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At least 1,279 records · Page 71Linked to original sources
PubMed2001

GC box-binding transcription factors control the neuronal specific transcription of the cyclin-dependent kinase 5 regulator p35.

Cyclin-dependent kinase 5 (cdk5)/p35 kinase activity is highest in post-mitotic neurons of the central nervous system and is critical for development and function of the brain. The neuronal specific activity of the cdk5/p35 kinase is achieved through the regulated expression of p35 mRNA. We have identified a small 200-bp fragment of the p35 promoter that is sufficient for high levels of neuronal specific expression. Mutational analysis of this TATA-less promoter has identified a 17-bp GC-rich element, present twice, that is both required for promoter activity and sufficient for neuronal specific transcription. A GC box within the 17-bp element is critical for both promoter activity and protein-DNA complex formation. The related transcription factors Sp1, Sp3, and Sp4 constitute most of the GC box DNA binding activity in neurons. We have found that both the relative contribution of the Sp family proteins to GC box binding and the transcriptional activity of these proteins is regulated during neuronal differentiation. Thus, our data show that the GC box-binding Sp proteins contribute to the regulation of p35 expression in neurons, suggesting changes in the Sp transcription factors level and activity may contribute to cell type-specific expression of many genes in the central nervous system.

Animals↗
PubMed2002

Induction of c-Myc expression suppresses insulin gene transcription by inhibiting NeuroD/BETA2-mediated transcriptional activation.

Insulin biosynthesis and secretion are critical for pancreatic beta-cell function, but both are impaired under diabetic conditions. We have found that hyperglycemia induces the expression of the basic helix-loop-helix transcription factor c-Myc in islets in several different diabetic models. To examine the possible implication of c-Myc in beta-cell dysfunction, c-Myc was overexpressed in isolated rat islets using adenovirus. Adenovirus-mediated c-Myc overexpression suppressed both insulin gene transcription and glucose-stimulated insulin secretion. Insulin protein content, determined by immunostaining, was markedly decreased in c-Myc-overexpressing cells. In gel-shift assays c-Myc bound to the E-box in the insulin gene promoter region. Furthermore, in betaTC1, MIN6, and HIT-T15 cells and primary rat islets, wild type insulin gene promoter activity was dramatically decreased by c-Myc overexpression, whereas the activity of an E-box mutated insulin promoter was not affected. In HeLa and HepG2 cells c-Myc exerted a suppressive effect on the insulin promoter activity only in the presence of NeuroD/BETA2 but not PDX-1. Both c-Myc and NeuroD can bind the E-box element in the insulin promoter, but unlike NeuroD, the c-Myc transactivation domain lacked the ability to activate insulin gene expression. Additionally p300, a co-activator of NeuroD, did not function as a co-activator of c-Myc. In conclusion, increased expression of c-Myc in beta-cells suppresses the insulin gene transcription by inhibiting NeuroD-mediated transcriptional activation. This mechanism may explain some of the beta-cell dysfunction found in diabetes.

Adenoviridae↗
PubMed2004

Transcriptional repressor DREAM interacts with thyroid transcription factor-1 and regulates thyroglobulin gene expression.

Tissue-specific gene expression depends on the interaction between tissue-specific and general transcription factors. DREAM is a Ca2+-dependent transcriptional repressor widely expressed in the brain where it participates in nociception through its control of prodynorphin gene expression. In the periphery, DREAM is highly expressed in the thyroid gland, the immune system, and the reproductive organs. Here, we show that DREAM interacts with thyroid-specific transcription factor TTF-1 and regulates the expression of the thyroglobulin (Tg) gene. The mechanism also involves binding of DREAM to the thyroglobulin promoter and blockage of TTF-1-mediated transactivation. The TSH/cAMP pathway and Ca2+ signaling regulate DREAM-mediated transcriptional repression of the thyroglobulin gene. Furthermore, chromatin immunoprecipitation experiments in FRTL-5 cells confirmed that Tg is a bona fide target gene for DREAM transrepression in thyroid follicular cells.

