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PubMed1989

Analysis of the upstream region of the Escherichia coli rnd gene encoding RNase D. Evidence for translational regulation of a putative tRNA processing enzyme.

The upstream region of the Escherichia coli rnd gene has been analyzed. A single promoter and transcription start site were identified in the 170-nucleotide upstream region of the clone based on deletion analysis, nuclease S1 mapping, primer extension, and quantitative dot-blots. The start site of transcription is 70 nucleotides upstream of the UUG initiation codon of the RNase D coding region. Between the RNA initiation site and the coding region is a GC-rich hairpin structure followed by 8 T residues that looks like a rho-independent transcription terminator. Surprisingly, deletion of the hairpin structure elevates rnd mRNA levels only slightly (less than 2-fold), but it dramatically decreases RNase D expression (greater than 95%), measured both by activity and immunoblotting. No evidence for an effect on mRNA stability or processing was observed. The uncommon UUG translation initiation codon was converted to AUG by site-directed mutagenesis. This alteration led to an 11-fold elevation of RNase D expression in single-copy plasmids. These data indicate that RNase D expression is negatively regulated at the translational level by the initiation codon, and that an upstream structure (or sequence) also influences translation of this gene.

Base Sequence↗
PubMed1988

Activation of the ompC gene by the OmpR protein in Escherichia coli. The cis-acting upstream sequence can function in both orientations with respect to the canonical promoter.

Expression of the ompC gene coding for an outer membrane protein of Escherichia coli is regulated by a transcriptional activation mechanism that requires the ompR gene product that acts on nucleotide sequences located upstream of the -35 and -10 regions of the ompC promoter. Using an ompC-lacZ fusion gene, the orientation of the cis-acting upstream sequence (OmpR-binding site) with respect to the canonical -35 and -10 regions (RNA polymerase-binding site) was changed. The ompC gene was activated in an OmpR-dependent manner even when the upstream sequence was in the reverse orientation with respect to the -35 and -10 regions, providing that these two DNA elements were aligned stereospecifically on the DNA helix. Evidence is presented that the upstream sequence can increase the transcription efficiency of the ompC promoter in a manner relatively independent of its orientation with respect to the -35 and -10 regions.

Bacterial Outer Membrane Proteins↗
PubMed1993

Mechanisms of upstream activation of the rrnD promoter P1 of Escherichia coli.

The rrn promoter regions of Escherichia coli contain a stretch of DNA, rich in An and Tn homopolymer sequences, which is located upstream of the tandem promoters P1 and P2. We have studied the effects of the upstream sequence of the rrnD operon on promoter function, using deletion variants for in vitro transcription. The presence of the upstream activating sequence (UAS) was found to increase P1 promoter strength without influencing P2. Two modes of P1 activation could be distinguished: a stimulation of P1 depending on the interaction of the factor of inversion stimulation (FIS) with the UAS (within the deletion boundaries of -50 and -112) and second, a factor-independent activation requiring the proximal part of the UAS (within the boundaries of -50 and -89). Both modes of activation were previously observed in the case of the rrnB operon and were ascribed to increased constants of RNA polymerase binding to P1 (Leirmo, S., and Gourse, R. L. (1991) J. Mol. Biol. 220, 555-568; Zacharias, M., Theissen, G., Bradaczek, C., and Wagner, R. (1991) Biochimie 73, 699-712). Our results show, however, that the mechanisms of upstream activation may vary with the reaction conditions. In a complex transcription system, originally designed for the use of cell extracts, FIS enhances first-order reactions that convert binary (open) complexes to transcribing complexes. Initiated complexes are stabilized by FIS. The factor-independent mode of P1 activation involves influences of the UAS on complex isomerizations as well as on binary complex formation. The results show that low molecular weight components of the complex transcription system change the function of the RNA polymerase at the rrn promoters (not at the reference promoter Ptac), so that the conversion of open to transcribing P1-complexes becomes dependent on the UAS and FIS.

