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PubMed1991

Sequences involved in brain-specific in vitro transcription from the core promoter of the mouse myelin basic protein gene.

In the previous study, we have shown that a short DNA stretch from -35 to -17 in the core promoter of the mouse myelin basic protein (MBP) gene is mainly responsible for brain-specificity in in vitro transcription. In this study, we found from intensive mutation analysis that the TATA-box sequence at -34 is not critical, but sequences downstream from the TATA-box especially around -20 were important for brain-specificity. The existence of a tissue-specific factor for the MBP core promoter different from the TATA-box-binding factor TFIID is implicated.

Animals↗
PubMed1994

Physical mapping at 6q27 of the locus for the TATA box-binding protein, the DNA-binding subunit of TFIID and a component of SL1 and TFIIIB, strongly suggests that it is single copy in the human genome.

The TATA box-binding protein (TBP) has a fundamental role in eukaryotic cell metabolism, since it is necessary for transcription of class I, class II, and class III genes; in fact, TBP is the DNA-binding subunit of TFIID and a component of SL1 and TFIIIB. Contrary to the previously hypothesized existence of a family of genes coding for DNA-binding proteins highly related to TBP, our experiments show that the segment coding for the evolutionarily well-conserved carboxyl-terminal domain, involved in DNA binding, is unique; accordingly, we conclude that the TBP locus itself, which we have localized to 6q27, is single copy in the human genome. On the other hand, a cDNA fragment coding for the evolutionarily variable amino-terminal domain detects multiple cross-hybridizing sequences in the genome of higher eukaryotes. We suggest that the common motif is represented by the long string of glutamine codons, which characterizes the amino-terminal segment of human TBP: in fact, other proteins involved in transcription, such as TAF II 110, Sp1, and some homeobox proteins, are known to contain glutamine-rich segments.

Animals↗
PubMed1997

The TATA-less promoter of hepatitis B virus S gene contains a TBP binding site and an active initiator.

The surface antigen (S) gene promoter, one of the major hepatitis B virus (HBV) promoters, directs the synthesis of a 2.1 kb mRNA which encodes the preS2 and S polypeptides. The preS2/S promoter does not contain a classical TATA box, and transcription regulation of the preS2/S gene has not been fully elucidated. We analysed two regions involved in preS2/S gene transcription of the HBV adw subtype: the diverged TATA box and a putative initiator element. We demonstrated sequence specific promoter activity of the putative TATA-like sequences in the preS2/S gene promoter (-25 to -32 bp). Using end labeled synthetic oligonucleotides we observed specific binding of nuclear extracts to the diverged TATA sequence, that was significantly reduced using a mutated oligonucleotide. Specific binding of yeast TBP to the diverged TATA sequence was shown which was increased in the mutant containing a classical TATA box. We analysed the proposed initiator (Inr) sequence of the preS2/S promoter region (-13 to -16 bp). Deletion of the inr element markedly reduced promoter activity as assessed by CAT expression. Gel shift assays showed specific binding of nuclear extracts to wild type but not to mutant Inr. Expression studies with double mutants of the diverged TATA and the Inr element established that both elements are active in transcription regulation.

Base Sequence↗
PubMed1994

Structural and functional characterization of the human decorin gene promoter. A homopurine-homopyrimidine S1 nuclease-sensitive region is involved in transcriptional control.

