transgenes can be silenced at both the transcriptional [1] and post-transcriptional levels [2]. Methylation of the transgene promoter correlates with transcriptional gene silencing (TGS) [3] whereas methylation of the coding sequence is associated with post-transcriptional gene silencing (PTGS) [4]. In animals, TGS requires methylation and changes in chromatin conformation [5]. The involvement of methylation during PTGS in plants is unclear and organisms with non-methylated genomes such as Caenorhabditis elegans or Drosophila can display RNA interference (RNAi), a silencing process mechanistically related to PTGS [6]. Here, we crossed Arabidopsis mutants impaired in a SWI2/SNF2 chromatin component (ddm1 [7]) or in the major DNA methyltransferase (met1 [8] and E. Richards, personal communication) with transgenic lines in which a reporter consisting of the cauliflower mosaic virus 35S promoter fused to the beta-glucuronidase (GUS) gene (35S-GUS) was silenced by TGS or PTGS. We observed an efficient release of 35S-GUS TGS by both the ddm1 and met1 mutations and stochastic release of 35S-GUS PTGS by these two mutations during development. These results show that DNA methylation and chromatin structure are common regulators of TGS and PTGS.

Animals↗
PubMed1999

Conversion of HPV 18 positive non-tumorigenic HeLa-fibroblast hybrids to invasive growth involves loss of TNF-alpha mediated repression of viral transcription and modification of the AP-1 transcription complex.

AP-1 represents a transcription factor, which plays a pivotal role in initiating and maintaining the expression of human papillomavirus (HPV) oncoproteins E6 and E7 during HPV-linked carcinogenesis of the uterine cervix. AP-1 stands as a synonym for different proteins such as c-Jun, JunB, JunD, c-Fos, FosB as well as the Fos-related antigens Fra-1 and Fra-2, which can either homo- or heterodimerize to build up a functional transcription complex. AP-1 is mainly considered as a positive regulator, which binds to cognate DNA sequences within the viral upstream regulatory region. By using non-tumorigenic HeLa-fibroblast hybrids ('444'), their tumorigenic segregants ('CGL3') as well as HPV 18 positive HeLa cells as a experimental model system, evidence is provided that AP-1 composition differs considerably between these cell lines. In nuclear extracts obtained from non-tumorigenic cells, Jun-family members (in the order c-Jun>JunD>JunB) were mainly heterodimerized with Fra-1, a protein, known to be involved in the abrogation of AP-1 activity under certain experimental conditions. In contrast, Fra-1 concentration is low in extracts from tumorigenic cells. Conversely, c-Fos, the canonical dimerization partner of Jun proteins is expressed in substantial quantity in HeLa- and 'CGL3' cells, but it is completely absent in AP-1 complexes from non-tumorigenic '444' cells. Ectopical expression of c-fos under a heterologous promoter in '444'-cells induces tumorigenicity and a change of the Jun/Fra-1 ratio towards a constellation initially detected in 'CGL3'-and HeLa cells. Furthermore, conversion to tumorigenicity is accompanied with a resistance against TNF-alpha, a cytokine, capable to selectively suppress HPV 18 transcription in formerly non-malignant cells. These data propose a novel role for AP-1 as an essential component of an inter- and intracellular surveillance mechanism negatively controlling HPV transcription in non-tumorigenic cells.

Blotting, Western↗
PubMed1996

Transcriptional regulation of the rat fatty acid synthase gene: identification and functional analysis of positive and negative effectors of basal transcription.

The gene for fatty acid synthase (FAS), which contains both GC-rich sequences and a TATA box in its promoter region, is expressed in a tissue-specific manner in response to developmental, nutritional and hormonal signals. Here we report the identification of sequence elements in the 5'-flanking region responsible for modulation of basal promoter activity. Transient transfection of H4IIE hepatoma cells and 3T3-30A5 preadipocytes with plasmids containing the chloroamphenicol acetyltransferase gene driven by FAS promoter sequences of different lengths revealed that two regions between nucleotides -249 and -30 contain elements capable of enhancing transcription. One of these positive regulatory elements was localized to nucleotides -241/-236 using DNase I footprinting, electrophoretic mobility-shift assays and mutagenesis. The sequence element is a typical GC box and the nuclear protein binding to this region appears immunochemically indistinguishable from Sp1. The second positive regulatory element, an inverted CCAAT box, was localized to nucleotides -98/-92 by electrophoretic mobility-shift assays and mutagenesis. A putative negative regulatory element, initially identified by reporter gene transfection experiments, was localized between nucleotides -319 and -301 by DNase I footprinting, electrophoretic mobility-shift assays and deletion mutagenesis; this region consists of 78% G residues. In conclusion, initiation of FAS transcription from a single start site is enhanced by the presence of an adjacent TATA motif, an inverted CCAAT box and an upstream binding site for the transcription factor Sp1; further modulation of transcription is achieved through complex interactions between these promoter elements and an upstream negative regulatory element.

