Showing papers on "Upstream activating sequence published in 2000"

A computational analysis of whole-genome expression data reveals chromosomal domains of gene expression.

Abstract: Chromosome correlation maps display correlations between the expression patterns of genes on the same chromosome. Using these maps, we show here that adjacent pairs of genes, as well as nearby non-adjacent pairs of genes, show correlated expression independent of their orientation. We present specific examples of adjacent pairs with highly correlated expression patterns, in which the promoter of only one of the two genes contains an upstream activating sequence (UAS) known to be associated with that expression pattern. Finally, we show that genes with similar functions tend to occur in adjacent positions along the chromosomes. Our results suggest that, in certain chromosomal expression domains, an UAS can affect the transcription of genes that are not immediately downstream from it.

Mediator of transcriptional regulation.

Abstract: ▪ Abstract Three lines of evidence have converged on a multiprotein Mediator complex as a conserved interface between gene-specific regulatory proteins and the general transcription apparatus of eukaryotes. Mediator was discovered as an activity required for transcriptional activation in a reconstituted system from yeast. Upon resolution to homogeneity, the activity proved to reside in a 20-protein complex, which could exist in a free state or in a complex with RNA polymerase II, termed holoenzyme. A second line of evidence came from screens in yeast for mutations affecting transcription. Two-thirds of Mediator subunits are encoded by genes revealed by these screens. Five of the genetically defined subunits, termed Srbs, were characterized as interacting with the C-terminal domain of RNA polymerase II in vivo, and were shown to bind polymerase in vitro. A third line of evidence has come recently from studies in mammalian transcription systems. Mammalian counterparts of yeast Mediator were shown to interac...

IL-10 gene expression is controlled by the transcription factors Sp1 and Sp3.

Abstract: IL-10 is an 18-kDa cytokine with a key role in homeostatic control of inflammatory and immune responses. We have investigated how transcription of the IL-10 gene is regulated, so as to be able to understand the circumstances of IL-10 expression in both health and disease. In the mouse, IL-10 gene expression is regulated by a TATA-type promoter with a critical cis-acting element containing GGA repeats located at −89 to −77. Its complementary sequence is similar to the cis-acting elements (TCC repeats) in the promoters of genes encoding epidermal growth factor receptor and CD58. All these elements comprise a common CCTCCT sequence with less conserved C + T-rich sequences. Eliminating this CCTCCT sequence results in a marked reduction in promoter activity, suggesting a necessary role in IL-10 gene expression. Despite its dissimilarity to the G + C-rich Sp1 consensus sequence (GC box), Sp1 and Sp3 transcription factors could be shown to bind to this motif. The requirement for Sp1 and Sp3 in transcription of IL-10 was confirmed using Drosophila SL2 cells, which lack endogenous Sp factors. These results suggest that the transcription of IL-10 is positively regulated by both Sp1 and Sp3.

Critical role of C/EBPδ and C/EBPβ factors in the stimulation of the cyclooxygenase‐2 gene transcription by interleukin‐1β in articular chondrocytes

Abstract: The activity of the [2831; 1103] promoter of the human cyclooxygenase-2 gene in cultured rabbit chondrocytes is stimulated 2.9 ^ 0.3-fold by interleukin-1b and this stimulation depends on [2132; 2124] C/EBP bindingand [2223; 2214] NF-kB binding-sites. The C/EBPb and C/EBPd factors bind to the [2132; 2124] sequence. The [261; 253] sequence is also recognized by C/EBPb and C/EBPd as well as USF. Mutation of the whole [261; 253] sequence abolished the stimulation of transcription but single mutations of the C/EBP or USF site did not alter the activity of the promoter, suggesting that the factors bound to the proximal [261; 253] sequence interact with different members of the general transcription machinery. The [2223; 2214] site binds only the p50/p50 homodimer and a non-rel-related protein, but not the transcriptionally active heterodimer p50/p65. The p50/p50 homodimer could interact with the C/EBP family members bound to the [2132; 2124] sequence for full stimulation of the COX-2 transcription by interleukin-1b in chondrocytes. By contrast, the [2448; 2449] sequence binds with a low affinity both the p50/p50 homodimeric and p50/p65 heterodimeric forms of NF-k Bb ut has no role in the regulation of the human COX-2 promoter in chondrocytes.

