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At least 1,351 records · Page 75Linked to original sources
PubMed2001

Specific Pax-6/microphthalmia transcription factor interactions involve their DNA-binding domains and inhibit transcriptional properties of both proteins.

Pax-6 and microphthalmia transcription factor (Mitf) are required for proper eye development. Pax-6, expressed in both the neuroretina and pigmented retina, has two DNA-binding domains: the paired domain and the homeodomain. Mice homozygous for Pax-6 mutations are anophthalmic. Mitf, a basic helix-loop-helix leucine zipper (b-HLH-LZ) transcription factor associated with the onset and maintenance of pigmentation, identifies the retinal pigmented epithelium during eye development. Loss of Mitf function results in the formation of an ectopic neuroretina at the expense of the dorsal retinal pigmented epithelium. In the present study, we investigated the interaction between Pax-6 and Mitf. In transient transfection-expression experiments, we found that transactivating effects of Pax-6 and Mitf on their respective target promoters were strongly inhibited by co-transfection of both transcription factors. This repression was due to direct protein/protein interactions involving both Pax-6 DNA-binding domains and the Mitf b-HLH-LZ domain. These results suggest that Pax-6/Mitf interactions may be critical for retinal pigmented epithelium development.

Animals↗
PubMed1996

pX, the HBV-encoded coactivator, interacts with components of the transcription machinery and stimulates transcription in a TAF-independent manner.

The X protein of hepatitis B virus (HBV) coactivates activators bearing potent (mostly acidic) activation domains. Here, we investigated the molecular mechanisms of this coactivation. We show that pX interacts with general transcription factors TFIIB and TFIIH, as well as with the potent activation domain of VP16. TFIIB interacts with both pX and VP16 simultaneously. In addition, the RNA polymerase II enzyme itself binds to pX. By reducing the activity of cellular coactivators, through squelching, we intensify the dependence of the activator on pX-mediated coactivation. Squelching is essentially diminished in the presence of pX, both in vivo and in vitro. The target of pX in this activity is the template-bound activator, and not the squelcher. Furthermore, by following transcription in a TAF-deprived reaction, we demonstrate absolute dependence of the activator on the activity of pX. We propose that pX coactivates transcription by substituting cellular coactivators in activator-preinitiation complex interactions.

Fungal Proteins↗
PubMed2006

Transcription regulation of the alpha-glucanase gene agn1 by cell separation transcription factor Ace2p in fission yeast.

During the final stage of the cell division cycle in the fission yeast Schizosaccharomyces pombe, transcription factor Ace2p activates expression of genes involved in the separation of newly formed daughter cells, such as agn1+, which encodes the alpha-glucanase Agn1p. The agn1 promoter contains three copies of the nucleotide sequence motif CCAGCC, whose presence seems to correlate with Ace2p-mediated transcription activation. Here, we describe a simple plate-based assay utilizing as a reporter the secreted glucoamylase of Arxula adeninivorans to investigate the function of this motif. We show that not all three repeats, but only the two most proximal to the transcription start point, act as an upstream activating sequence (UAS). Finally, we demonstrate that this UAS is essential for agn1 promoter activity in vivo.

Base Sequence↗
PubMed2002

Transcriptional activation of the human stress-inducible transcriptional repressor ATF3 gene promoter by p53.

Activating transcription factor 3 (ATF3) is an immediate early response gene that is induced in cells exposed to a variety of stress stimuli. In this report, upon exposure of cells to ultraviolet (UV) or proteasome inhibitor MG132, ATF3 protein was induced more efficiently in cells with intact p53 allele than in those with null mutant p53 allele. In Saos-2 cells harboring the temperature-sensitive mutant p53(Val-138), the expression of ATF3 gene was more significant at permissive temperature of 32.5 degrees C than at non-permissive 37.5 degrees C. Reporter assay of the human ATF3 gene promoter identified two p53-responsive elements at -379 to -370 and -351 to -342 from the transcriptional start site. These elements were capable of conferring p53 responsiveness to a heterologous promoter and specifically bound p53 protein in electrophoretic mobility shift assay. Furthermore, ATF3 gene promoter was more significantly activated by UV in cells with wild p53 allele. These results clearly show that the human ATF3 gene is one of the target genes directly activated by p53 and may suggest a functional link between stress-inducible transcriptional repressor ATF3 and p53.