Animals↗
PubMed2005

Drosophila mitochondrial transcription factor B1 modulates mitochondrial translation but not transcription or DNA copy number in Schneider cells.

We report the cloning and molecular analysis of Drosophila mitochondrial transcription factor (d-mtTF) B1. An RNA interference (RNAi) construct was designed that reduces expression of d-mtTFB1 to 5% of its normal level in Schneider cells. In striking contrast with our previous study on d-mtTFB2, we found that RNAi knock-down of d-mtTFB1 does not change the abundance of specific mitochondrial RNA transcripts, nor does it affect the copy number of mitochondrial DNA. In a corollary manner, overexpression of d-mtTFB1 did not increase either the abundance of mitochondrial RNA transcripts or mitochondrial DNA copy number. Our data suggest that, unlike d-mtTFB2, d-mtTFB1 does not have a critical role in either transcription or regulation of the copy number of mitochondrial DNA. Instead, because we found that RNAi knockdown of d-mtTFB1 reduces mitochondrial protein synthesis, we propose that it serves its primary role in modulating translation. Our work represents the first study to document the role of mtTFB1 in vivo and establishes clearly functional differences between mtTFB1 and mtTFB2.

Amino Acid Sequence↗
PubMed2005

Analysis of RovA, a transcriptional regulator of Yersinia pseudotuberculosis virulence that acts through antirepression and direct transcriptional activation.

The transcription factor RovA of Yersinia pseudotuberculosis and analogous proteins in other Enterobacteriaceae activate the expression of virulence genes that play a crucial role in stress adaptation and pathogenesis. In this study, we demonstrate that the RovA protein forms dimers independent of DNA binding, stimulates RNA polymerase, most likely via its C-terminal domain, and counteracts transcriptional repression by the histone-like protein H-NS. As the molecular function of the RovA family is largely uncharacterized, random mutagenesis and terminal deletions were used to identify functionally important domains. Our analysis showed that a winged-helix motif in the center of the molecule is essential and directly involved in DNA binding. Terminal deletions and amino acid changes within both termini also abrogate RovA activation and DNA-binding functions, most likely due to their implication in dimer formation. Finally, we show that the last four amino acids of RovA are crucial for activation of gene transcription. Successive deletions of these residues result in a continuous loss of RovA activity. Their removal reduced the capacity of RovA to activate RNA polymerase and abolished transcription of RovA-activated promoters in the presence of H-NS, although dimerization and DNA binding functions were retained. Our structural model implies that the final amino acids of RovA play a role in protein-protein interactions, adjusting RovA activity.

Bacterial Proteins↗
PubMed2006

Leptin transcriptionally enhances peptide transporter (hPepT1) expression and activity via the cAMP-response element-binding protein and Cdx2 transcription factors.

PepT1 is an intestinal epithelial apical membrane transporter that is expressed in the small intestine, with little or no expression in the normal colon. However, we previously demonstrated that colonic PepT1 may be expressed during chronic inflammation. To begin elucidating inflammatory hPepT1 signaling, we herein investigated the long term leptin treatments, on PepT1 expression and activity in Caco2-BBE cells, and began to reveal the involved signaling pathways. We successfully cloned the 723-bp hPepT1 promoter region and identified the human transcription initiation site 86 bp upstream from the translation start site. Leptin treatment dose- and time-dependently increased hPepT1 promoter and transport activities in Caco2-BBE cells, with maximal activity observed in cells treated with 100 nM leptin for 8 h. Under these conditions, we observed 2-fold increases in hPepT1 mRNA and protein expression, as well as increased transport activity. Our molecular analyses of possible signal-transduction pathways revealed that leptin treatment enhanced the intracellular levels of cAMP and phosphorylated cAMP-response element-binding protein (CREB) protein in Caco2-BBE cells, whereas our deletion, mutation, and CDX2 overexpression analyses demonstrated that interaction of the Cdx2 and phosphorylated CREB transcription factors was essential for leptin-induced hPepT1 transcription in Caco2-BBE cells. Our results indicate that leptin, which is increased in inflamed colonic mucosa, triggers colonic expression of hPepT1 via the CREB and Cdx2 transcription factors. These findings provide important new insights into the mechanisms of intestinal inflammation and may suggest new therapeutic modalities in the future.

