Showing papers on "Upstream activating sequence published in 1996"
TL;DR: The results suggest that AP-1 tethers c-Ets-1 to the TIMP-1 promoter via protein-protein interaction to achieve Ets-dependent transcriptional regulation.
189 citations
TL;DR: In a conserved but poorly studied upstream region, several novel sites of DNase I hypersensitivity in vivo are mapped that constitutively distinguish IL-2 producer type T cells from cell types that cannot express IL-1 and what interaction(s) conveys T-lineage specificity.
Abstract: The interleukin 2 (IL-2) gene is subject to two types of regulation: its expression is T-lymphocyte-specific and it is acutely dependent on specific activation signals. The IL-2 transcriptional apparatus integrates multiple types of biochemical information in determining whether or not the gene will be expressed, using multiple diverse transcription factors that are each optimally activated or inhibited by different signaling pathways. When activation of one or two of these factors is blocked IL-2 expression is completely inhibited. The inability of the other, unaffected factors to work is explained by the striking finding that none of the factors interacts stably with its target site in the IL-2 enhancer unless all the factors are present. Coordinate occupancy of all the sites in the minimal enhancer is apparently maintained by continuous assembly and disassembly cycles that respond to the instantaneous levels of each factor in the nuclear compartment. In addition, the minimal enhancer undergoes specific increases in DNase I accessibility, consistent with dramatic changes in chromatin structure upon activation. Still to be resolved is what interaction(s) conveys T-lineage specificity. In the absence of activating signals, the minimal IL-2 enhancer region in mature T cells is apparently unoccupied, exactly as in non-T lineage cells. However, in a conserved but poorly studied upstream region, we have now mapped several novel sites of DNase I hypersensitivity in vivo that constitutively distinguish IL-2 producer type T cells from cell types that cannot express IL-2. Thus a distinct domain of the IL-2 regulatory sequence may contain sites for competence- or lineage-marking protein contacts.
178 citations
TL;DR: Comparison with GenBank sequences revealed a fourth FBP family member encoded by Caenorhabditis elegans chromosome III, illustrating the high degree of homology in this evolutionarily ancient and conserved family.
171 citations
TL;DR: It is demonstrated that both upstream regions function as promoters in hematopoietic (Jurkat) and nonhematopoetic (HEK) cell lines and was enhanced, in a cell-type specific manner, by a heterologous enhancer sequence.
Abstract: The human chromosome 21 AML1 gene is expressed predominantly in the hematopoietic system. In several leukemia-associated translocations AML1 is fused to other genes and transcription of the fused regions is mediated by upstream sequences that normally regulate the expression of AML1. The 5' genomic region of AML1 was cloned and sequenced. The two 5' untranslated regions (UTRs) previously identified in AML1 cDNAs were located in this region and the distance between them was established. The distal 5' UTR maps over 7 kb upstream of the proximal one. Using primer extension with mRNA, transcription start sites were identified at two distinct sites above these 5' uTRs. Sequence analysis revealed the absence of a TATA motif and the presence of Sp1, PU.1, Oct, CRE, Myb, Ets, and Ets-like binding sites in both upstream regions. Several initiator elements (Inr) that overlap the transcription start sites were also identified. These proximal and distal upstream regions and their deletion mutants were cloned in front of a luciferase reporter gene and used in transfection assays. We demonstrate that both upstream regions function as promoters in hematopoietic (Jurkat) and nonhematopoietic (HEK) cell lines. The activity of both promoters was orientation dependent and was enhanced, in a cell-type specific manner, by a heterologous enhancer sequence. These results indicate that additional control elements, either negative or positive, regulate the tissue-specific expression of AML1.
159 citations
TL;DR: It is demonstrated that another positive regulator, designated Gat1p, participates in the transcription of NCR-sensitive genes and is able to weakly activate transcription when tethered upstream of a reporter gene devoid of upstream activation sequence elements.