Activating Transcription Factor 3↗
PubMed2001

Spot 14 protein interacts and co-operates with chicken ovalbumin upstream promoter-transcription factor 1 in the transcription of the L-type pyruvate kinase gene through a specificity protein 1 (Sp1) binding site.

In hepatocytes, the amount of the Spot 14 (S14) protein is closely related to the full expression of enzymes involved in the glycolytic and lipogenic pathways. In the present study we address the role played by this protein in the control of transcription of the L-type pyruvate kinase (L-PK) gene in primary hepatocytes. We show that human S14, which by itself does not bind to the L-PK promoter, physically interacts with the human chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) and induces the switch of this factor from a repressor to an activator. However, the enhancing activity of S14 and COUP-TF1 depends on the presence of a proximal GC-rich box (the L0 element) that specifically binds nuclear proteins from the livers of rats fed a glucose-rich diet. Moreover, the L0 element, which strongly binds dephosphorylated specificity protein 1 (Sp1), loses all affinity when this factor is phosphorylated by cAMP-dependent protein kinase. Mutations that affect binding of Sp1 and nuclear proteins to the L0 box also decrease basal transcription and impair glucose responsiveness of the promoter. These results therefore shed light on the mechanism by which the S14 protein, whose concentration rapidly rises after glucose intake, contributes to the full activity of the L-PK promoter.

Animals↗
PubMed2006

Transcriptional stimulation of the human NHE3 promoter activity by PMA: PKC independence and involvement of the transcription factor EGR-1.

NHE3 (Na+/H+ exchanger 3) is essential for Na+ absorption in the ileum and is expressed in a cell-specific manner in the apical membrane of the intestinal epithelial cells. In the present study, we report the stimulatory effect of PMA on the hNHE3 (human NHE3) transcription. Pretreatment with actinomycin D or cycloheximide blocked the up-regulation of the NHE3 mRNA by PMA, indicating that the increased level of NHE3 mRNA expression is regulated by transcriptional activation and is dependent on de novo protein synthesis. 5'-Deletion of the promoter region and transfection analysis in C2BBe1 cells revealed that the PMA effect is mediated through a GC-rich DNA region between nt -88 and -69. Gel mobility-shift assays demonstrated that in nuclear extracts from C2BBe1 cells grown under the basal growth conditions, Sp1 (stimulating protein-1) and Sp3 interact with this GC-rich DNA region, while, in PMA-treated nuclear extracts, PMA-induced EGR-1 (early growth response gene product 1) transcription factor binds to the same site. Binding of EGR-1 diminished the Sp1 and Sp3 interactions with this promoter region significantly. Co-transfection of Sp1 or Sp3 into SL2 cells activated the NHE3-reporter constructs, suggesting that Sp1 and Sp3 act as positive regulators of the NHE3 expression. In addition, overexpression of EGR-1 was sufficient to transactivate the NHE3-reporter gene activity, and knockdown of EGR-1 with gene-specific small interfering RNA resulted in inhibition of the PMA-induced up-regulation of the endogenous NHE3 mRNA expression. Furthermore, the PKC (protein kinase C) inhibitor chelerythrine chloride did not affect PMA-induced NHE3 promoter activity, suggesting that PMA stimulation of the hNHE3 gene expression may be PKC-independent.

Amino Acid Motifs↗
PubMed2000

Regulation of mouse delta-opioid receptor gene transcription: involvement of the transcription factors AP-1 and AP-2.

