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PubMed1990

Selective inhibition of activated but not basal transcription by the acidic activation domain of VP16: evidence for transcriptional adaptors.

The interaction between the chimeric activator GAL4-VP16, consisting of the DNA binding domain of GAL4 and the acidic activation domain of VP16, and its target in the transcriptional machinery was studied in vitro. GAL4-VP16 stimulated transcription from a promoter bearing GAL4 sites, and greatly inhibited transcription from a promoter bearing binding sites for the dA:dT activator and from a basal promoter bearing only a TATA box. Mutations in the acidic domain that reduced activation from the GAL4 site promoter also reduced inhibition from the dA:dT promoter, indicating a similar interaction between VP16 and its target in both processes. Strikingly, if the DNA binding domain of GAL4-VP16 was occupied by a GAL4 site oligonucleotide, the protein inhibited activation by the dA:dT activator but did not inhibit basal transcription. We propose that, under these conditions, GAL4-VP16 acted to titrate an "adaptor" that bridges an interaction between the upstream activator and the basic transcriptional machinery at the TATA box.

Binding Sites↗
PubMed2001

Crystal structure of negative cofactor 2 recognizing the TBP-DNA transcription complex.

The X-ray structure of a ternary complex of Negative Cofactor 2 (NC2), the TATA box binding protein (TBP), and DNA has been determined at 2.6 A resolution. The N termini of NC2 alpha and beta resemble histones H2A and H2B, respectively, and form a heterodimer that binds to the bent DNA double helix on the underside of the preformed TBP-DNA complex via electrostatic interactions. NC2beta contributes to inhibition of TATA-dependent transcription through interactions of its C-terminal alpha helix with a conserved hydrophobic feature on the upper surface of TBP, which in turn positions the penultimate alpha helix of NC2beta to block recognition of the TBP-DNA complex by transcription factor IIB. Further regulatory implications of the NC2 heterodimer structure are discussed.

Amino Acid Sequence↗
PubMed1993

Interaction between the tobacco DNA-binding activity CBF and the cyt-1 promoter element of the Agrobacterium tumefaciens T-DNA gene T-CYT correlates with cyt-1 directed gene expression in multiple tobacco tissue types.

A novel DNA-binding activity, designated CBF, has been identified in nuclear extracts from tobacco leaf, stem and root tissue. CBF interacts specifically with a 30 bp promoter fragment, referred to as cyt-1, of the Agrobacterium tumefaciens T-DNA cytokinin (T-cyt) gene. The T-cyt promoter, although of bacterial origin is active in planta and the 30 bp cyt-1 element is located within a region that is essential for T-cyt promotor activity in leaf, stem and root cells of tobacco plants. Gel retardation assays using different synthetic oligonucleotides and methylation interference experiments pinpointed the binding site of CBF to a GC-rich sequence ATGCCCCACA within the cyt-1 element. Site-directed mutagenesis of the CBF binding site within the T-cyt promoter by using PCR resulted in an almost complete loss of T-cyt promoter activity in transgenic tobacco plants. In a gain-of-function experiment a hexamer of cyt-1 was shown to be able to confer leaf, stem and root expression when fused upstream of a TATA box containing -55 derivative of the T-cyt promoter. A mutant cyt-1 hexamer, defective in CBF binding, did not show activity above background levels. These results indicate that binding of CBF to the cyt-1 element is required for cyt-1 directed gene expression, suggesting that CBF might act as a transcriptional activator. Apart from the ASF-1 binding site of the CaMV 35S promoter, which is also present in the T-DNA nopaline and octopine synthase genes, the cyt-1 element is the only other identified element reported until now that in combination with a TATA box is sufficient to drive gene expression in multiple tobacco tissue types.

Agrobacterium tumefaciens↗
PubMed1996

The Arabidopsis thaliana ACT4/ACT12 actin gene subclass is strongly expressed throughout pollen development.