Abstract: Saccharomyces cerevisiae cells selectively use nitrogen sources in their environment. Nitrogen catabolite repression (NCR) is the basis of this selectivity. Until recently NCR was thought to be accomplished exclusively through the negative regulation of Gln3p function by Ure2p. The demonstration that NCR-sensitive expression of multiple nitrogen-catabolic genes occurs in a gln3 delta ure2 delta dal80::hisG triple mutant indicated that the prevailing view of the nitrogen regulatory circuit was in need of revision; additional components clearly existed. Here we demonstrate that another positive regulator, designated Gat1p, participates in the transcription of NCR-sensitive genes and is able to weakly activate transcription when tethered upstream of a reporter gene devoid of upstream activation sequence elements. Expression of GAT1 is shown to be NCR sensitive, partially Gln3p dependent, and Dal80p regulated. In agreement with this pattern of regulation, we also demonstrate the existence of Gln3p and Dal80p binding sites upstream of GAT1.
140 citations
TL;DR: Three sites in the PDR5 5′-noncoding region that are closely related to one another and are bound by both Pdr1p and Pdr3p are found and each element is required for wild-type expression and drug resistance.
133 citations
TL;DR: It is proposed that the ATF-2 bZIP and activation domain are engaged in an inhibitory intramolecular interaction and that inducers of ATF- 2 disrupt this interaction to activate transcription.
Abstract: ATF-2 is a cellular basic region-leucine zipper (bZIP) transcription factor that can mediate diverse transcriptional responses, including activation by the adenovirus Ela protein. ATF-2 contains an activation domain, required for transcriptional activity, but in the absence of an appropriate inducer, full-length ATF-2 is transcriptionally inactive. Here we have investigated the mechanism underlying this regulated inhibition of ATF-2 transcriptional activity. We show that the region of ATF-2 that suppresses the activation region is the bZIP DNA-binding domain and that maximal inhibition requires both the basic region and leucine zipper subdomains. Inhibition is activation domain specific: The ATF-2 bZIP suppresses the ATF-2 and the related Ela activation domains but not acidic- and glutamine-rich activation domains. In vitro protein interaction assays demonstrate that the ATF-2 activation domain and bZIP specifically bind to one another. Finally, we show that bZIP-mediated inhibition can be modulated in a cell-type-specific manner by another sequence element within ATF-2. On the basis of these and other data, we propose that the ATF-2 bZIP and activation domain are engaged in an inhibitory intramolecular interaction and that inducers of ATF-2 disrupt this interaction to activate transcription.
120 citations
TL;DR: The results of this screen and the characterization of GAL4 lines that show distinct patterns of gene expression during Drosophila development demonstrate both the strengths and weaknesses of the GAL 4 enhancer trap technique for identifying unique patterns of genes expression during development.
Abstract: To identify genes that are expressed in specific cell types or tissues during development, we generated enhancer-trap lines in which the yeast transcriptional activator, GAL4, was mobilized throughout the Drosophila genome. The GAL4 lines are part of a two-part system involving GAL4 and its target, the upstream activating sequence (UAS). Detection of GAL4 expression patterns was achieved by crossing individual GAL4 lines with flies carrying the reporter gene lacZ under the transcriptional control of the UAS followed by histochemical and immunocytochemical staining. Here, we present the results of this screen and the characterization of GAL4 lines that show distinct patterns of gene expression during Drosophila development, including embryogenesis, oogenesis, and imaginal disc development. However, we were unable to identify GAL4 lines that were expressed within the germ line or during early embryogenesis. Furthermore, consistent with previous results, we found that the GAL4 enhancer trap technique had a much lower frequency of transposition than has been reported for lacZ enhancer trap screens. Taken together, these results demonstrate both the strengths and weaknesses of the GAL4 enhancer trap technique for identifying unique patterns of gene expression during development.
120 citations
TL;DR: By template competition experiments, it was shown that TBP is required for UAF-dependent recruitment of CF to the rDNA promoter, explaining the TBP requirement for stimulatory activity of the upstream element.