The aim of this study was to examine the effects of the phorbol ester O-tetradecanoylphorbol 13-acetate (TPA) and forskolin on delta-opioid receptor gene transcription. Treatment of NG108-15 cells with TPA (100 nM) for 48 h increased delta-opioid receptor mRNA levels, whereas different concentrations of forskolin induced a transient down-regulation of mRNA 5 h after treatment, followed by increased mRNA levels after 48 h. Reporter gene assays in transiently transfected NG108-15 cells in combination with electrophoretic mobility shift assays indicate that the increase of delta-opioid receptor mRNA after stimulation with TPA is mediated by transcription factor AP-1, which binds 355 bp upstream of the start codon within the gene promoter. The forskolin-induced mRNA increase is mediated neither by a cyclic AMP-response element nor indirectly by AP-1 up-regulation. Reporter gene assays, mutational analysis, and electrophoretic mobility shift assays revealed that delta-opioid receptor gene regulation by forskolin is mediated by transcription factor AP-2, which binds to an element 157 bp upstream of the start codon.

1-Methyl-3-isobutylxanthine↗
PubMed2001

The transcription factor E2F1 promotes dopamine-evoked neuronal apoptosis by a mechanism independent of transcriptional activation.

The E2F1 transcription factor plays an important role in promoting neuronal apoptosis; however, it is not clear how E2F1 does this. Here we show that E2F1 is involved in dopamine (DA)-evoked apoptosis in cerebellar granule neurons (CGNs). E2F1 -/- CGNs and CGNs expressing an antisense E2F1 cDNA were significantly protected from DA-toxicity relative to controls. The neuronal protection was accompanied by significantly reduced caspase 3 activity. E2F1-mediated neuronal apoptosis did not require activation of gene transcription because: (1) ectopic expression of E2F1 or its mutants lacking the transactivation domain induced neuronal apoptosis, whereas an E2F1 mutant lacking the DNA-binding domain did not; (2) under all of these conditions, known E2F1 target genes including cyclin A, cdc2 and p19(ARF) were not induced; and (3) DA-evoked neuronal apoptosis was associated with up-regulated E2F1, but not transcription of its target genes. Finally, E2F1-mediated neuronal apoptosis was associated with reduced nuclear factor (NF)-kappaB DNA-binding activity. Taken together, these data suggest that E2F1 promotes DA-evoked caspase 3-dependent neuronal apoptosis by a mechanism independent of gene transactivation, and this may possibly occur through inhibition of anti-apoptotic genes including NF-kappaB.

Animals↗
PubMed2004

A dominant negative mutant of microphthalmia transcription factor (MITF) lacking two transactivation domains suppresses transcription mediated by wild type MITF and a hyperactive MITF derivative.

Microphthalmia transcription factor (MITF) positively regulates transcription of differentiation-related genes in several cell lineages, including melanocytes. Recent data also indicate a new important role for MITF as a factor that appears to be required for survival of melanoma cells, suggesting a possibility that abrogation of MITF function in transformed melanocytes could lead to a decreased survival via attenuating anti-apoptotic signals. Therefore, to gain a better understanding of the role which MITF plays in melanoma cell survival, it is important to find efficient means of abolishing the transactivation of its target genes. Recently, a dominant negative MITF lacking the N-terminus has been shown to down-regulate tyrosinase and Trp1 expression in normal melanocytes and mouse B16 melanoma cells. Here, a dominant negative mutant of the melanocyte-specific isoform of MITF is described carrying deletions of both N- and C-terminal transactivation domains. Cotransfection of this mutant resulted in a complete inhibition of the wild type MITF function as tested on both the reporter-linked tyrosinase promoter and an endogenous, ectopic MITF-triggered tyrosinase gene in U2-OS cells. The dominant negative construct also strongly repressed the activity of a hyperactive MITF-Vp16 chimera. Importantly, deletion of both activation domains was necessary to eliminate the residual transcription activity observed when only the N-terminal domain was removed and to achieve the repressive effect in human melanoma cells. If the activity of MITF plays a role in the long-term survival of malignant melanocytes, overexpression of a strong dominant negative MITF mutant might be a useful strategy to suppress its transactivation function.

Animals↗
PubMed1995

Dual transcriptional control by Ear3/COUP: negative regulation through the DR1 direct repeat and positive regulation through a sequence downstream of the transcriptional start site of the mouse mammary tumor virus promoter.