Cell-Specific Transcriptional Regulation of Human Leukotriene B4 Receptor Gene

Abstract: Leukotriene B4 (LTB4) is a lipid mediator that activates leukocytes and is involved in host defense and inflammation. BLT1, a high-affinity receptor for LTB4 (originally termed BLT), is expressed exclusively in inflammatory cells and is inducible in macrophages upon activation. The mechanisms of tissue-specific expression and induction of BLT1 are important for the understanding of mechanism of onset and the potential treatment of inflammatory disorders. Here, we report the genomic structure and a promoter analysis of the human BLT1 gene, with an emphasis on the mechanism of cell-specific transcription. No TATA or CAAT elements exist around the transcription initiation sites, but a GC-rich sequence is observed in this region. A reporter gene assay revealed that a region ∼80 basepair upstream from the initiator sequence is required for the basal transcription of the BLT1 gene. Sp1 was found to be a major activator of basal transcription by electrophoretic mobility shift assays and site-directed mutagenesis. The CpG sites of the BLT1 promoter region were highly methylated in BLT1-nonexpressing cells, but not methylated in BLT1-expressing cells. Further, methylation of this region in vitro inhibited the promoter activity to ∼15% of the control. Thus, methylation at CpG sites in the promoter region is important for cell-specific transcription of the BLT1 gene. The promoter region of the BLT1 gene is localized within the open reading frame (ORF) of the BLT2 gene, which encodes a low-affinity receptor for LTB4 (Yokomizo, T., K. Kato, K. Terawaki, T. Izumi, and T. Shimizu. 2000. J. Exp. Med. 192:421–431). To our knowledge, this is the first example of “promoter in ORF” in higher eukaryotes.

Sequence of the 5′-flanking region and promoter activity of the human mucin gene MUC5B in different phenotypes of colon cancer cells

Abstract: Control of gene expression in intestinal cells is poorly understood. Molecular mechanisms that regulate transcription of cellular genes are the foundation for understanding developmental and differentiation events. Mucin gene expression has been shown to be altered in many intestinal diseases and especially cancers of the gastrointestinal tract. Towards understanding the transcriptional regulation of a member of the 11p15.5 human mucin gene cluster, we have characterized 3.55 kb of the 5'-flanking region of the human mucin gene MUC5B, including the promoter, the first two exons and the first intron. We report here the promoter activity of successively 5'-truncated sections of 956 bases of this region by fusing it to the coding region of a luciferase reporter gene. The transcription start site was determined by primer-extension analysis. The region upstream of the transcription start site is characterized by the presence of a TATA box at bases -32/-26, DNA-binding elements for transcription factors c-Myc, N-Myc, Sp1 and nuclear factor kappaB as well as putative activator protein (AP)-1-, cAMP-response-element-binding protein (CREB)-, hepatocyte nuclear factor (HNF)-1-, HNF-3-, TGT3-, gut-enriched Kruppel factor (GKLF)-, thyroid transcription factor (TTF)-1- and glucocorticoid receptor element (GRE)-binding sites. Intron 1 of MUC5B was also characterized, it is 2511 nucleotides long and contains a DNA segment of 259 bp in which are clustered eight tandemly repeated GA boxes and a CACCC box that bind Sp1. AP-2alpha and GATA-1 nuclear factors were also shown to bind to their respective cognate elements in intron 1. In transfection studies the MUC5B promoter showed a cell-specific activity as it is very active in mucus-secreting LS174T cells, whereas it is inactive in Caco-2 enterocytes and HT-29 STD (standard) undifferentiated cells. Within the promoter, maximal transcription activity was found in a segment covering the first 223 bp upstream of the transcription start site. Finally, in co-transfection experiments a transactivating effect of Sp1 on to MUC5B promoter was seen in LS174T and Caco-2 cells.