5' Flanking Region↗
PubMed1994

Interleukin 2 transcription factors as molecular targets of cAMP inhibition: delayed inhibition kinetics and combinatorial transcription roles.

Elevation of cAMP can cause gene-specific inhibition of interleukin 2 (IL-2) expression. To investigate the mechanism of this effect, we have combined electrophoretic mobility shift assays and in vivo genomic footprinting to assess both the availability of putative IL-2 transcription factors in forskolin-treated cells and the functional capacity of these factors to engage their sites in vivo. All observed effects of forskolin depended upon protein kinase A, for they were blocked by introduction of a dominant negative mutant subunit of protein kinase A. In the EL4.E1 cell line, we report specific inhibitory effects of cAMP elevation both on NF-kappa B/Rel family factors binding at -200 bp, and on a novel, biochemically distinct "TGGGC" factor binding at -225 bp with respect to the IL-2 transcriptional start site. Neither NF-AT nor AP-1 binding activities are detectably inhibited in gel mobility shift assays. Elevation of cAMP inhibits NF-kappa B activity with delayed kinetics in association with a delayed inhibition of IL-2 RNA accumulation. Activation of cells in the presence of forskolin prevents the maintenance of stable protein-DNA interactions in vivo, not only at the NF-kappa B and TGGGC sites of the IL-2 enhancer, but also at the NF-AT, AP-1, and other sites. This result, and similar results in cyclosporin A-treated cells, imply that individual IL-2 transcription factors cannot stably bind their target sequences in vivo without coengagement of all other distinct factors at neighboring sites. It is proposed that nonhierarchical, cooperative enhancement of binding is a structural basis of combinatorial transcription factor action at the IL-2 locus.

Base Sequence↗
PubMed1997

Three classes of mammalian transcription activation domain stimulate transcription in Schizosaccharomyces pombe.

Representatives of three distinct classes of mammalian protein domain activating RNA polymerase II were fused to the yeast GAL4p DNA-binding domain. The resulting fusion proteins were tested in the fission yeast Schizosaccharomyces pombe for their ability to activate transcription of different reporter constructs containing GAL4-binding sites in positions close to or far from the TATA box. The acidic-rich activation domain of VP16 stimulates transcription in S.pombe from proximal and distal positions, suggesting that the mechanism of activation is conserved from man to budding and fission yeasts. Unlike in Saccharomyces cerevisiae, the glutamine-rich activation domains of Sp1, Oct1 and Oct2 activate transcription in S. pombe when tested in a proximal TATA box context. Similarly to mammalian cells, these domains are inactive or weakly active when tested in a distal position. Moreover, the proline-rich activation domains of AP-2 and CTF/NF1 display strong transcriptional activities from a TATA box-proximal position, and weak activities when tested in a remote position. Consequently, proline-rich and glutamine-rich activation domains act differently in S.cerevisiae and mammalian cells, but similarly in S.pombe and mammalian cells.

Animals↗
PubMed2001

Transcription of chromosomal rRNA genes by both RNA polymerase I and II in yeast uaf30 mutants lacking the 30 kDa subunit of transcription factor UAF.

UAF, a yeast RNA polymerase I transcription factor, contains Rrn5p, Rrn9p, Rrn10p, histones H3 and H4, and uncharacterized protein p30. Mutants defective in RRN5, RRN9 or RRN10 are unable to transcribe rDNA by polymerase I and grow extremely slowly, but give rise to variants able to grow by transcribing chromosomal rDNA by polymerase II. Thus, UAF functions as both an activator of polymerase I and a silencer of polymerase II for rDNA transcription. We have now identified the gene for subunit p30. This gene, UAF30, is not essential for growth, but its deletion decreases the cellular growth rate. Remarkably, the deletion mutants use both polymerase I and II for rDNA transcription, indicating that the silencer function of UAF is impaired, even though rDNA transcription by polymerase I is still occurring. A UAF complex isolated from the uaf30 deletion mutant was found to retain the in vitro polymerase I activator function to a large extent. Thus, Uaf30p plays only a minor role in its activator function. Possible reasons for slow growth caused by uaf30 mutations are discussed.