Ear3/COUP is an orphan member of the steroid/thyroid hormone receptor superfamily of transcription factors and binds most tightly to a direct repeat of AGGTCA with 1 nucleotide in between (DR1). Ear3/COUP also binds with a similar affinity to the palindromic thyroid hormone response element (TRE). This binding preference of Ear3/COUP is same as that of the retinoid X receptor (RXR), which is another member of the superfamily. In the present study, we identified a sequence responsible for Ear3/COUP-mediated transactivation in the region downstream of the transcription start site of the mouse mammary tumor virus promoter. This cis-acting sequence was unresponsive to RXR. When the DR1 or TRE sequence was added upstream of the promoter, transactivation by Ear3/COUP was completely abolished, whereas RXR enhanced transcription from the promoter. The mode of action of Ear3/COUP could be utilized to control complex gene expressions in morphogenesis, homeostasis, and development.

Animals↗
PubMed1995

Characterization of a HeLa cell factor which negatively regulates transcriptional activation in vitro by transcriptional enhancer factor-1 (TEF-1).

A novel negatively acting factor has been identified and partially purified from HeLa and BJA-B cell extracts by chromatographic fractionation. Addition of this factor to HeLa cell extracts or to a reconstituted HeLa cell transcription system repressed transcriptional activation by a chimeric activator, GAL-TEF-1, containing the activation function of transcriptional enhancer factor-1 (TEF-1). In contrast, this factor did not repress transactivation by the chimeric GAL-VP16 activator. Repression of transactivation by GAL-TEF-1 could be alleviated by the addition of immunopurified HeLa cell TFIID, but not by increased quantities of GAL-TEF-1. These observations suggest that this negatively acting factor represses transactivation by interfering with the function of, or competing for, the TATA-binding protein-associated coactivators which mediate the activity TEF-1.

Cell Line↗
PubMed1997

Functional analysis of microphthalmia-associated transcription factor in pigment cell-specific transcription of the human tyrosinase family genes.

Tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2 are the enzymes involved in melanin biosynthesis and are preferentially expressed in pigment cells. Their human gene promoters share the 11-base pair M box containing a CATGTG motif, which was shown here to be bound in vitro by microphthalmia-associated transcription factor (MITF). Transient cotransfection analysis showed that MITF overexpression increased the expression of a reporter gene under the control of the human tyrosinase or TRP-1 gene promoter but not the TRP-2 promoter. The promoter activation caused by MITF is dependent on each CATGTG motif of the distal enhancer element, the M box, and the initiator E box of the tyrosinase gene and the TRP-1 M box. Furthermore, a truncated MITF lacking the carboxyl-terminal 125 amino acid residues transactivated the tyrosinase promoter less efficiently than did MITF, suggesting that MITF's carboxyl terminus contains a transcriptional activation domain, but unexpectedly such a truncated MITF remarkably transactivated the TRP-2 gene promoter. These results suggest that MITF is sufficient to direct pigment cell-specific transcription of the tyrosinase and TRP-1 genes but not the TRP-2 gene.

Base Sequence↗
PubMed1997

Synergy between interferon-gamma and tumor necrosis factor-alpha in transcriptional activation is mediated by cooperation between signal transducer and activator of transcription 1 and nuclear factor kappaB.

Interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) cooperate to induce the expression of many gene products during inflammation. The present report demonstrates that a portion of this cooperativity is mediated by synergism between two distinct transcription factors: signal transducer and activator of transcription 1 (STAT1) and nuclear factor kappaB (NF-kappaB). IFNgamma and TNFalpha synergistically induce expression of mRNAs encoding interferon regulatory factor-1 (IRF-1), intercellular adhesion molecule-1, Mig (monokine induced by gamma-interferon), and RANTES (regulated on activation normal T cell expressed and secreted) in normal but not STAT1-deficient mouse fibroblasts, indicating a requirement for STAT1. Transient transfection assays in fibroblasts using site-directed mutants of a 1.3-kilobase pair sequence of the IRF-1 gene promoter revealed that the synergy was dependent upon two sequence elements; a STAT binding element and a kappaB motif. Artificial constructs containing a single copy of both a STAT binding element and a kappaB motif linked to the herpes virus thymidine kinase promoter were able to mediate synergistic response to IFNgamma and TNFalpha; such response varied with both the relative spacing and the specific sequence of the regions between these two sites. Cooperatively responsive sequence constructs bound both STAT1alpha and NF-kappaB in nuclear extracts prepared from IFNgamma- and/or TNFalpha-stimulated fibroblasts, although binding of individual factors was not cooperative. Thus, the frequently observed synergy between IFNgamma and TNFalpha in promoting inflammatory response depends in part upon cooperation between STAT1alpha and NF-kappaB, which is most likely mediated by their independent interaction with one or more components of the basal transcription complex.