Abstract: Transcription of Saccharomyces cerevisiae rDNA by RNA polymerase I involves at least two transcription factors characterized previously: upstream activation factor (UAF) consisting of Rrn5p, Rrn9p, Rrn10p, and two more uncharacterized proteins; and core factor (CF) consisting of Rrn6p, Rrn7p, and Rrn11p. UAF interacts directly with an upstream element of the promoter and mediates its stimulatory function, and CF subsequently joins a stable preinitiation complex. The TATA-binding protein (TBP) has been known to be involved in transcription by all three nuclear RNA polymerases. We found that TBP interacts specifically with both UAF and CF, the interaction with UAF being stronger than that with CF. Using extracts from a TBP (I143N) mutant, it was shown that TBP is required for stimulation of transcription mediated by the upstream element, but not for basal transcription directed by a template without the upstream element. By template competition experiments, it was shown that TBP is required for UAF-dependent recruitment of CF to the rDNA promoter, explaining the TBP requirement for stimulatory activity of the upstream element. We also studied protein-protein interactions and found specific interactions of TBP with Rrn6p and with Rrn9p both in vitro and in the yeast two-hybrid system in vivo. Thus, these two interactions may be involved in the interactions of TBP with CF and UAF, respectively, contributing to the recruitment of CF to the rDNA promoter. Additionally, we observed an interaction between Rrn9p and Rrn7p both in vitro and in the two-hybrid system; thus, this interaction might also contribute to the recruitment of CF.
113 citations
TL;DR: This connection between biochemical and genetic studies reveals that mediator is important for both activation and repression of transcription, and that mediators plays a role in transcriptional regulation in vivo as well as in vitro.
111 citations
TL;DR: A model derived from the current data suggests that regulation of renin expression is achieved through cooperation of transcription factors binding at the proximal promoter element and a distal enhancer element to abrogate or override the effects of an intervening negative regulatory region.
TL;DR: It is suggested that IHF activates transcription by forming a higher-order protein-DNA complex in the UAS1 region that structurally alters the DNA helix in a way that facilitates open complex formation at the downstream ilvPG promoter site.
Abstract: Integration host factor (IHF) is a DNA-bending protein that binds to an upstream activating sequence (UAS1) and, on a negatively supercoiled DNA template, activates transcription from the ilvPG promoter of the ilvG-MEDA operon of Escherichia coli. The transcriptional initiation site of the ilvGMEDA operon is located 92 bp downstream of UAS1. Activation is still observed when the orientation of the upstream IHF binding site is reversed. This manipulation places the IHF binding site on the opposite face of the DNA helix, directs the IHF-induced DNA bend in the opposite direction, and presents the opposite face of the nonsymmetrical, heterodimeric, IHF molecule to the downstream RNA polymerase. Lymphoid enhancer-binding factor, LEF-1, is a DNA-bending, lymphoid-specific, mammalian transcription factor that shares no amino acid sequence similarity with IHF. When the IHF site in UAS1 is replaced with a LEF-1 site, LEF-1 activates transcription from the downstream ilvPG promoter in E. coli as well as it is activated by its natural activator, IHF. These results suggest that specific interactions between IHF and RNA polymerase are not required for activation. The results of DNA structural studies show that IHF forms a protein-DNA complex in the UAS1 region that, in the absence of RNA polymerase, alters the structure of the DNA helix in the -10 hexanucleotide region of the downstream ilvPG promoter. The results of in vitro abortive transcription assays show that IIIF also increases the apparent rate of RNA polymerase isomerization from a closed to an open complex. We suggest, therefore, that IHF activates transcription by forming a higher-order protein-DNA complex in the UAS1 region that structurally alters the DNA helix in a way that facilitates open complex formation at the downstream ilvPG promoter site.
TL;DR: The gene structures, nucleotide sequences and chromosomal assignment for the p35 and p40 subunits of mouse‐IL‐12 are provided and an AGTTTCTACTTT sequence, which acts as an interferon‐γ response element in the promoter of the major histocompatibility complex class I gene, was also found upstream of the p40 gene.
Abstract: Interleukin-12 (IL-12) is a heterodimeric cytokine composed of p35 and p40 subunits and is required for induction of T helper 1 (Th1) responses. Knowledge of how the IL-12 gene is regulated will permit an understanding of susceptibility and resistance to pathogenic microbes and to autoiummune diseases. In this report, we provide the gene structures, nucleotide sequences and chromosomal assignment for the p35 and p40 subunits of mouse IL-12. The p35 and p40 subunit genes are distributed over 8 kb and 14 kb, and map to chromosomes 3 and 11, respectively. The p35 subunit gene consists of eight exons, including a 5'-noncoding exon that was defined by sequence comparison of genomic DNA with the 5'ends of novel cDNA molecules. Transcription of p35 mRNA can start from the first exon but can also initiate further downstream. Potential transcription regulatory elements, AP1, AP2, AP3, NF-kB and GATA recognition sequences, are located within 523 bp upstream of the p35 gene; however, no TATA box was identified. The p40 subunit gene consists of eight exons. A TATA box is located 30 bp upstream from the transcription start site, and AP1, AP3, GATA, and Pu.1 recognition sequences are located within 690 bp upstream of the p40 gene. An AGTTTCTACTTT sequence, which acts as an interferon-gamma response element in the promoter of the major histocompatibility complex class I gene, was also found upstream of the p40 gene.