Amino Acid Sequence↗
PubMed1989

Regulation of phage Mu repressor transcription by IHF depends on the level of the early transcription.

Integration Host Factor (IHF) of E. coli can stimulate both early and repressor transcription of bacteriophage Mu. We introduced several mutations in the early promoter (Pe) and studied the effect of these mutations on the stimulation of early and repressor transcription by IHF. All mutant promoters are still positive regulated by IHF, but the level of stimulation is dependent on the strength of the promoter. The strength of the early promoter has an even greater impact on the regulation of the repressor promoter by IHF: stimulation is observed in the presence of a relatively weak Pe, whereas with a strong Pe the repressor promoter Pc is inhibited by IHF. This inhibition is most probably due to an interference of the early transcription with the opposing repressor transcription. The implication of this type of regulation for the Mu life cycle is discussed.

Bacterial Proteins↗
PubMed1990

An archaebacterial cell-free transcription system. The expression of tRNA genes from Methanococcus vannielii is mediated by a transcription factor.

Our understanding of the mechanism of RNA biosynthesis in archaebacteria is limited, due in part to the inability of purified RNA polymerases to transcribe purified genes accurately in vitro. In the present study, we show that cell extracts of Methanococcus vannielii and Methanococcus thermolithotrophicus purified by gradient centrifugation synthesize a distinct transcript from templates harboring a cloned homologous tRNA(Val) and tRNA(Arg) gene. The in vitro transcripts initiate with GTP at the same sites as in Methanococcus cells. About 60% of the sequence of the in vitro RNA products was analyzed by dideoxyterminated primer extension and found to be identical with that of the precursors of tRNA(Val) and tRNA(Arg). This finding indicates that this RNA polymerase fraction both initiates and terminates transcription faithfully in vitro. After purification of a cell-free extract (S-100) of M. thermolithotrophicus by phosphocellulose chromatography, the endogenous RNA polymerase has lost its ability to transcribe the tRNA(Val) gene accurately. The activity directing specific expression of this template was reconstituted by the addition of a protein-fraction devoid of RNA polymerase activity. Thus, a transcription factor appears to be required for accurate cell-free expression of tRNA genes from M. vannielii.

Base Sequence↗
PubMed1991

USF-related transcription factor, HIV-TF1, stimulates transcription of human immunodeficiency virus-1.

The transcription factor HIV-TF1, which binds to a region about 60 bp upstream from the enhancer of the human immunodeficiency virus-1 (HIV-1), was purified from human B cells. HIV-TF1 had a molecular weight of 39,000. Binding of HIV-TF1 to the HIV long terminal repeat (LTR) activated transcription from the HIV promoter in vitro. The HIV-TF1-binding site in HIV LTR was similar to the site recognized by upstream stimulatory factor (USF) in the adenovirus major late promoter. DNA-binding properties of HIV-TF1 suggested that HIV-TF1 might be identical or related to USF. Interestingly, treatment of purified HIV-TF1 by phosphatase greatly reduced its DNA-binding activity, suggesting that phosphorylation of HIV-TF1 was essential for DNA binding. The disruption of HIV-TF1-binding site induced a 60% decrease in the level of transcription from the HIV promoter in vivo. These results suggest that HIV-TF1 is involved in transcriptional regulation of HIV-1.

B-Lymphocytes↗
PubMed1997

A multiplicity of mediators: alternative forms of transcription complexes communicate with transcriptional regulators.