Plants contain complex actin gene families composed of several diverse and ancient subclasses of genes. One Arabidopsis actin gene subclass represented by the ACT4 and ACT12 genes has been isolated and characterized. Both actin genes have typical plant actin gene structures, including three small introns interrupting the coding region and an intron within the mRNA leader. Their encoded proteins differ from each other in only one amino acid, whereas they differ in 3-10% of their amino acids from the other five Arabidopsis actin subclasses. They also share a few small blocks of DNA sequence homology in the 5' flanking region near their TATA boxs, but not in their introns, 3' flanking regions, or degenerate positions within codons. Southern analysis with gene-specific probes from 5' flanking sequences showed that both were single copy genes in the genome. Both RNA gel blot analysis with 3' gene-specific probes and reverse transcriptase-mediated polymerase chain reactions (RT-PCR) with gene-specific primers detected low levels of ACT4 and ACT12 mRNAs in flowers and very high levels in pollen. The RT-PCR detected very low levels of these mRNAs in the vegetative organs. The 5' region from both genes, including the promoter region, TATA box, the sequence for the mRNA leader and its intron, and the first 19 actin codons, was fused to a beta-glucuronidase (GUS) reporter gene. Expression of the GUS fusions were examined histochemically in 40 independent transgenic Arabidopsis plants. Expression of the ACT4/GUS fusion was restricted to young vascular tissues, tapetum, and developing and mature pollen. Similar expression patterns in these tissues and cell types were observed for ACT12/GUS fusion, yet unlike ACT4, ACT12 was also strongly expressed in the root cap and in a ring of pericycle tissues during lateral root initiation and early development. The unique expression patterns of the ACT4/ACT12 actin gene subclass are discussed in light of recent data on the other expressed members of the Arabidopsis actin gene family.

Actins↗
PubMed1996

Early B cell factor interacts with a subset of kappa promoters.

A conserved sequence element situated between the decamer and TATA box in V kappa II and V kappa V promoters has a high homology to the binding site for early B cell factor (EBF). The kappa promoter element was shown to bind EBF specifically using both in vitro-translated protein and nuclear extract. Concomitant binding of EBF and Oct proteins to a wild-type kappa promoter template was observed at low efficiency, and such dual occupancy was dependent on an intact amino terminus of the Oct protein. When the two binding sites were separated by a 10-bp spacer, this dependency disappeared. A single kappa promoter EBF site together with a TATA box and an Ig heavy chain enhancer showed marginal transcriptional stimulatory activity. In contrast, the EBF site acted synergistically with a decamer element in EBF-negative plasmacytoma cells, but not in B cells of an earlier differentiation stage. In these cells, a distinct protein was observed that interacted with the EBF binding motif, while overexpression of EBF down-regulated the expression of a reporter construct containing Ig control elements.

Animals↗
PubMed1988

Cell-specific activity of a GGTCA half-palindromic oestrogen-responsive element in the chicken ovalbumin gene promoter.

Using a transient co-transfection system we have identified a functional oestrogen responsive element (ERE) in the near vicinity of the TATA box of the chicken ovalbumin gene promoter. Oestrogen induced activation of transcription was observed only in chicken embryo fibroblast cells, and not in human HeLa cells, suggesting the existence of a cell-specific transcription factor(s) that is necessary for the hormonal response. By 5'-deletion mapping and site-directed mutagenesis, a sequence encompassing the more proximal of two GGTCA half-palindromic motifs was identified as a functional ovalbumin gene ERE. Both motifs correspond to oestrogen receptor binding sites in vitro as revealed by DNase I footprinting. Optimal response to the hormonal stimulus apparently requires stereoalignment of the ERE and the TATA box.

Animals↗
PubMed1992

Identification and transcriptional analyses of the UL3 and UL4 genes of equine herpesvirus 1, homologs of the ICP27 and glycoprotein K genes of herpes simplex virus.