TL;DR: Five sites of DNA-protein interaction within the first 260 bp upstream of the transcription start site are identified, which involve at least three different families of transcription factors and two sites, a distal DR-1 motif and a proximal octamer motif, are the most important for promoter activity.
Abstract: vHNF1 (also termed HNF1 beta) is a member of the hepatocyte nuclear fa ctor 1 (HNF1; also termed HNF1 alpha) family of homeodomain-containing transcription factors that interact with a sequence motif found in the regulatory regions of a large number of genes expressed mainly in the liver. It has been suggested that vHNF1 plays a role in early differentiation of specialized epithelia of several endoderm- and mesoderm-derived organs, with HNF1 playing a role in later stages. In support of this idea, expression of vHNF1 but not HNF1 is induced upon treatment of the embryonal carcinoma cell line F9 with retinoic acid. We have cloned and analyzed the vHNF1 promoter to gain a better understanding of the regulation of vHNF1 expression and how it relates to the expression of HNF1. We have identified five sites of DNA-protein interaction within the first 260 bp upstream of the transcription start site, which involve at least three different families of transcription factors. Two sites, a distal DR-1 motif and a proximal octamer motif, are the most important for promoter activity. The DR-1 motif interacts with several members of the steroid hormone receptor superfamily including HNF4, COUP-TFI/Ear3, COUP-TFII/Arp1, and RAR alpha/RXR alpha heterodimers. The vHNF1 promoter is transactivated by COUP-TFI/Ear3 and COUP-TFII/Arp1 and, unlike the HNF1 promoter, is virtually unaffected by HNF4. Interestingly, the proximal octamer site and not the DR-1 site is required for COUP-TFI/Ear3 and COUP-TFII/Arp1 transactivation of the vHNF1 promoter. COUP-TFI/Ear3 does not bind directly to this proximal octamer site. We present evidence of an interaction between COUP-TFI/Ear3 and the octamer-binding proteins in vitro and in the cell, suggesting that COUP-TFI and COUP-TFII activate the vHNF1 promoter via an indirect mechanism.
TL;DR: An oleate-activated transcription factor (Oaf1p) that binds to the activating sequence (UAS1) in the POX1 gene is purified and identified, with a predicted molecular mass of approximately 118 kDa.
TL;DR: Different subunits of TBP/TAF complexes perform a variety of functions critical for transcriptional regulation in animal cells, and it is found that TAFs are responsible for at least two regulatory functions.
Abstract: The control of gene expression during development, differentiation and maintenance of cellular function is governed by a complex array of transcription factors. We have undertaken a molecular dissection of the regulatory factors that direct transcription of protein coding genes by RNA polymerase II. Our early studies identified sequence-specific transcriptional activators that bind to enhancer and promoter sequences to modulate the transcriptional initiation event. However, the mechanism by which activators enhance transcription and mediate promoter selectivity remained unknown. Combining biochemical purification and in vitro assays, we have recently identified an essential class of transcription factors called TAFs that are tightly associated with the basal factor TBP (TATA-binding protein). We have found that TAFs are responsible for at least two regulatory functions. Some TAFs serve as coactivators capable of binding activators and mediating enhancing function. Other TAFs have been shown to confer template selectivity by binding directly to core DNA elements of the promoter. Thus different subunits of TBP/TAF complexes perform a variety of functions critical for transcriptional regulation in animal cells.
TL;DR: The pituitary-specific transcription factor Pit-1/GHF-1 is a member of the POU domain family of regulatory proteins that is involved in the commitment and expansion of the somatotropic cell lineage and activates the transcription of a set of anterior pituitsary genes.
TL;DR: It is argued that Gln3p is capable of direct UAS(NTR) binding and participates in transcriptional activation of NCR-sensitive genes.