Decorin is a leucine-rich, chondroitin/dermatan sulfate proteoglycan which binds collagen and growth factors. We have recently completed the genomic organization of human decorin and discovered two alternatively spliced leader exons, designated exon Ia and Ib, in the 5'-untranslated region. Initial analysis of the sequences upstream to these two exons showed that promoter Ia contained only two GC boxes while promoter Ib contained a CAAT and two TATA boxes in close proximity to the transcription start site. To determine if these 5'-flanking sequences exhibited promoter activity, chimeric chloramphenicol acetyltransferase expression plasmids containing the promoter region of either exon Ia or Ib were transfected into HeLa and MG-63 osteosarcoma cells. The results showed that only the region flanking exon Ib was functional. In vitro transcription assay generated two transcripts of 92 and 82 base pairs (bp) indicating that both TATA boxes could be used. Using stepwise 5' deletion analysis we found that the minimum promoter region at -140 bp from the transcription start site, which contained only the CAAT and the two TATA boxes, exhibited strong promoter activity. When a larger construct containing an additional 800 bp of upstream region was tested, a significant increase in transcriptional activity was observed. Interestingly, this promoter region contained several putative binding sites for ubiquitous factors (AP1, AP5, and NF-kappa B) and for transforming growth factor-beta and a 150-bp homopurine/homopyrimidine element with several mirror repeats. When contained in a supercoiled plasmid, this sequence exhibited sensitivity to endonuclease S1, an enzyme that preferentially digests single-stranded DNA. Precise S1 mapping, obtained by direct sequencing of nine distinct S1-generated clones, revealed that in all cases the borders of the sensitive sequence resided within the pur/pyr segment. We propose that this region of the promoter could adopt an intramolecular hairpin triplex structure in vivo and may play a role in the chromatin organization at the decorin gene locus. In addition, this region was able to up-regulate a minimal heterologous promoter in transient transfection assays. The results show that the structure of the decorin gene promoter is different from that of any other proteoglycan promoter characterized so far and indicate that the pur/pyr segment plays a role in the regulation of gene transcription.

Base Sequence↗
PubMed1996

The architecture of the heat-inducible Drosophila hsp27 promoter in nuclei.

Transcriptional activation of the Drosophila hsp27 gene in response to heat shock critically relies on binding sites for heat shock factor (HSF) about 300 bp upstream of the transcription start site. In contrast to the well-characterised heat-inducible hsp70 and hsp26 promoters, no other transcription factor binding sites have been identified closer to the TATA box. In order to understand the structural requirements for activation from a distance we studied the protein-DNA interactions at the hsp27 promoter in Drosophila embryos and tissue culture cells before and after heat induction. Genomic footprinting with nucleases and a chemical probe, the 1,10-phenanthroline cuprous complex (OP-Cu), suggests that the DNA between the TATA box and the heat shock elements (HSEs) is constitutively organised by a positioned nucleosome, effectively shortening the distance between the distal HSEs and the TATA box. Protection of the TATA element from nuclease attack and the OP-Cu reactivity pattern around the start site of transcription is consistent with the constitutive presence of TFIID and the "poised polymerase", a transcription machinery blocked in an early phase of elongation. The general transcription factors at the TATA box and the positioned nucleosome are separated by a stable structure, presumably a protein bound to a palindromic sequence. These constitutive features define the "preset" architecture of the promoter within which the induced binding of HSF in vivo is observed. Our study highlights the importance of positioned nucleosomes as architectural elements within promoters and identifies a new regulatory sequence that may function either to direct a nucleosome boundary or to mediate signals of distant activator proteins.

Animals↗
PubMed1993

In vivo characterization of site-directed mutations in the promoter of the herpes simplex virus type 1 latency-associated transcripts.

Transient expression assays in PC12 cells showed that the cAMP response element (CRE) and the TATA box of the herpes simplex virus type 1 latency-associated transcripts (LATs) promoter are essential for basal expression. Recombinant viruses were generated containing site-specific mutations in these motifs. The abilities of these recombinants to replicate, express LATs and reactivate from latency were compared with wild-type and marker-rescued viruses in a murine ocular model. The acute replication of these TATA and CRE mutant viruses was at a level equivalent to their respective marker-rescued viruses. The reactivation of virus was unaffected by mutation in the TATA box as compared with wild-type or marker-rescued viruses. In situ hybridization of TATA box mutant virus-infected ganglia, however, showed threefold fewer LAT-positive neurons than wild-type virus-infected ganglia, with consistently weaker hybridization signals. Thus, this TATA box is required for normal expression of the LATs but not for efficient reactivation. The LATs CRE mutant reactivated with slightly but reproducibly reduced frequency and delayed kinetics relative to marker-rescued virus. By in situ hybridization, however, the percentage and intensity of LATs-positive neurons were found to be comparable for the CRE mutant- and wild-type virus-infected ganglia, suggesting that the CRE is dispensable for abundant LATs expression but that a reactivation function of the LATs may depend upon the presence of the CRE. Finally, using a modified assay for examining the timing of reactivation, we showed that the induction of viral reactivation by addition of exogenous cAMP can occur independently of the LATs.