Stp1p, Stp2p and Abf1p are involved in regulation of expression of the amino acid transporter gene BAP3 of Saccharomyces cerevisiae

Abstract: Expression of the BAP3 gene of Saccharomyces cerevisiae, encoding a branched chain amino acid permease, is induced in response to the availability of several naturally occurring amino acids in the medium. This induction is mediated via an upstream activating sequence (called UAS aa )i n theBAP3 promoter, and dependent on Stp1p, a nuclear protein with zinc finger domains, suggesting that Stp1p is a transcription factor involved in BAP3 expression. In this paper, we show that Stp2p, a protein with considerable similarity to Stp1p, is also involved in the induction of BAP3 expression. To gain more insight into the roles of STP1 and STP2, we have overexpressed both Stp1p and Stp2p in yeast cells. Gel shift assays with the UAS aa as a probe show that the UAS aa can form two major complexes. One complex is dependent on Stp2p overexpression and the other is formed independently of STP1 or STP2, suggesting that the UAS aa is also bound by another factor. Here we show that the other factor is Abf1p, which binds specifically to the UAS aa of BAP3.

Expression of the nifA gene of Herbaspirillum seropedicae: role of the NtrC and NifA binding sites and of the -24/-12 promoter element.

Abstract: The nifA promoter of Herbaspirillum seropedicae contains potential NtrC, NifA and IHF binding sites together with a −12/−24 σN-dependent promoter. This region has now been investigated by deletion mutagenesis for the effect of NtrC and NifA on the expression of a nifA::lacZ fusion. A 5’ end to the RNA was identified at position 641, 12 bp downstream from the −12/−24 promoter. Footprinting experiments showed that the G residues at positions −26 and −9 are hypermethylated, and that the region from −10 to +10 is partially melted under nitrogen-fixing conditions, confirming that this is the active nifA promoter. In H. seropedicae nifA expression from the σN-dependent promoter is repressed by fixed nitrogen but not by oxygen and is probably activated by the NtrC protein. NifA protein is apparently not essential for nifA expression but it can still bind the NifA upstream activating sequence.

An imperfect inverted repeat is critical for DNA binding of the response regulator RegR of Bradyrhizobium japonicum.

Abstract: RegR is the response regulator of the RegSR two-component regulatory system in Bradyrhizobium japonicum. The only target known so far is the fixR-nifA operon, encoding the redox-responsive transcription factor NifA, which activates many genes required for symbiotic nitrogen fixation in soybean nodules. In previous in vivo studies, we identified a 32 bp upstream activating sequence located around position –68, which is essential for RegR-dependent expression of the fixR-nifA operon. Here, we used an in vitro binding-site selection assay (SELEX) to more precisely define the DNA-binding specificity of RegR. The selected sequences comprised an imperfect inverted repeat (GCGGC-N5-GTCGC) which is highly similar to an imperfect inverted repeat in the fixR UAS (GCGAC-N5-GACGC). In a parallel approach, band-shift experiments were performed with oligonucleotides comprising defined point or deletion mutations in the fixR UAS. This led to the identification of 11 critical nucleotides within a 17 bp minimal RegR binding site centered at position –64 upstream of the fixR-nifA transcription start site. Notably, all 11 critical nucleotides were located either within the half sites of the inverted repeat (four nucleotides in each half site) or in the 5 bp spacer that separates the half sites (three nucleotides). Based on these results, we defined a DNA motif comprising those nucleotides that are critical for RegR binding (RegR box; 5′-GNGAGCAGTTNNGNCGC-3′). A comparison of the RegR box with functional binding sites of the RegR-like regulator RegA of Rhodobacter capsulatus revealed considerable similarities. Thus, the RegR box may assist in the identification of new RegR target genes not only in B.japonicum but also in other α-proteobacteria possessing RegR-like response regulators.