The DNA sequence of 3,240 nucleotides of the XbaI G fragment located in the unique long (UL) region of the equine herpesvirus 1 genome revealed two major open reading frames (ORFs) designated UL3 and UL4. The UL3 ORF of 470 amino acids (aa) maps at nucleotides (nt) 4450 to 3038 from the long terminus, and its predicted 51.4-kDa protein product exhibits significant homology to the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1; 32% identity) and to the ORF4 protein of varicella-zoster virus (13% identity). Interestingly, a zinc finger motif is conserved in the C-terminal domains of both ICP27 of HSV-1 (aa 483 to 508) and UL3 of equine herpesvirus 1 (aa 441 to 466). The UL4 ORF of 343 aa maps at nt 5618 to 4587 and could encode a protein of 38.1 kDa which exhibits significant homology to the UL53 protein (cell fusion protein or glycoprotein K) of HSV-1 (26% identity) and to the ORF5 protein of varicella-zoster virus (33% identity). Analyses of the UL4 amino acid sequence revealed domains characteristic of a membrane-bound glycoprotein and included potential signature sequences for (i) a signal sequence, (ii) two N-linked glycosylation sites, and (iii) four transmembrane domains. Nucleotide sequence analyses also revealed potential TATA boxes located upstream of the UL3 and UL4 ORFs. However, only a single polyadenylation signal (nt 2988 to 2983) was detected downstream of the UL3 ORF. Northern (RNA) blot hybridization and S1 nuclease analyses were used to map and characterize the UL3 and UL4 mRNAs. Metabolic inhibitors were used to identify the kinetic class of these two genes. The data revealed that UL3 is an early gene that encodes a 1.6-kb mRNA, while UL4 is a late gene encoding a 3.8-kb mRNA that overlaps the UL3 transcript. Both transcripts were shown by S1 nuclease analyses to initiate 24 to 26 nt downstream of their respective TATA boxes and to have a common transcription termination signal as a pair of 3'-coterminal mRNAs.

Amino Acid Sequence↗
PubMed1993

Eukaryotic activators function during multiple steps of preinitiation complex assembly.

Eukaryotic activator proteins (activators) stimulate transcription by increasing assembly of the preinitiation complex. We have developed methods to quantify the stable assembly of general transcription factors into transcriptional complexes in response to activators. We show that activators function during at least two stages of preinitiation complex assembly: first, to recruit the general transcription factor TFIIB, and then at a second step, after TFIIB entry. It is at this second step that the TATA-box binding protein associated factors act. This step also seems to be critical for activators to stimulate transcription synergistically.

Cell Nucleus↗
PubMed2003

Characterization of the S-RNase promoters from sweet cherry (Prunus avium L.).

Genomic DNA fragments containing the S(3)-, S(4)-, and S(6)-RNase genes were isolated from the sweet cherry (Prunus avium L.) and sequenced. Comparison of the 5'-flanking sequences of these three S-RNases indicated that a highly conserved region (designated CR) existed just upstream from the putative TATA boxes. We postulate that CR contains cis-regulatory element(s) involved in pistil expression. To examine the activity of the isolated S-RNase promoters of sweet cherry in the pistil, we transiently introduced approximately 650-bp fragments of the S(4)- and S(6)-RNase promoters fused to beta-glucuronidase (GUS) gene into the pistil of the petunia using a particle bombardment technique. Histochemical analysis showed that the 5'-flanking region of each S-RNase was active in the pistil. This suggests that cis-regulatory element(s) for pistil-specific expression may exist(s) within the 650-bp region upstream from the TATA box in the sweet cherry S-RNase promoter.

5' Flanking Region↗
PubMed2000

Influence of HMG-1 and adenovirus oncoprotein E1A on early stages of transcriptional preinitiation complex assembly.