The already complex process of transcription by RNA polymerase II has become even more complicated in the last few years with the identification of auxiliary factors in addition to the essential general initiation factors. In many cases these factors, which have been termed mediators or co-activators, are only required for activated or repressed transcription. In some cases the effects are specific for certain activators and repressors. Recently some of these auxiliary factors have been found in large complexes with either TBP, as TBP-associated factors (TAFs) in the general factor TFIID, or with pol II and a subset of the general factors, referred to as the 'holoenzyme'. Although the exact composition of these huge assemblies is still a matter of some debate, it is becoming clear that the complexes themselves come in more than one form. In particular, at least four forms of TFIID have been described, including one that contains a tissue-specific TAF and another with a cell type-specific form of TBP. In addition, in yeast there are at least two forms of the 'holoenzyme' distinguished by their mediator composition and by the spectrum of transcripts whose expression they affect. Genetic and biochemical analyses have begun to identify the interactions between the components of these complexes and the ever increasing family of DNA binding regulatory factors. These studies are complicated by the fact that individual regulatory factors often appear to have redundant interactions with multiple mediators. The existence of these different forms of transcription complexes defines a new target for regulation of subsets of eukaryotic genes.

Animals↗
PubMed1999

Orientation and positional mapping of the subunits of the multicomponent transcription factors RFX and X2BP to the major histocompatibility complex class II transcriptional enhancer.

Major histocompatibility complex class II genes contain a common complex enhancer that allows for their coordinate regulation. The X box element of the enhancer cooperatively binds the multisubunit transcription factors RFX and X2BP. RFX is an essential class II transcription factor and contains three distinct proteins: RFX5, RFX-B/Ank and RFXAP. X2BP, a CREB/ATF family transcription factor, most likely binds as a homodimer. A site-specific protein-DNA photocrosslinking assay was used to investigate the interactions of the subunits of RFX and X2BP with X box DNA. Two of the RFX subunits, RFX5 and RFX-B/Ank, were found to bind defined sites within the X1 half of the X box. The third RFX subunit, RFXAP, made extensive X1 box contacts. The subunits of X2BP made contacts with the edges of the X2 half of the X box in a manner consistent with other bZIP transcription factor contact patterns. The resulting map provides specific base pair contacts and subunit orientation with respect to the DNA sequence of the RFX-X2BP-X box complex. Our results suggest possible stoichiometry of the RFX subunits and potential interaction between RFX-B/Ank and RFXAP with one of the subunits of X2BP.

Base Sequence↗
PubMed2003

Import of mitochondrial transcription factor A (TFAM) into rat liver mitochondria stimulates transcription of mitochondrial DNA.

Mitochondrial transcription factor A (TFAM) has been shown to stimulate transcription from mitochondrial DNA promoters in vitro. In order to determine whether changes in TFAM levels also regulate RNA synthesis in situ, recombinant human precursor proteins were imported into the matrix of rat liver mitochondria. After uptake of wt-TFAM, incorporation of [alpha-32P]UTP into mitochondrial mRNAs as well as rRNAs was increased 2-fold (P < 0.05), whereas import of truncated TFAM lacking 25 amino acids at the C-terminus had no effect. Import of wt-TFAM into liver mitochondria from hypothyroid rats stimulated RNA synthesis up to 4-fold. We conclude that the rate of transcription is submaximal in freshly isolated rat liver mitochondria and that increasing intra-mitochondrial TFAM levels is sufficient for stimulation. The low transcription rate associated with the hypothyroid state observed in vivo as well as in organello seems to be a result of low TFAM levels, which can be recovered by treating animals with T3 in vivo or by importing TFAM in organello. Thus, this protein meets the criteria for being a key factor in regulating mitochondrial gene expression in vivo.

Animals↗
PubMed2006

Mutagenic properties of oxidized GTP and ATP in in vitro transcription-reverse transcription.