Animals↗
PubMed1997

Transcriptional and post-transcriptional mechanisms can regulate cell-specific expression of the human Pi-class glutathione S-transferase gene.

Previous studies from this laboratory have identified transcriptional mechanisms that are utilized to increase expression of the human glutathione S-transferase gene GSTP1 in a multidrug-resistant derivative (VCREMS) of the human mammary carcinoma cell line MCF7 [Moffat, McLaren and Wolf (1994) J. Biol. Chem. 269, 16397-16402]. The data presented here provide strong evidence that post-transcriptional mechanisms can also play an important role in determining cell-specific expression of the GSTP1 gene. GSTP1 mRNA levels were shown to be elevated 3.1-fold in the human bladder carcinoma cell line EJ compared with VCREMS cells. Despite this observation, transient transfection assays revealed a decreased rate of GSTP1 promoter activity in EJ cells. Indeed, GSTP1 transcriptional repressor activity, mediated by a region located between nucleotides -105 and -86 (as we have previously described in MCF7 cells), was observed in EJ cells. However, in contrast with our results in MCF7 cells, the EJ repressor activity did not displace the essential nuclear complex bound to the C1 promoter element (-73 to -54) in vitro. In addition, competition experiments indicated that an AP-1-like protein is an integral component of the C1-bound complex in EJ cells. Interestingly, experiments utilizing actinomycin D to inhibit transcription demonstrated significantly greater stability of GSTP1 mRNA in EJ cells than in VCREMS cells. These findings suggest that cell-specific differences in the rates of GSTP1 mRNA decay provide the predominant mechanism responsible for elevated expression of the GSTP1 gene in EJ cells.

Base Sequence↗
PubMed1998

Regulation of transcription initiation at the Escherichia coli nir operon promoter: a new mechanism to account for co-dependence on two transcription factors.

Expression from the Escherichia coli nir promoter is co-dependent on Fnr (a transcription factor triggered by oxygen starvation) and on NarL or NarP (transcription factors triggered by nitrite and nitrate ions). Fnr binds to a single DNA site centred between basepairs 41 and 42 upstream from the nir transcript start, whereas NarL and NarP bind to a site upstream, centred between basepairs 69 and 70. A novel mechanism to account for co-dependence on Fnr and NarL/NarP is suggested from experiments in which the spacing between the DNA sites for Fnr and NarL/NarP was altered. DNA sequence elements located upstream of the NarL/NarP-binding site are the targets for two or more proteins that act to repress Fnr-dependent activation of the nir promoter. This inhibition is counteracted by NarL or NarP. The model has been corroborated by the effects of several deletions and single base substitutions in the nir promoter upstream sequences: these deletions and substitutions prevent the binding of the repressor proteins. One of these repressors has been identified as the Fis protein, that binds to a site located 135-149bp upstream of the nir transcript start: the binding of Fis is suppressed by a single base substitution at position -146. The other repressor protein(s) have yet to be identified, but appear to bind downstream of the DNA site for Fis: binding is suppressed by a single base substitution at position -99.

Bacterial Proteins↗
PubMed2002

Basic transcription element binding protein is a thyroid hormone-regulated transcription factor expressed during metamorphosis in Xenopus laevis.

Basic transcription element binding protein (BTEB) is a member of the Krüppel family of zinc finger transcription factors. It has been shown that BTEB plays a role in promoting neuronal process formation during postembryonic development. In the present study, the biochemical properties, transactivation function, and the developmental and hormone-regulated expression of BTEB in Xenopus laevis (xBTEB) are described. xBTEB binds the GC-rich basic transcription element (BTE) with high affinity and functions as a transcriptional activator on promoters containing multiple or single GC boxes. xBTEB mRNA levels increase in the tadpole brain, intestine and tail during metamorphosis, and are correlated with tissue-specific morphological and biochemical transformations. xBTEB mRNA expression can be induced precociously in premetamorphic tadpole tissues by treatment with thyroid hormone. In situ hybridization histochemistry showed that thyroid hormone upregulates xBTEB mRNA throughout the brain of premetamorphic tadpoles, with the highest expression found in the subventricular zones of the telencephalon, diencephalon, optic tectum, cerebellum and spinal cord. xBTEB protein parallels changes in its mRNA, and it was found that xBTEB is not expressed in mitotic cells in the developing brain, but is expressed just distal to the proliferative zone, supporting the hypothesis that this protein plays a role in neural cell differentiation.

Animals↗
PubMed2001

CREB/CBP and SRE-interacting transcriptional regulators are fast on-off switches: duration of calcium transients specifies the magnitude of transcriptional responses.