3T3 Cells↗
PubMed1997

Functional analysis of surfactant protein B (SP-B) promoter. Sp1, Sp3, TTF-1, and HNF-3alpha transcription factors are necessary for lung cell-specific activation of SP-B gene transcription.

Surfactant protein B (SP-B) is essential for maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B mRNA expression is restricted to alveolar type II epithelial cells and bronchiolar epithelial cells (Clara cells) of adult lung. We previously (Margana, R. K., and Boggaram, V. (1996) Am. J. Physiol. 270, L601-L612) found that a minimal promoter region (-236 to +39) of rabbit SP-B gene is sufficient for high level expression of chloramphenicol acetyltransferase reporter gene in NCI-H441 cells, a cell line with characteristics of Clara cells. In the present study we used mutational analysis, electrophoretic mobility shift assays, and DNase I footprinting to identify cis-DNA regulatory elements and trans-acting protein factors required for lung cell-specific expression of SP-B gene. We found that in addition to thyroid transcription factor 1 (TTF-1) and hepatocyte nuclear factor 3alpha (HNF-3alpha) binding sites, two spatially separate DNA sequences that bind Sp1 and Sp3 factors are necessary for the maintenance of SP-B promoter activity. Mutation of any one of the transcription factor binding sites caused a significant reduction in SP-B promoter activity suggesting that Sp1, Sp3, and TTF-1 and HNF-3alpha interact cooperatively with SP-B promoter to activate gene transcription.

Animals↗
PubMed1999

The adenovirus oncoprotein E1a stimulates binding of transcription factor ETF to transcriptionally activate the p53 gene.

Expression of the tumor suppressor protein p53 plays an important role in regulating the cellular response to DNA damage. During adenovirus infection, levels of p53 protein also increase. It has been shown that this increase is due not only to increased stability of the p53 protein but to the transcriptional activation of the p53 gene during infection. We demonstrate here that the E1a proteins of adenovirus are responsible for activating the mouse p53 gene and that both major E1a proteins, 243R and 289R, are required for complete activation. E1a brings about the binding of two cellular transcription factors to the mouse p53 promoter. One of these, ETF, binds to three upstream sites in the p53 promoter and one downstream site, whereas E2F binds to one upstream site in the presence of E1a. Our studies indicate that E2F binding is not essential for activation of the p53 promoter but that ETF is. Our data indicate the ETF site located downstream of the start site of transcription is the key site in conferring E1a responsiveness on the p53 promoter.

Adenovirus E1A Proteins↗
PubMed2000

Transcription factor ZBP-89 cooperates with histone acetyltransferase p300 during butyrate activation of p21waf1 transcription in human cells.