Animals↗
PubMed1995

The glutamine-rich activation domains of human Sp1 do not stimulate transcription in Saccharomyces cerevisiae.

Eukaryotic transcriptional activators have been classified on the basis of the characteristics of their activation domains. Acidic activation domains, such as those in the yeast GAL4 or GNC4 proteins and the herpes simplex virus activator VP16, stimulate RNA polymerase II transcription when introduced into a variety of eukaryotic cells. This species interchangeability demonstrates that the mechanism by which acidic activation domains function is highly conserved in the eukaryotic kingdom. To determine whether such a conservation of function exists for a different class of activation domain, we have tested whether the glutamine-rich activation domains of the human transcriptional activator Sp1 function in the yeast Saccharomyces cerevisiae. We report here that the glutamine-rich domains of Sp1 do not stimulate transcription in S. cerevisiae, even when accompanied by human TATA-box binding protein (TBP) or human-yeast TATA-box binding protein hybrids. Thus, in contrast to the case for acidic activation domains, the mechanism by which glutamine-rich domains stimulate transcription is not conserved between S. cerevisiae and humans.

Amino Acid Sequence↗
PubMed2001

Species-specific mechanisms control the activity of the Pit1/PIT1 phosphate transporter gene promoter in mouse and human.

The Pit1 phosphate transporter is involved in regulated phosphate handling in bone forming cells. In this study, we compared the structure of the murine and human Pit1/PIT1 promoters and characterized cis-acting elements controlling Pit1/PIT1 expression. The Pit1/PIT1 promoter sequence and its location relative to the first transcribed exon are conserved and similar transcription factor binding sites are found at identical positions in mouse and human. Luciferase reporter gene assays in transiently transfected mouse ATDC5 chondrocytes and human SaOS-2 osteoblasts indicated that the activity of the mouse Pit1 promoter depends on several cis-acting elements, including ATF/CREB, Sp1 and AP-1 sites, an E-box and a TATA box. In contrast, the activity of the human promoter essentially requires a TATA-like sequence and one single Sp1 site. This Sp1 site binds Sp1, Sp3, as well as unidentified proteins present in SaOS-2 nuclear extracts and co-transfection experiments in SL2 cells indicate that Sp1 and Sp3 activate transcription from the human PIT1 promoter. These data suggest that, despite similarities in promoter structure, changes in the relative importance of conserved transcription factor binding sites cause species-dependent differences in Pit1 promoter function, which allow Sp1-related proteins to play a particularly important role in human.

5' Flanking Region↗
PubMed2005

The cellular TATA binding protein is required for rep-dependent replication of a minimal adeno-associated virus type 2 p5 element.

The p5 promoter region of adeno-associated virus type 2 (AAV-2) is a multifunctional element involved in rep gene expression, Rep-dependent replication, and site-specific integration. We initially characterized a 350-bp p5 region by its ability to behave like a cis-acting replication element in the presence of Rep proteins and adenoviral factors. The objective of this study was to define the minimal elements within the p5 region required for Rep-dependent replication. Assays performed in transfected cells (in vivo) indicated that the minimal p5 element was composed by a 55-bp sequence (nucleotides 250 to 304 of wild-type AAV-2) containing the TATA box, the Rep binding site, the terminal resolution site present at the transcription initiation site (trs(+1)), and a downstream 17-bp region that could potentially form a hairpin structure localizing the trs(+1) at the top of the loop. Interestingly, the TATA box was absolutely required for in vivo but dispensable for in vitro, i.e., cell-free, replication. We also demonstrated that Rep binding and nicking at the trs(+1) was enhanced in the presence of the cellular TATA binding protein, and that overexpression of this cellular factor increased in vivo replication of the minimal p5 element. Together, these studies identified the minimal replication origin present within the AAV-2 p5 promoter region and demonstrated for the first time the involvement of the TATA box, in cis, and of the TATA binding protein, in trans, for Rep-dependent replication of this viral element.