The TATA-binding protein (TBP) in the TFIID complex binds specifically to the TATA-box to initiate the stepwise assembly of the preinitiation complex (PIC) for RNA polymerase II transcription. Transcriptional activators and repressors compete with general transcription factors at each step to influence the course of the assembly. To investigate this process, the TBP.TATA complex was titrated with HMG-1 and the interaction monitored by electrophoretic mobility shift assays. The titration produced a ternary HMG-1.TBP. TATA complex, which exhibits increased mobility relative to the TBP. TATA complex. The addition of increasing levels of TFIIB to this complex results in the formation of the TFIIB.TBP.TATA complex. However, in the reverse titration, with very high mole ratios of HMG-1 present, TFIIB is not dissociated off and a complex is formed that contains all factors. The simultaneous addition of E1A to a mixture of TBP and TATA; or HMG-1, TBP, and TATA; or TFIIB, TBP, and TATA inhibits complex formation. On the other hand, E1A added to the pre-established complexes shows a significantly reduced capability to disrupt the complex. In add-back experiments with all complexes, increased levels of TBP re-established the complexes, indicating that the primary target for E1A in all complexes is TBP.

Adenovirus E1A Proteins↗
PubMed1989

The function of plant heat shock promoter elements in the regulated expression of chimaeric genes in transgenic tobacco.

A series of deletion mutants of a soybean heat shock (hs) gene promoter was generated and linked to the chloramphenicol acetyl transferase (CAT) coding sequence. These chimaeric promoter/reporter gene constructs were introduced into tobacco and thermoregulated expression of CAT activity was examined in leaf extracts. Three different types of gene fusions were tested using two different BIN19 vector constructions: (1) translational fusion between the N-terminus of the protein coding sequence of the heat shock gene Gmhsp17.3-B and CAT; (2) transcriptional fusions between the 5' nontranslated RNA regions of Gmhsp17.3-B and CAT; and (3) promoter fusions joining the hs promoter upstream sequences to the TATA box sequence of the delta CaMV 35S-CATter vector. Alternatively, multiple copies of a synthetic deoxyoligonucleotide with the soybean hs consensus element (HSE2) were used. Heat inducible CAT activities were detected except in plants containing a transcriptional fusion devoid of all but 18 nucleotides at the 5' terminus of the hs gene transcript. CAT activity was detectable in these plants only during the recovery at 25 degrees C after a hs (40 degrees C). Overlapping HSE-like promoter sequences seem to be necessary for the induction of heat inducible transcription of linked genes; synthetic HSE2 sequences have the capacity to reconstitute a hs promoter in combination with a TATA box sequence. Effective translation during hs seems to require sequences in the 5' non-translated leader of the hs protein mRNA; these sequences can be functionally replaced by the 5' leader sequence of the delta CaMV 35S promoter.

Amino Acid Sequence↗
PubMed1988

Activation of the adenovirus EIIa late promoter by a single-point mutation which enhances binding of transcription factor IID.

We have converted the TACAAA sequence present at position -29 of the adenovirus EIIa late promoter into a canonical TATA box by oligonucleotide-directed mutagenesis. When linear templates were analyzed in nuclear extracts, transcription of the mutant promoter showed a 10-fold higher level of activity than that of the wild-type promoter. This increase was correlated with an increased affinity of the mutant promoter for transcription initiation factor IID. Further analyses demonstrated that the activating functions of three EIIa late upstream promoter elements (Huang, D.-H., and Roeder, R.G. (1988) Mol. Cell. Biol. 8, 1906-1914) were maintained in the mutant promoter background. These observations indicated, first, that the upstream elements did not act merely to overcome a rate-limiting initiation step imposed by an inefficient TATA element and, second, that the strength of the interaction between transcription initiation factor IID and the TATA box was directly related to promoter activity.

Adenoviridae↗
PubMed2000

Cloning and characterization of human estrogen receptor beta promoter.