Abstract: When readily used nitrogen sources are available, the expression of genes encoding proteins needed to transport and metabolize poorly used nitrogen sources is repressed to low levels; this physiological response has been designated nitrogen catabolite repression (NCR). The cis-acting upstream activation sequence (UAS) element UAS(NTR) mediates Gln3p-dependent, NCR-sensitive transcription and consists of two separated dodecanucleotides, each containing the core sequence GATAA. Gln3p, produced in Escherichia coli and hence free of all other yeast proteins, specifically binds to wild-type UAS(NTR) sequences and DNA fragments derived from a variety of NCR-sensitive promoters (GDH2, CAR11 DAL3, PUT1, UGA4, and GLN1). A LexA-Gln3 fusion protein supported transcriptional activation when bound to one or more LexAp binding sites upstream of a minimal CYC1-derived promoter devoid of UAS elements. LexAp-Gln3p activation of transcription was largely independent of the nitrogen source used for growth. These data argue that Gln3p is capable of direct UAS(NTR) binding and participates in transcriptional activation of NCR-sensitive genes.
TL;DR: To determine which nucleotides were critical for promoter function, oligonucleotide-directed mutagenesis and mutagenic PCR amplification were used to produce point mutations in the region upstream of the start point of transcription, indicating that in the absence of a functional -35 region, the rpsL promoter is dependent on the TGn sequence upstream from the -10 hexamer.
Abstract: The DNA sequence of the promoter region of the Mycobacterium smegmatis rpsL gene, which encodes the S12 ribosomal protein, was determined. Primer extension analysis and S1 nuclease protection experiments identified the 5' end of the rpsL mRNA to be 199 bp upstream of the translation initiation codon. The rpsL promoter contained sequences upstream of this start point for transcription that were similar to the canonical hexamers found at the -10 and -35 regions of promoters recognized by Esigma70, the major form of RNA polymerase in Escherichia coli. To define the promoter of the rpsL gene, DNA fragments containing progressive deletions of the upstream region of the rpsL gene were inserted into a plasmid vector containing a promoterless xylE gene. These insertions revealed that the 200 bp of DNA sequence immediately upstream from the translation initiation codon was not essential for promoter function. In addition, 5' deletions removing all but 34 bp upstream of the transcription start point retained greater than 90% promoter activity, suggesting that the -35 hexamer was not essential for promoter activity. To determine which nucleotides were critical for promoter function, oligonucleotide-directed mutagenesis and mutagenic PCR amplification were used to produce point mutations in the region upstream of the start point of transcription. Single base substitutions in the -10 hexamer, but not in the -35 hexamer, severely reduced rpsL promoter activity in vivo. Within the -10 hexamer, nucleotide substitutions causing divergence from the E. Coli sigma70 consensus reduced promoter activity. The DNA sequence immediately upstream from the - 10 hexamer contained the TGn motif described as an extended -10 region in prokaryotic promoters. Mutations in this motif, in combination with a transition at either the -38 or -37 position within the -35 hexamer, severely reduced promoter activity, indicating that in the absence of a functional -35 region, the rpsL promoter is dependent on the TGn sequence upstream from the -10 hexamer. Comparison of the nucleotide sequence of the rpsL promoter region of M. smegmatis with the homologous sequences from Mycobacterium leprae, Mycobacterium bovis, and Mycobacterium tuberculosis showed the presence in these slowly growing mycobacterial species of conserved promoter elements a similar distance upstream of the translation initiation codon of the rpsL gene, but these other mycobacterial promoters did not contain the extended -10 motif.
TL;DR: These substitutions identify two separate regions on the surface of CRP that appear to be able to interact with RNA polymerase specifically at class II promoters and it is shown that these alternative activating regions are functional in the downstream subunit of the bound CRP dimer.
Abstract: At class II CRP-dependent promoters the DNA site for CRP overlaps the DNA site for RNA polymerase, covering the -35 region. Transcription activation at class II CRP- dependent promoters requires a contact between an activating region in the upstream subunit of the bound CRP dimer and a contact site in the C-terminal domain of the alpha-subunit of RNA polymerase. Transcription activation is suppressed by amino acid substitutions in the activating region, but activation can be restored by second site substitutions at K52 or E96. These substitutions identify two separate regions on the surface of CRP that appear to be able to interact with RNA polymerase specifically at class II promoters. Using the method of 'oriented heterodimers' we show that these alternative activating regions are functional in the downstream subunit of the bound CRP dimer.