Soggy, a spermatocyte-specific gene, lies 3.8 kb upstream of and antipodal to TEAD-2, a transcription factor expressed at the beginning of mouse development

Abstract: Investigation of the regulatory region of mTEAD-2, a gene expressed at the beginning of mouse pre-implantation development, led to the surprising discovery of another gene only 3.8 kb upstream of mTEAD-2. Here we show that this new gene is a single copy, testis-specific gene called SOGGY: (mSgy) that produces a single, dominant mRNA approximately 1.3 kb in length. It is transcribed in the direction opposite to mTEAD-2, thus placing the regulatory elements of these two genes in close proximity. mSgy contains three methionine codons that could potentially act as translation start sites, but most mSGY protein synthesis in vitro was initiated from the first Met codon to produce a full-length protein, suggesting that mSGY normally consists of 230 amino acids (26.7 kDa). Transcription began at a cluster of nucleotides approximately 150 bp upstream of the first Met codon using a TATA-less promoter contained within the first 0.9 kb upstream. The activity of this promoter was repressed by upstream sequences between -0.9 and -2.5 kb in cells that did not express mSgy, but this repression was relieved in cells that did express mSgy. mSgy mRNA was detected in embryos only after day 15 and in adult tissues only in the developing spermatocytes of seminiferous tubules, suggesting that mSgy is a spermatocyte-specific gene. Since mTEAD-2 and mSgy were not expressed in the same cells, the mSgy/mTEAD-2 locus provides a unique paradigm for differential regulation of gene expression during mammalian development.

Analysis of the dormancy-inducible narK2 promoter in Mycobacterium bovis BCG

Abstract: Upon depletion of oxygen, the obligate aerobe mycobacteria switch from growth to a state of non-replicating persistence or dormancy. Here, we report the first functional analysis of a dormancy-dependent mycobacterial promoter in Mycobacterium bovis BCG. Promoter probing using a ′lacZ reporter detected a dormancy-inducible promoter activity upstream of the coding sequence for the putative nitrite extrusion protein NarK2. Primer extension analysis mapped a transcriptional start point 47 bp upstream of the narK2 start codon. Deletion analysis revealed that the sequence −222 to −133 bp upstream from the transcriptional start point was required for basal and dormancy-inducible reporter expression. The sequence +1 to +47 downstream of the transcriptional start point had a strong inhibitory effect on the level of dormancy-induced β-galactosidase activity. The identification of apparent activating and inhibiting regions suggests that the narK2 promoter is at least under dual control.

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

Abstract: 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.

A unique, short sequence determines p53 gene basal and UV-inducible expression in normal human cells.

Abstract: A unique, short sequence determines p53 gene basal and UV-inducible expression in normal human cells

Cloning and characterization of the 5′-upstream sequence governing the cell cycle-dependent transcription of mouse DNA polymerase α 68 kDa subunit gene

Abstract: We have isolated the genomic DNA fragment spanning the 5-end and the first four exons encoding the 68 kDa subunit (p68) of the mouse DNA polymerase alpha-primase complex [corrected]. The p68 promoter region lacks TATA and CAAT boxes, but contains a GC-rich sequence, two palindrome sequences and two putative E2F-binding sites [corrected]. A series of transient expression assays using a luciferase reporter gene indicated that a region from nucleotide position -89 to -30 (-89/-30) with respect to the transcription initiation site is crucial for basal transcription of the p68 gene in proliferating NIH 3T3 cells. In particular, part of the GC-rich sequence (-57/-46) and the palindrome (-81/-62) elements were necessary for promoter activity, both of which share homology with the E-box sequence. Gel mobility shift assays using NIH 3T3 nuclear extracts revealed that the upstream stimulatory factor, known as an E-box-binding protein, binds to these sites. Moreover, we observed binding of E2F to two sites near the transcription initiation site (-11/-3 and +9/+16). A transient luciferase expression assay using synchronized NIH 3T3 cells in G(0)phase revealed that these E2F sites are essential for transcription induction of the p68 gene after serum stimulation, but are dispensable for basal transcription. These results indicate that growth-dependent regulation of transcription of the mouse p68 and p180 genes is mediated by a common factor, E2F; however, basal transcription of the genes, interestingly, is regulated by different transcription factors.