Estrogen receptors beta (ERbeta) belong to the nuclear receptor superfamily of ligand-dependent transcription factors that play critical roles in regulating genes involved in a wide array of biological processes. To investigate regulation of tissue-specific expression of ERbeta, we cloned and characterized a 2.1-kilobase 5'-flanking region of the human ERbeta gene. Two major transcription start sites were identified by primer extension and rapid amplification of 5'-cDNA end. The human ERbeta proximal promoter contains both TATA box and initiator element (Inr) and is GC-rich with a GC content of 65%. An Alu repeat sequence containing an ER-dependent transcription enhancer exists between -1416 and -1703. The full-length 5'-flanking sequence of ERbeta fused to a luciferase reporter exhibited functional promoter activity in ERbeta-positive TSUPr1 cell, but not in ERbeta-negative DU145 cells. In addition, DNase I protection assays of the proximal promoter showed unique protection patterns with nuclear extracts from TSUPr1 cells and ERbeta negative HeLa cells, suggesting presence of cell-specific trans-acting factors that mediate tissue/cell-specific ERbeta expression. Serial deletion analysis revealed that a 293-bp region encompassing the TATA box and Inr element possesses basal promoter activity.

Base Sequence↗
PubMed1998

Analysis of a cis-Acting Element Involved in Regulation by Estrogen of Human Angiotensinogen Gene Expression.

The work was aimed to identify the estrogen responsive element in the human angiotensinogen gene. The nucleotide sequence between the transcription initiation site and TATA box in angiotensinogen gene promoter was found to be strongly homologous with the consensus estrogen responsive element. This sequence was confirmed as the estrogen responsive element (HAG ERE) by electrophoretic mobility shift assay. The recombinant expression vectors were constructed in which chloramphenicol acetyltransferase (CAT) reporter gene was driven by angiotensinogen core promoter with HAG ERE of by TK core promoter with multiplied HAG ERE, and were used in cotransfection with the human estrogen receptor expression vector into HepG(2) cells; CAT assays showed an increase of the CAT activity on 17beta-estradiol treatment in those transfectants. These results suggest that the human angiotensinogen gene is transcriptionally up-regulated by estrogen through the estrogen responsive element near TATA box of the promoter.

Journal Article↗
PubMed2003

Regulation of the intronic promoter of rat estrogen receptor alpha gene, responsible for truncated estrogen receptor product-1 expression.

We have characterized the intronic promoter of the rat estrogen receptor (ER) alpha gene, responsible for the lactotrope-specific truncated ER product (TERP)-1 isoform expression. Transcriptional regulation was investigated by transient transfections using 5'-deletion constructs. TERP promoter constructs were highly active in MMQ cells, a pure lactotrope cell line, whereas a low basal activity was detected in alphaT3-1 gonadotrope cells or in COS-7 monkey kidney cells. Serial deletion analysis revealed that 1) a minimal -693-bp region encompassing the TATA box is sufficient to allow lactotrope-specific expression; 2) the promoter contains strong positive cis-acting elements both in the distal and proximal regions, and 3) the region spanning the -1698/-1194 region includes repressor elements. Transient transfection studies, EMSAs, and gel shifts demonstrated that estrogen activates the TERP promoter via an estrogen-responsive element (ERE1) located within the proximal region. Mutation of ERE1 site completely abolishes the estradiol-dependent transcription, indicating that ERE1 site is sufficient to confer estrogen responsiveness to TERP promoter. In addition, ERalpha action was synergized by transfection of the pituitary-specific factor Pit-1. EMSAs showed that a single Pit-1 DNA binding element in the vicinity of the TATA box is sufficient to confer response by the TERP promoter. In conclusion, we demonstrated, for the first time, that TERP promoter regulation involves ERE and Pit-1 cis-elements and corresponding trans-acting factors, which could play a role in the physiological changes that occur in TERP-1 transcription in lactotrope cells.

5' Flanking Region↗
PubMed1987

OVEC, a versatile system to study transcription in mammalian cells and cell-free extracts.