Base Sequence↗
PubMed1999

Enhancement of TBP binding by activators and general transcription factors.

Eukaryotic transcriptional activators function, at least in part, by promoting assembly of the preinitiation complex, which comprises RNA polymerase II and its general transcription factors (GTFs). Activator-mediated stimulation of the assembly of the preinitiation complex has been studied in vitro but has been relatively refractory to in vivo analysis. Here we use a DNA-crosslinking/immunoprecipitation assay to study in living cells the first step in the assembly of the preinitiation complex, the interaction between the TATA-box-binding protein (TBP) and its binding site, the TATA box. Analysis of a variety of endogenous yeast genes, and of a series of activators of differing strength, reveals a general correlation between TBP binding and transcriptional activity. Using mutant yeast strains, we show that Mot1 prevents the binding of TBP to inactive promoters and that activator-mediated stimulation of TBP binding requires additional GTFs, including TFIIB and Srb4. Taken together, our results indicate that TBP binding in vivo is stringently controlled, and that the ability of activators to stimulate this step in the assembly of the preinitiation complex is a highly cooperative process involving multiple transcription factors.

Adenosine Triphosphatases↗
PubMed2004

The MTE, a new core promoter element for transcription by RNA polymerase II.

The core promoter is the ultimate target of the vast network of regulatory factors that contribute to the initiation of transcription by RNA polymerase II. Here we describe the MTE (motif ten element), a new core promoter element that appears to be conserved from Drosophila to humans. The MTE promotes transcription by RNA polymerase II when it is located precisely at positions +18 to +27 relative to A(+1) in the initiator (Inr) element. MTE sequences from +18 to +22 relative to A(+1) are important for basal transcription, and a region from +18 to +27 is sufficient to confer MTE activity to heterologous core promoters. The MTE requires the Inr, but functions independently of the TATA-box and DPE. Notably, the loss of transcriptional activity upon mutation of a TATA-box or DPE can be compensated by the addition of an MTE. In addition, the MTE exhibits strong synergism with the TATA-box as well as the DPE. These findings indicate that the MTE is a novel downstream core promoter element that is important for transcription by RNA polymerase II.

Base Sequence↗
PubMed1995

Genomic structure and expression of the ADH7 gene encoding human class IV alcohol dehydrogenase, the form most efficient for retinol metabolism in vitro.

Human alcohol dehydrogenase (ADH) consists of a family of five evolutionarily related classes of enzymes that collectively function in the metabolism of a wide variety of alcohols including ethanol and retinol. Class IV ADH has been found to be the most active as a retinol dehydrogenase, thus it may participate in retinoic acid synthesis. The gene encoding class IV ADH (ADH7) has now been cloned and subjected to molecular examination. Southern blot analysis indicated that class IV ADH is encoded by a single unique gene and has no related pseudogenes. The class IV ADH gene is divided into nine exons, consistent with the highly conserved intron/exon structure of other mammalian ADH genes. The predicted amino acid sequence of the exon coding regions indicates that a protein of 373 amino acids, excluding the amino-terminal methionine, would be translated, sharing greater sequence identity with class I ADH (69%) than with classes II, III or V (59-61%). Expression of class IV ADH mRNA was detected in human stomach but not liver. This correlates with previous protein studies, which have indicated that class IV ADH is the major stomach ADH but unlike other ADHs is absent from liver. Primer extension studies using human stomach RNA were performed to identify the transcription initiation site lying 100 base pairs upstream of the ATG translation start codon. Nucleotide sequence analysis of the promoter region indicated the absence of a TATA box sequence often located about 25 base pairs upstream of the start site as well as the absence of GC boxes, which are quite often seen in promoters lacking a TATA box. The class IV ADH promoter thus differs from the other ADH promoters, which contain either a TATA box (classes I and II) or GC-boxes (class III), suggesting a fundamentally different form of transcriptional regulation.