TL;DR: The role of the Rap1p in the activation of glycolytic enzyme genes is to bind in their UAS elements and to facilitate the binding of Gcr1p at adjacent binding sites.
Abstract: Transcriptional activation in eukaryotic organisms normally requires combinatorial interactions of multiple transcription factors. In most cases, the precise role played by each transcription factor is not known. The upstream activating sequence (UAS) elements of glycolytic enzyme genes in Saccharomyces cerevisiae are excellent model systems for the study of combinatorial interactions. The yeast protein known as Rap1p acts as both a transcriptional repressor and an activator, depending on sequence context. Rap1p-binding sites are found adjacent to Gcr1p-binding sites in the UAS elements of glycolytic enzyme genes. These UAS elements constitute some of the strongest activating sequences known in S. cerevisiae. In this study, we have investigated the relationship between Rap1p- and Gcr1p-binding sites and the proteins that bind them. In vivo DNA-binding studies with rap1ts mutant strains demonstrated that the inability of Rap1p to bind at its site resulted in the inability of Gcr1p to bind at adjacent binding sites. Synthetic oligonucleotides, modeled on the UAS element of PYK1, in which the relative positions of the Rap1p- and Gcr1p-binding sites were varied prepared and tested for their ability to function as UAS elements. The ability of the oligonucleotides to function as UAS elements was dependent not only on the presence of both binding sites but also on the relative distance between the binding sites. In vivo DNA-binding studies showed that the ability of Rap1p bind its site was independent of Gcr1p but that the ability of Gcr1p to bind its site was dependent on the presence of an appropriately spaced and bound Rap1p-binding site. In vitro binding studies showed Rap1p-enhanced binding of Gcr1p on oligonucleotides modeled after the native PYK1 UAS element but not when the Rap1p- and Gcr1p-binding sites were displaced by 5 nucleotides. This work demonstrates that the role of the Rap1p in the activation of glycolytic enzyme genes is to bind in their UAS elements and to facilitate the binding of Gcr1p at adjacent binding sites.
TL;DR: The results suggest that the multiple upstream elements that are identified may act as a backup regulatory region in the event of disruption of the HNF-1 element in the 5' untranslated region of the factor VIII gene.
Abstract: Coagulation factor VIII is an essential cofactor required for normal hemostatic function. A deficiency in factor VIII results in the bleeding disorder hemophilia A. Despite the fact that the factor VIII gene was cloned a decade ago, the mechanisms which control its transcription remain unresolved. In our studies, we have characterized 12 protein binding sites within the factor VIII promoter by DNase I protection assays performed with rat liver nuclear extracts. Three of these elements (sites 1 to 3) are situated within the 5' untranslated region of the gene, while three other sites (sites 4 to 6) lie within the first 100 bp upstream of the transcriptional start site. We have identified an additional site (site 7) approximately 300 bp upstream from site 6, as well as a cluster of five sites in a 250-bp region which terminates approximately 1 kb from the transcriptional start site. Seven of these binding sites (sites 2, 3, 4, 6, 7, 9, and 10) bind members of the C/EBP family of transcription factors. DBP also binds to five of these sites (sites 3, 4, 6, 7, and 9). Utilizing transient transfection studies in HepG2 cells, we have shown that deletion of the factor VIII promoter sequences distal to nucleotide -44 results in a significant but small increase in promoter activity. The activity of each of the various 5' deletion constructs is significantly enhanced by cotransfection of C/EBPalpha and D-site-binding protein expression plasmids, while cotransfection of both C/EBPalpha and C/EBPbeta plasmids resulted in a further enhancement of transactivation. These studies also provide evidence of a repressor element located between nucleotides -740 and -1002. Since the minimal promoter sequence (-44 to +148) maintains the transcriptional activity of the full-length promoter sequence, we proceeded to identify additional factors binding to sites 1 to 4. Competition studies revealed that a ubiquitous transcription factor, NF-Y, binds to site 4, while the liver-enriched transcription factor hepatocyte nuclear factor I (HNF-1) binds to site 1. Mutation analysis of the minimal promoter demonstrated that HNF-1 is critical for activating transcription of the factor VIII gene in vitro. Our results also suggest that the multiple upstream elements that we have identified may act as a backup regulatory region in the event of disruption of the HNF-1 element in the 5' untranslated region.