We have developed a vector, OVEC ("oligonucleotide vector") to study DNA sequences involved in the regulation of transcription in mammalian cells. This vector is equally suitable for studying expression in vivo after transfection into cells, or for transcription studies in vitro with cell-free extracts. Putative cis-acting DNA segments from enhancers or promoters can be inserted at a position immediately upstream of the TATA box and coding sequence of the rabbit beta-globin gene. A regulatory DNA segment can be tested by itself or in conjunction with an enhancer located either in an adjacent upstream position, or downstream of the beta-globin gene. S1 nuclease mapping can be used to study transcription from circular and linear templates and run-off transcription in vitro is also feasible. Transcripts from a reference globin gene with a small deletion around the transcription initiation site can be measured with the same S1 nuclease probe and thus serve as an internal standard. We demonstrate the usefulness of OVEC by inserting either short oligonucleotides comprising a metal responsive enhancer element, or the SV40 enhancer, directly upstream of the TATA box. Both constructs yield high levels of correctly initiated transcripts in a transient expression assay in HeLa cells. In a HeLa cell nuclear extract the SV40 enhancer stimulates transcription 40-fold.

Animals↗
PubMed2002

Suppression of promoter activity of the LAT gene by IE180 of pseudorabies virus.

The latency-associated transcript (LAT) gene is the only viral genomic region that is abundantly transcribed during pseudorabies virus (PrV) latent infection. The mechanism of reactivation of PrV from latency remains unknown. To analyze the regulation mechanism of the LAT promoter, we constructed a series of recombinant vectors in which various sequences upstream of LAT were linked to the chloramphenicol acetyltransferase (CAT) gene. Transcriptional efficiency was examined by cotransfection with plasmids carrying the PrV IE, EP0, or gD gene, respectively. Results showed that the activity of PrV LAT promoter was dramatically repressed by the IE180 protein and a TATA box and a putative IE180 binding site within the promoter were involved in this repression. To dissect the functional domains of IE180, we compared the relative repressive abilities of IE180 variants to the LAT promoter by transient transfection assays. Mutational analysis demonstrated that almost the whole IE180 (amino acid residues 1-1440) are essential for its repression to LAT promoter. To explore the possible mechanism of repression, an electrophoretic mobility shift assay (EMSA) using nuclear extracts from neuronal cells was performed and formation of protein-DNA complexes between IE180 and the oligonucleotide probe (-46 to -19, relative to the start site of LAT transcription) was demonstrated. The association of IE180 with the region encompassing the putative IE180 binding site and the TATA box upstream of PrV LAT gene was further confirmed by supershift of EMSA complexes using IE180 specific antibody. Thus, our results suggested that IE180 repressed the LAT promoter via an interaction between IE180, LAT promoter and cellular protein(s).

Animals↗
PubMed1993

Characterization of the transcriptional regulatory region of the human WT1 gene.

Specific control of the expression of the Wilms tumor gene WT1 is important for normal development of the kidney. In order to characterise the transcriptional control region of the WT1 gene we have isolated genomic clones spanning the upstream region, the first WT1 exon and the 5' end of the Wit1 gene. DNA sequencing revealed that the WT1 promoter lacks a TATA box or CCAAT motif and has a GC content of 71%. Four transcriptional start sites are clustered within a 32 bp region. GC-boxes are present at nucleotide positions -413, -160, +84 and +158. DNAase I protection assays with purified Sp1 protein revealed the existence of 11 different binding sites in the WT1 promoter. WT1 and Wit1 promoter activities were tested in COS-7 cells with luciferase reporter gene constructs either containing or lacking an SV40 enhancer. WT1 promoter activity was found in a fragment extending from 449 bp upstream to 201 bp downstream of the WT1 start site. It was 26 fold lower in the absence of the SV40 enhancer than in the presence. Cotransfection with a Sp1 expression vector stimulated both constructs 3-4 fold. Wit1 promoter activity was identified in a DNA fragment extending from 200 bp upstream of the putative Wit1 TATA box to 130 bp downstream. Several potential recognition sites for WT1/EGR, Pax-8, and GAGA-like transcription factors are present in the WT1 promoter.

Base Sequence↗