The Role of Sp Family Members, Basic Krupple-Like Factor, and E Box Factors in the Basal and IFN-γ Regulated Expression of the Human Complement C4 Promoter

Abstract: The fourth component of human complement (C4) is a serum protein that is expressed in the liver and other organs. The promoter region of the C4 gene has been analyzed in reporter gene assays in two cell lines that represent hepatic (HepG2) and monocytic (U937) lineages. Analysis indicated that regions important for basal transcription in HepG2 cells included Sp1 and E box sites within the first 100 bp upstream of the transcription initiation site but not the nuclear factor-1 site important in the control of the mouse C4 gene. Also, a region encompassing −468 to −310 was able to repress activity 2-fold. However, when a CACCC or GT box sequence at −140 was mutated the repressive activity of the upstream region resulted in almost no activity. The −140 region consists of a series of four closely positioned GT boxes that were shown to bind Sp1, Sp3, and basic Krupple-like factor in EMSA. This novel two-part regulatory element may be involved in the regulated expression of C4. However, IFN-γ a major activator of C4 expression did not signal through this two-part regulatory element. We were able to map the position of an IFN-γ responsive element in U937. IFN-γ was able to increase transcription by up to 20-fold with mutations in the E box sequence at −78 to −73, thus completely abolishing induction. We conclude that the E box binding factors, which appear to be distinct from upstream stimulatory factors 1 and 2, are totally responsible for IFN-γ induction of C4.

HNF‐4 plays a pivotal role in the liver‐specific transcription of thechipmunk HP‐25 gene

Abstract: The gene for chipmunk hibernation-specific protein HP-25 is expressed specifically in the liver. To understand the transcriptional regulation of HP-25 gene expression, we isolated its genomic clones, and characterized its structural organization and 5′ flanking region. The gene spans approximately 7 kb and consists of three exons. The transcription start site, as determined by primer extension analysis, is located at 113 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 80 bp 5′ flanking sequence was sufficient for the liver-specific promoter activity. In a gel retardation assay using HepG2 nuclear extracts, the 5′ flanking sequence from −74 to −46 showed a shifted band. All cDNA clones isolated by a yeast one-hybrid system for a protein capable of binding to this 5′ flanking sequence encoded HNF-4. HNF-4 synthesized in vitro bound to this sequence in a gel retardation assay. Furthermore, supershift assays with anti-(HNF-4) Ig confirmed that the protein in HepG2 or chipmunk liver nuclear extracts that bound to this sequence was HNF-4. When transfected into HeLa cells, HNF-4 transactivated transcription from the HP-25 gene promoter, and mutation of the HNF-4 binding site abolished transactivation by HNF-4, indicating that HNF-4 plays an important role in HP-25 gene expression.

YHP1 encodes a new homeoprotein that binds to the IME1 promoter in Saccharomyces cerevisiae.

Abstract: The IME1 gene is essential for initiation of meiosis in the yeast Saccharomyces cerevisiae. Transcription of IME1 is detected under conditions of starvation for nitrogen and glucose, and in the presence of the MATa1 and MATα2 gene products. In our previous work, we have shown that there are two elements acting as TUP1-dependent upstream repression sequence (URS) and tup1 mutation-dependent upstream activation sequence (UAS) between nt −915 and −621 of the IME1 promoter under nutritional conditions. The region from −915 to −621 has also been reported to harbour meiotic URS and UAS when a/α cells were transferred to sporulation conditions. To identify proteins that are able to bind to the region, we screened a cDNA library fused with the Gal4-activation domain by means of the one-hybrid system. We identified a previously unknown gene (YDR451c), which we designated YHP1, encoding a homeodomain protein of the Drosophila antennapedia type. The region for binding of Yhp1 was delimited to the 28 bp region between nt −702 and −675 of the IME1 promoter in vivo and in vitro, and the 28 bp region harboured a URS activity in a Yhp1-dependent manner under nutrient growth conditions. Although a yhp1 single-disruption mutation did not give rise to a scorable phenotype under nutritional and sporulation conditions, the level of the YHP1 transcript was significantly lower in the cells grown in acetate medium (presporulation medium) and sporulation medium than those grown in glucose medium, and the reduction of YHP1 transcription in acetate medium coincided with an increment of the IME1 transcript. We suggest that the homeoprotein Yhp1 that binds directly to the 28 bp region of the IME1 promoter is a new repressor acting under glucose growth conditions. Copyright © 2000 John Wiley & Sons, Ltd.