Inducible p53-independent regulation of the cyclin-dependent kinase inhibitor p21(waf1) transcription is mediated through proximal GC-rich sites. Prior studies have shown that Sp1, Sp3, and the histone acetylase co-activator p300 are components of the complexes binding to these sites. Although Sp1 and Sp3 collaborate with p300, a direct interaction between Sp1 and p300 does not occur. This study sought to determine whether ZBP-89 rather than Sp1 is the direct target of p300 during butyrate induction of p21(waf1). ZBP-89 (BFCOL1, BERF-1, ZNF 148) is a Krüppel-type zinc finger transcription factor that binds to GC-rich elements and represses or activates known target genes. Adenoviral-mediated expression of ZBP-89 in HT-29 cells revealed that ZBP-89 potentiates butyrate induction of endogenous p21(waf1) gene expression. Further, cotransfection of a ZBP-89 expression vector with a 2.3-kilobase p21(waf1) reporter recapitulated the potentiation by butyrate. DNase I footprinting analysis of the human p21(waf1) promoter with recombinant ZBP-89 identified a binding site at -245 to -215. Electrophoretic mobility shift assays confirmed that both recombinant and endogenous ZBP-89 and Sp1 bind to this element. The potentiation was abolished in the presence of adenoviral protein E1A. Deletion of the N-terminal domain of ZBP-89 abolished the potentiation mediated by butyrate treatment. This same deletion mutant abolished the ZBP-89 interaction with p300. Cotransfection of p300 with ZBP-89 stimulated the p21(waf1) promoter in the absence of butyrate. p300 co-precipitated with ZBP-89 but not with Sp1, whereas ZBP-89 co-precipitated with Sp1. Together, these findings demonstrate that ZBP-89 also plays a critical role in butyrate activation of the p21(waf1) promoter and reveals preferential cooperation of this four-zinc finger transcription factor with p300.

Acetyltransferases↗
PubMed2000

Transcriptional regulation of the human acid alpha-glucosidase gene. Identification of a repressor element and its transcription factors Hes-1 and YY1.

Acid alpha-glucosidase, the product of a housekeeping gene, is a lysosomal enzyme that degrades glycogen. A deficiency of this enzyme is responsible for a recessively inherited myopathy and cardiomyopathy, glycogenesis type II. We have previously demonstrated that the human acid alpha-glucosidase gene expression is regulated by a silencer within intron 1, which is located in the 5'-untranslated region. In this study, we have used deletion analysis, electrophoretic mobility shift assay, and footprint analysis to further localize the silencer to a 25-base pair element. The repressive effect on the TK promoter was about 50% in both orientations in expression plasmid, and two transcriptional factors were identified with antibodies binding specifically to the element. Mutagenesis and functional analyses of the element demonstrated that the mammalian homologue 1 of Drosophila hairy and Enhancer of split (Hes-1) binding to an E box (CACGCG) and global transcription factor-YY1 binding to its core site function as a transcriptional repressor. Furthermore, the overexpression of Hes-1 significantly enhanced the repressive effect of the silencer element. The data should be helpful in understanding the expression and regulation of the human acid alpha-glucosidase gene as well as other lysosomal enzyme genes.

Base Sequence↗
PubMed2001

An IgH enhancer that drives transcription through basic helix-loop-helix and Oct transcription factor binding motifs. Functional analysis of the E(mu)3' enhancer of the catfish.

The transcriptional enhancer (E(mu)3') of the IgH locus of the channel catfish, Ictalurus punctatus, shows strong B cell-specific activity and differs from the mammalian E(mu) enhancer in both location and structure. It occurs between the mu and delta genes and contains numerous transcription factor binding sites, predominantly octamer and muE5 motifs of consensus and variant sequences. It lacks the classical muA-muE3(CBF)-muB core array of binding motifs seen within mammalian IgH E(mu) enhancers. To determine the functionally important motifs, a series of mutant enhancers was created using sequence-targeted polymerase chain reaction. Whereas the mutation of consensus and variant octamer motifs (individually or in multiples) decreased enhancer function, mutation of a single consensus muE5 motif destroyed the function of this enhancer in mammalian plasmacytomas. Mutation of this consensus muE5 site, combined with mutations of certain octamer sites, destroyed function in catfish B cells. Experiments using artificial enhancers containing multimers of motifs or short regions of the native enhancer suggested that the minimal E(mu)3' enhancer (a) contains a consensus muE5 site and two octamer sites, (b) is B cell-specific, and (c) is active across species. The dependence of an Ig enhancer on sites that bind basic helix-loop-helix and Oct transcription factors has not previously been observed and confirms large differences in structure and function between fish and mammalian IgH enhancers.

Amino Acid Motifs↗