TL;DR: It is argued that HSF, independent of and dominant among sequence-specific factors binding to the HSC82 upstream region, antagonizes nucleosomal repression and creates an accessible chromatin structure conducive to preinitiation complex assembly and transcriptional activation.
Abstract: Transcription in eukaryotic cells occurs in the context of chromatin. Binding of sequence-specific regulatory factors must contend with the presence of nucleosomes for establishment of a committed preinitiation complex. Here we demonstrate that the high-affinity binding site for heat shock transcription factor (HSF) is occupied independently of other cis-regulatory elements and is critically required for preventing nucleosomal assembly over the yeast HSC82 core promoter under both noninducing (basal) and inducing conditions. Chromosomal mutation of this sequence, termed HSE1, erases the HSF footprint and abolishes both transcription and in vivo occupancy of the TATA box. Moreover, it dramatically reduces promoter chromatin accessibility to DNase I and TaqI, as the nuclease-hypersensitive region is replaced by a localized nucleosome. By comparison, in situ mutagenesis of two other promoter elements engaged in stable protein-DNA interactions in vivo, the GRF2/REB1 site and the TATA box, despite reducing transcription three- to fivefold, does not compromise the nucleosome-free state of the promoter. The GRF2-binding factor appears to facilitate the binding of proteins to both HSE1 and TATA, as these sequences, while still occupied, are less protected from in vivo dimethyl sulfate methylation in a deltaGRF2 strain. Finally, deletion of a consensus upstream repressor sequence (URS1), positioned immediately upstream of the GRF2-HSE1 region and only weakly occupied in chromatin, has no expression phenotype, even under meiotic conditions. However, deletion of URS1, like mutation of GRF2, shifts the translational setting of an upstream nucleosomal array flanking the promoter region. Taken together, our results argue that HSF, independent of and dominant among sequence-specific factors binding to the HSC82 upstream region, antagonizes nucleosomal repression and creates an accessible chromatin structure conducive to preinitiation complex assembly and transcriptional activation.
TL;DR: Transcriptional regulation by glucocorticoids and chronic acidosis was demonstrated and promoter activity was present in OKP cells, a renal proximal tubule cell line, but not in fibroblasts, suggesting that the NHE3 promoter contains elements conferring epithelial cell-specific expression.
Abstract: NHE3, a transmembrane protein involved in transcellular ion transport, is expressed in the apical membrane of renal and gastrointestinal epithelia. Chronic regulation by multiple stimuli, including glucocorticoid-induced transcriptional regulation, has been demonstrated. To study the tissue-specific expression and transcriptional regulation of NHE3, the 5' flanking region of the rat NHE3 gene was cloned. Two genomic libraries were screened with the 5' end of the NHE3 cDNA. The 5' flanking region and first exon were isolated. Primer extension mapped a single transcription start site in stomach, colon and kidney. The NHE3 promoter near the transcription initiation site is characterized by the absence of TATA and CAAT sequences. Two Sp1 sites, one Egr-1 site, and an initiator with the sequence GGGATTAAA mark the area of transcription initiation. Upstream sequences include multiple DNA sequence elements recognized by the glucocorticoid and thyroid receptors, Sp1, atriopeptin-2, and several other transcription factors. Transcriptional regulation by glucocorticoids and chronic acidosis was demonstrated. Promoter activity was present in OKP cells, a renal proximal tubule cell line, but not in fibroblasts. This suggests that the NHE3 promoter contains elements conferring epithelial cell-specific expression.
TL;DR: The presence of a divergently transcribed gene (phhR) encoding an activator protein in the flanking upstream region of the E. coli system is reported, and it is proposed that one or both boxes may be the target of PhhR acting as an autogenous repressor at a 70 promoter in one direction.