HLA-B locus-specific downregulation in human melanoma requires enhancer A as well as a sequence element located downstream of the transcription initiation site.

Abstract: Human MHC class I molecules are encoded by three different loci (HLA-A, -B, and -C), which are regulated at the transcriptional level through several conserved cis-acting promoter elements. The presence of locus-specific residues throughout the entire promoter region strongly suggests that the various HLA class I loci are differentially regulated. To identify regulatory sequences involved in locus-specific HLA class I gene transcription, a series of truncated HLA-A2 and HLA-B7 promoter-reporter constructs were transfected into melanoma cell lines expressing high and low levels of endogenous HLA-B, but comparable levels of HLA-A. These experiments showed that differential regulation of HLA-B expression in melanoma cell lines is mediated by a previously unidentified co-operative action of enhancer A, located 175 bp upstream of the transcription initiation site (+1), and a specific region of 20 nucleotides located at +13 to +33 bp downstream of the transcription initiation site. Furthermore, we demonstrated binding of transcription factor Yin Yang 1 to the HLA-A +13/+33 bp region, but not to the equivalent HLA-B region. Based on these results, we present a model suggesting that YY1 displaces either activating or repressing transcription factors, thereby making the HLA-A gene resistant to differential regulation.

Identification of functional TTF-1 and Sp1/Sp3 sites in the upstream promoter region of rabbit SP-B gene

Abstract: Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B mRNA is expressed in a cell type-restricted manner in...

Far upstream sequences regulate the human prolactin promoter transcription.

Abstract: The human prolactin gene is mainly expressed in pituitary lactotrope cells, but transcription from an alternative, far upstream promoter was detected in lymphoid, placental and mammary cells. We describe the transcriptional activity in rat pituitary cells of the complete region separating the two promoters, using transient transfection experiments. A far upstream activating region was only functional in combination with the prolactin promoter. DNaseI protection experiments revealed, in addition to binding sites for the pituitary-specific factor Pit-1, sites (e.g. SD1) for several ubiquitous factors and one lymphoid-specific factor (SD4). A single copy of the ubiquitous site SD1 or the lymphoid-specific site SD4 was unable to activate transcription of a heterologous promoter in pituitary cells. However, SD1 activated transcription in nonpituitary cells and SD4 was functional specifically in lymphoid cells. Five copies of a distal site (D8) activated transcription in each cell type tested. Gel retardation experiments show that this site binds the specific factor C/EBP in liver and a distinct factor in other cell types. Our results suggest that different elements within this large region direct specific expression from each promoter via a complex interplay between cell-specific and ubiquitous transcription factors.

Transcriptional regulation of the gene encoding the StAR protein in the human adrenocortical cell line, H295R by cAMP and TGFbeta1.

Abstract: In the present work, we have analyzed the transcriptional regulation of StAR expression in the human adrenocortical cell line H295R. We observed that StAR mRNA levels rapidly increased in response to forskolin, starting after 2 h of treatment and reaching a maximum by 12 h. Deletion analysis of the human StAR promoter demonstrated that the first 150 bp upstream of the transcription start were sufficient for both basal and cAMP-induced expression of a reporter gene. We demonstrate that out of the three binding sequences for the steroidogenic factor 1 (SF-1) only the central one (-105 to -96) is implicated in both basal and cAMP-induced StAR expression. In addition, another important regulatory element for both basal and cAMP-induced StAR expression is present between -62 and -24 upstream of the transcription start. We also show that TGFβ1 inhibits StAR expression at the transcriptional level. We found that the TGFβ-inhibitory element lies between -150 and -85 upstream of the transcription start and that th...