Oxidation of RNA precursors may disturb genetic information by mispair formations. In this study, effects of oxidized ribonucleoside triphosphates on in vitro transcription catalyzed by T7 RNA polymerase were examined. 8-Hydroxyguanosine 5'-triphosphate (8-OH-GTP) and 2-hydroxyadenosine 5'-triphosphate (2-OH-ATP) reduced amount of RNA. In addition, mRNA was converted to cDNA by reverse transcriptase, and the cDNA was then amplified by PCR. The PCR product was subsequently cloned into plasmid DNA and sequence of DNA was analyzed for each bacterial colony. The two oxidized ribonucleotides induced mutations in cDNA, suggesting disturbance of genetic information during transcription and/or reverse transcription. 8-OH-GTP induced T-->G plus T-->C mutations and 2-OH-ATP caused T-->C mutations. These results indicate that formation of these oxidized RNA precursors in cells affects transcription quantitatively and qualitatively. In addition, they are potential antiviral drugs that cause mutations in genomic RNA.

Adenosine Triphosphate↗
PubMed1997

Cell-type expression, immunolocalization, and deoxyribonucleic acid-binding activity of basic transcription element binding transcription factor, an Sp-related family member, in porcine endometrium of pregnancy.

Basic transcription element binding (BTEB) protein is a newly identified member of the C2H2 zinc finger family that also includes the transcription factors, Sp1, Sp2, Sp3, and Sp4. This family of proteins binds GC-rich motifs widely distributed in gene promoters, resulting in distinct activation or repression of transcriptional activities. Whereas Sp proteins are ubiquitously expressed, expression of BTEB appears more limited and has not been documented in the female reproductive tract of any mammalian species. This study was designed to identify and characterize the cellular distribution of BTEB in the porcine endometrium and placenta at known stages of pregnancy. Northern analysis of uterine endometrium detected BTEB mRNA that corresponds in size (5 kilobases) to that of the major BTEB transcript in rat brain. The steady-state levels of BTEB mRNA were higher (p < 0.05) in endometrium than placenta at corresponding days of pregnancy, although for each tissue, the levels did not change with pregnancy stage (p > 0.05). Luminal epithelial (LE), glandular epithelial (GE), and stromal (ST) cells isolated from pregnancy endometrium expressed the BTEB gene, but mRNA abundance varied with cell type (LE, GE > ST). Western blot analysis using an antiserum generated against the N-terminal region of a porcine BTEB fusion protein produced in Escherichia coli revealed the presence of BTEB protein only in endometrium, not in placenta. Immunohistochemical studies localized BTEB predominantly to the nuclei of endometrial GE and LE cells. Consistent with the presence of functional BTEB protein, binding to a double-stranded oligonucleotide containing multiple GC motifs was demonstrated in nuclear extracts prepared from endometrium and from endometrial LE and GE, but not ST, cells by electrophoretic mobility shift assay. These results demonstrate the preferential endometrial cell-type expression of BTEB and suggest its regulatory role in pregnancy-associated endometrial epithelial gene expression.

Animals↗
PubMed2005

Tsix transcription across the Xist gene alters chromatin conformation without affecting Xist transcription: implications for X-chromosome inactivation.

X-chromosome inactivation (XCI) is highly dynamic during early mouse embryogenesis and strictly depends on the Xist noncoding RNA. The regulation of Xist and its antisense partner Tsix remains however poorly understood. We provide here the first evidence of transcriptional control of Xist expression. We show that RNA polymerase II (RNAPolII) preinitiation complex recruitment and H3 Lys 4 (H3-K4) methylation at the Xist promoter form the basis of the Xist expression profiles that drives both imprinted and random XCI. In embryonic stem (ES) cells, which are derived from the inner cell mass where imprinted XCI is reversed and both Xs are active, we show that Xist is repressed at the level of preinitiation complex (PIC) recruitment. We further demonstrate that Tsix, although highly transcribed in ES cells, is not itself responsible for the transcriptional down-regulation of Xist. Rather, Tsix induces efficient H3-K4 methylation over the entire Xist/Tsix unit. We suggest that chromatin remodeling of the Xist locus induced by biallelic Tsix transcription renders both Xist loci epigenetically equivalent and equally competent for transcription. In this model, Tsix, by resetting the epigenetic state of the Xist/Tsix locus, mediates the transition from imprinted to random XCI.

Alleles↗