Abstract: Pseudomonas aeruginosa was recently found to possess a cluster of structural genes encoding phenylalanine hydroxylase (PhhA), carbinolamine dehydratase (PhhB), and aromatic aminotransferase (PhhC). We now report the presence, in the flanking upstream region, of a divergently transcribed gene (phhR) encoding an activator protein. Inactivation of phhR markedly reduced expression of the structural genes. PhhR belongs to the large prokaryote family of sigma 54 enhancer-binding proteins, and activation of the phh operon by PhhR in P. aeruginosa required rpoN. The closest homologues of PhhR are the TyrR proteins from Escherichia coli and Haemophilus influenzae. E. coli TyrR is an unusual member of the homologue family in that the transcriptional units regulated by tyrR are driven by sigma 70 promoters. P. aeruginosa phhR was able to replace E. coli tyrR as a repressor of the aroF-tyrA operon (but not as an activator of mtr) in the heterologous E. coli system. Two regions that resemble E. coli TyrR boxes were identified in the intervening region between phhR and phhA. We propose that one or both boxes may be the target of PhhR acting as an autogenous repressor at a sigma 70 promoter in one direction. In the other direction, one or both boxes may be the upstream activator sequence targeted by PhhR to facilitate expression of the phh operon from a sigma 54 promoter. The phh operon was strongly induced in fructose- or glucose-based minimal medium by L-phenylalanine. Inactivation of phhR in P. aeruginosa abolished ability to utilize either L-phenylalanine or L-tyrosine as a sole source of carbon for growth.
TL;DR: The T7 RNA polymerase-dependent transcription was studied as a function of nucleotide sequence structures positioned upstream of the T7 promoter as well as model double-stranded DNA templates constructed for this purpose.
Abstract: The T7 RNA polymerase-dependent transcription was studied as a function of nucleotide sequence structures positioned upstream of the T7 promoter Model double-stranded DNA templates were constructed for this purpose They contained a target sequence of 485 base pairs (cDNA fragment of Venesuelian encephalomyelitis equine virus genome), T7 promoter consensus and different extra base sequences upstream of the T7 promoter The level of the target sequence transcription was clearly determined by the extra base sequence The presence of one extra base pair GC ensured the most pronounced effect, transcription was increased one order of magnitude in comparison with template which has only a canonical T7 promoter sequence at the 5'-end
TL;DR: Results suggest that the cooperation of different transcription factors is essential for IL-9 gene expression in T cells, and that NF-κB, c-Jun, and potentially novel proteins can bind to this important sequence.
TL;DR: Analysis of the human genomic DNA sequence upstream of the B29 ATG start codon indicates that B29 gene expression is controlled by the complex interplay of positive and negative regulatory elements.
TL;DR: The above results correlate the asymmetric arrangement of cis-acting elements with a differential expression of the reiterated nifH genes, both in culture and during symbiosis with bean plants.
Abstract: In Rhizobium etli the nitrogenase reductase genes are reiterated. Strain CE3 has three copies; nifHa and nifHb form part of nifHDK operons with the nitrogenase structural genes, while nifHc is linked to a truncated nifD homolog. Their sequences are identical up to 6 residues upstream from a sigma54-dependent promoter. A remarkable difference among them is the absence of canonical NifA binding sites upstream of nifHc while a canonical binding site is located 200 bp upstream of nifHa and nifHb. To evaluate the transcriptional regulation of the reiterated nifH genes, we constructed fusions of nifHa and nifHc with the lacZ gene of Escherichia coli. Both genes were expressed at maximum levels under 1% oxygen in free-living cultures, and their expression declined as the oxygen concentration was increased. This expression was dependent on the integrity of nifA, and nifHc was expressed at higher levels than nifHa. The same pattern was observed with root nodule bacteroids. Expression of both genes in E. coli required sigma54 in addition to NifA bound to the upstream activator sequence. In vivo dimethyl sulfate footprinting analyses showed that NifA binds to the canonical site upstream of nifHa and to a TGT half-site 6 nucleotides further upstream. NifA protected an imperfect binding site upstream of nijHc at position 85 from the promoter. The integration host factor stimulated each gene differently, nifHa being more dependent on this protein. The above results correlate the asymmetric arrangement of cis-acting elements with a differential expression of the reiterated nifH genes, both in culture and during symbiosis with bean plants.
TL;DR: The mutational analysis suggests that the unique architecture of the E2-early promoter necessitates the concerted interaction of all three host transcription factors with their cognate recognition elements to form a stable and functional transcription complex.