Alcohol Dehydrogenase↗
PubMed2000

Saturation mutagenesis of the haloarchaeal bop gene promoter: identification of DNA supercoiling sensitivity sites and absence of TFB recognition element and UAS enhancer activity.

Transcription from the bop promoter in the haloarchaeon Halobacterium NRC-1, is highly induced under oxygen-limiting conditions. A DNA gyrase inhibitor, novobiocin, was previously shown to block bop gene induction and suggested that DNA supercoiling mediates transcriptional induction. A region of non-B structure was found 3' to the TATA box within an 11 bp alternating purine-pyrimidine sequence (RY box), which correlated to both increased DNA supercoiling and transcriptional induction. Here, saturation mutagenesis of the RY box region has been used to show that single-base substitutions of A(r)G either 23 or 19 bp 5' to the transcription start site temper the effect of DNA supercoiling based on novobiocin insensitivity of transcription. Mutagenesis of the region 5' to the TATA box showed its involvement in DNA supercoiling modulation of transcription, defined the 3' end of the upstream activator sequence (UAS) regulatory element, and ruled out the requirement for a TFB (TFIIB) Recognition Element. Spacing between the TATA box and UAS was found to be critical for promoter activity because insertion of partial or whole helical turns between the two elements completely inhibited transcription indicating that the UAS element does not function as a transcriptional enhancer. The results are discussed in the context of DNA melting and flexibility around the TATA box region and the involvement of multiple regulatory and transcription factors in bop promoter activity.

Archaeal Proteins↗
PubMed2006

The initiator motif is preferentially used as the core promoter element in ionizing radiation-responsive genes.

Recent improvements in DNA microarray technologies and bioinformatics have made it possible to look for common features of ionizing radiation-responsive genes and their regulatory regions. We analyzed the promoters of 217 radiation-responsive human genes, compiled from microarray databases available in the literature. Using the DBTSS database, the transcriptional start sites were determined, and the core promoter elements, such as the TATA-box, initiator (Inr), GC-box and CCAAT-box, were searched for in the -1000 bp/ +200 bp region of each gene by using MATCH. It was found that the frequency of Inr in radiation-responsive genes was higher than that in general genes, and the frequencies of the GC-box and CCAAT-box were significantly lower than those in general genes. Use of the GC-box and the CCAAT-box in radiation-responsive genes was found to be dependent on the TATA-box status; that is, GC-box frequency was low in TATA box-containing genes, and CCAAT-box frequency was also low in TATA-less genes. When correlations between gene functions and frequencies of core promoter elements were examined, no apparent biased use of the core promoter element in association with a specific gene function was observed. It may be speculated that use of Inr in the core promoter correlates with appearance of radiation-responsive enhancer (silencer) elements in the upstream (downstream) regulatory region.

Blood Proteins↗
PubMed1989

Identification of the latency-associated transcript promoter by expression of rabbit beta-globin mRNA in mouse sensory nerve ganglia latently infected with a recombinant herpes simplex virus.

The herpes simplex virus type 1 latency-associated transcript (LAT) is expressed as a major species in latently infected mouse neurons. Previous sequence analysis revealed no obvious promoter elements near the 5' end of the LAT, but a TATA box and other potential promoter elements were found 700 base pairs upstream. A recombinant virus in which the rabbit beta-globin gene was inserted immediately downstream of the TATA box expressed globin mRNA and did not express the LAT. A second recombinant virus, in which this TATA box was removed, was negative for LAT expression in a latent infection. The location of the LAT promoter suggested that RNA upstream of the LAT was synthesized and degraded during latent-phase transcription. Low levels of this RNA were observed by in situ hybridization. In other experiments, RNA from a productive infection was used to detect a transcript extending from the LAT promoter to a polyadenylation signal approximately 8.5 kilobase downstream. These data suggest that the LAT may be processed from a larger transcription unit which begins distal to the TATA box 700 base pairs upstream of the LAT and extends to a polyadenylation signal almost 5 kilobases downstream of the 3' end of the LAT.