Transient increases in the intracellular calcium concentration, which are associated with electrical activation of neurones, control synapse-to-nucleus communication. Calcium signals differ in time and space but it is unclear exactly how this translates into stimulus-specific gene expression. Analysis of transcription induced by calcium transients with defined durations revealed that the evoked genomic responses, unlike those following neurotrophin exposure, are not all-or-none but graded events. The CRE-binding protein CREB, its coactivator CREB-binding protein (CBP), and SRE-interacting transcriptional regulators are fast on-off switches: their activities are induced by short-lasting calcium signals, remain active for the duration of the signal and are rapidly shut-off after calcium concentrations have returned to basal levels. CREB is switched on by a fast, nuclear calmodulin (CaM) kinase-dependent mechanism that mediates CREB phosphorylation on serine 133 within 30 s of calcium entry. The second calcium-activated route to CREB involves the MAP kinase/extracellular signal-regulated kinase (ERK1/2) cascade. This pathway can be triggered by brief, 30-60 s calcium transients. ERK1/2 activity peaks several minutes after calcium entry and can outlast the calcium transient. The shut-off of CREB and ERK1/2 involves rapid dephosphorylation of their activator sites. These properties of transcription factors and their regulating kinases and phosphatases provide a mechanism through which the duration of calcium signals specifies the magnitude of the transcriptional response. The decoding of temporal features of calcium transients is likely to contribute to impulse-specific gene expression.

Animals↗
PubMed2000

Pleiotropic action of aldosterone in epithelia mediated by transcription and post-transcription mechanisms.

The aldosterone-induced increase in sodium reabsorption across tight epithelia can be divided schematically into two functional phases: an early regulatory phase starting after a lag period of 20 to 60 minutes, during which the pre-existing transport machinery is activated, and a late phase (>2.5 h), which can be viewed as an anabolic action leading to a further amplification/differentiation of the Na+ transport machinery. At the transcriptional level, both early and late responses are initiated during the lag period, but the functional impact of newly synthesized regulatory proteins is faster than that of the structural ones. K-Ras2 and SGK were identified as the first early aldosterone-induced regulatory proteins in A6 epithelia. Their mRNAs also were shown to be regulated in vivo by aldosterone, and their expression (constitutively active K-Ras2 and wild-type SGK) was shown to increase the function of ENaC coexpressed in Xenopus oocytes. Recently, aldosterone was also shown to act on transcription factors in A6 epithelia: It down-regulates the mRNAs of the proliferation-promoting c-Myc, c-Jun, and c-Fos by a post-transcriptional mechanism, whereas it up-regulates that of Fra-2 (c-Fos antagonist) at the transcriptional level. Together, these new data illustrate the complexity of the regulatory network controlled by aldosterone and support the view that its early action is mediated by the induction of key regulatory proteins such as K-Ras2 and SGK. These early induced proteins are sites of convergence for different regulatory inputs, and thus, their aldosterone-regulated expression level tunes the impact of other regulatory cascades on sodium transport. This suggests mechanisms for the escape from aldosterone action.

Aldosterone↗
PubMed2002

RNA polymerase II-dependent transcription in trypanosomes is associated with a SNAP complex-like transcription factor.

Spliced leader RNA transcription is essential for cell viability in trypanosomes. The SL RNA genes are expressed from the only defined RNA polymerase II-dependent promoter identified to date in the trypanosome genome. The SL RNA gene promoter has been shown by in vitro and in vivo analyses to have a tripartite architecture. The upstream most cis-acting element, called PBP-1E, is located between 70 and 60 bp upstream from the transcription start site. This essential element functions along with two downstream elements to direct efficient and proper initiation of transcription. Electrophoretic mobility-shift studies detected a 122-kDa protein, called PBP-1, which interacts with PBP-1E. This protein is the first sequence-specific, double-stranded DNA-binding protein isolated in trypanosomes. Three polypeptides copurify with PBP-1 activity, suggesting that PBP-1 is composed of 57-, 46-, and 36-kDa subunits. We have cloned the genes that encode the 57- and 46-kDa subunits. The 46-kDa protein is a previously uncharacterized protein and may be unique to trypanosomes. Its predicted tertiary structure suggests it binds DNA as part of a complex. The 57-kDa subunit is orthologous to the human small nuclear RNA-activating protein (SNAP)50, which is an essential subunit of the SNAP complex (SNAPc). In human cells, SNAPc binds to the proximal sequence element in both RNA polymerase II- and III-dependent small nuclear RNA gene promoters. These findings identify a surprising link in the transcriptional machinery across a large evolutionary distance in the regulation of small nuclear RNA genes in eukaryotes.

Amino Acid Sequence↗