Animals↗
PubMed1997

Functional characterization of the major late promoter of mouse adenovirus type 1.

During the late phase of adenovirus infection, the major late promoter (MLP) controls the regulated expression of the genes that encode most viral structural proteins. Recently, the region of the genome of mouse adenovirus type 1 (MAV-1), predicted to contain the MLP, was sequenced and compared to that of the human virus MLP. The general organization of the transcriptional elements of the putative MAV-1 MLP is similar to that of the human virus counterpart, with some interesting differences. We wished to investigate the function of the predicted MLP of MAV-1 and to determine the significance of the differences found in the MAV-1 MLP. To test the activity of the predicted MLP of MAV-1, both Northern blot and primer extension analyses were performed on intracellular RNA isolated from cells infected with MAV-1. The results show that late RNA can be detected 48 hr postinfection and increases up to 6 days p.i. Primer extension analysis revealed that the major start sites of transcription are 28 and 31 nt downstream of the first T residue of the predicted TATA box. To analyze the functional significance of the predicted transcriptional elements, a transient transfection system, using the firefly luciferase gene controlled by the MAV-1 MLP sequence, was established. The predicted MLP sequence was capable of directing luciferase gene expression, to a level some 60% of that of the human virus MLP. Mutations were created in the inverted CAAT box, the SP1 site, and the TATA box, either singly or in combination. Each single-element mutation causes a marked reduction in luciferase gene expression, with the SP1 mutation showing the greatest effect. Double mutations were even more deficient, suggesting a level of functional redundancy among the various transcriptional elements. Finally, the putative SP1-binding site was examined by gel mobility shift assay and shown to interact with purified SP1 protein specifically, supporting the functional significance of this transcriptional element. These findings contribute to a better understanding of gene expression in MAV-1 and to its development as an appropriate model for the study of the molecular basis of pathogenesis in a natural host animal.

Adenoviridae↗
PubMed2001

The downstream activation sequence of the strict late Herpes Simplex Virus Type 1 U(L)38 promoter interacts with hTAF(II)70, a component of TFIID.

A class of strict late Herpes Simplex Virus Type 1 (HSV-1) promoters contains a conserved sequence element (termed the downstream activation sequence, DAS) located downstream of the transcription start site. These DAS-containing promoters also require both a TATA box and an initiator element for maximal levels of transcription. In this communication, we demonstrate that the downstream promoter element (DPE) found on a class of Drosophila TATA-less promoters and known to bind the homologue of human TAF(II)70 (a component of TFIID), can functionally substitute for DAS in the context of the strict late UL38 promoter in spite of no obvious sequence similarity. Although Drosophila DPE-containing promoters do not require a TATA box, the element does not remove the requirement for a TATA box when functioning in the HSV promoter. Next, we demonstrate that hTAF(II)70, interacts in a sequence specific manner with DAS as predicted from the fact that DPE binds Drosophila TBP. These results suggest that multiple TFIID/promoter interactions are important in the activation of HSV-1 late gene expression upon viral DNA replication. We propose that such interactions could be favored upon viral DNA replication since TFIID concentrates to viral transcription foci that form during the later stages of infection.

Amino Acid Sequence↗
PubMed1984

Nucleotide sequences of the retroviral long terminal repeats and their adjacent regions.

The nucleotide sequences of the LTRs and their adjacent regions from 19 type C and one type B retrovirus were compared. Salient features are: (a) The R regions in the genomes of most of the type C retroviruses begin with GC and end with CA. (b) The mammalian type C retroviruses have a polyadenylation signal "AATAAA" in the R region, and most have a "CAT" box and a "TATA" box in the U3 region. (c) The avian type C retroviruses have an AATAAA sequence, and some also have "CAT-like" and "TATA-like" boxes, in the U3 region. (d) As with many transposable elements, the IR regions of the proviruses begin with TG and end with CA, and the DR sequences in the host genomes flanking the proviruses are different from one another. Although SNV is an avian retrovirus, the nucleotide sequences in the R, U5, TBS, and PU region are more similar to the mammalian type C than to the avian type C retroviruses.

Animals↗