Base Sequence↗
PubMed1995

Cooperatively between an upstream TATA-like sequence and a CAA repeated element mediates E1A-dependent negative repression of the H-2Kb class I gene.

In primary rodent cells transformed by the E1A region of the highly oncogenic adenovirus type 12, repression of transcription mediated by the far upstream TATA-like element was observed only in conjunction with either possible juxtaposition of a CAA repeated element in the presence of E1A and was dependent upon the relative arrangement of both the TATA-like and CAA repeated motifs in both homologous and heterologous promoter constructs. A gel shift competition study demonstrated that the TATA-binding protein (TBP) or a TBP-like protein can bind to both the upstream TATA-like sequence and the regular TATA box on the H-2Kb basal promoter. Moreover, employing immunoselection and cyclic amplification and selection of targets (CASTing) methods with nuclear extracts derived from Ad12-E1A transformants, we have identified a high affinity binding site in the H-2Kb class I promoter for E1A-associated DNA-binding proteins. The sequences of the binding sites were identified and were found to contain both the upstream TATA-like motif and the CAA repeated motifs. Our results suggest that the TATA-like sequence in the far upstream region of the H-2Kb gene is one of the elements that is required for Ad12-E1A-mediated negative repression.

Adenovirus E1A Proteins↗
PubMed1994

The upstream region is required but not sufficient to control mutually exclusive expression of Paramecium surface antigen genes.

Paramecium tetraurelia stock 51 can express at least 11 different surface antigens, yet only one type is found on the surface of a cell at any given time. The mechanism that controls this mutually exclusive expression is unknown. A previous study has shown that the 51A surface antigen gene is regulated at the level of transcription (Gilley, D., Rudman, B. M., Preer, J. R., Jr., and Polisky, B. (1990) Mol. Cell. Biol. 10, 1538-1544). We show here that the 51B surface antigen gene is also transcriptionally regulated, and when the 51A and 51B genes are cotransformed into an A-, B- mutant, the 51A antigen is dominant at 27 degrees C just as in wild type cells. We have utilized this cotransformation system to experimentally determine that 273 base pairs of DNA upstream of the 51A gene is sufficient to allow the dominant expression of A, but 150 base pairs is not adequate. A hybrid gene that contains the upstream region of 51B attached to the 51A transcribed region was cotransformed with the 51B gene into the A-, B- mutant. Despite containing the same upstream sequences, only the hybrid 51B/A was transcribed at 27 degrees C. These results suggest the upstream region is required but not sufficient for mutually exclusive transcriptional control.

Animals↗
PubMed1994

Genomic organization, 5'-upstream sequence, and chromosomal localization of an insulinoma-associated intronless gene, IA-1.

IA-1 is a novel cDNA originally isolated from a human insulinoma subtraction library (ISL-153). It encodes a protein containing both a zinc finger DNA-binding domain and a putative prohormone domain. IA-1 transcripts have been found thus far only in tumors of neuroendocrine origin. Clinical studies have shown that IA-1 is a sensitive marker for neuroendocrine differentiation of human lung tumors. In this study, we cloned and sequenced the entire IA-1 gene and its 5'-upstream region from a human liver genomic library. In situ hybridization localized the IA-1 gene to the short arm of human chromosome 20. Sequence analysis and restriction enzyme mapping showed that the IA-1 gene is uninterrupted and appears to be intronless. Evidence that IA-1 is an intronless gene that can translate into protein was obtained from in vitro translation studies that showed that both IA-1 cDNA and IA-1 genomic DNA yielded identical protein products of approximately 61,000 daltons. Examination of the 5'-upstream region (2090 base pairs) revealed several tissue-specific regulatory elements, including glucokinase upstream promoter elements and a Pit-1 factor binding site. The presence of several different upstream regulatory elements may account for IA-1 gene expression in different neuroendocrine tumors.

Base Sequence↗
PubMed1995

A far-upstream sequence of the wheat histone H3 promoter functions differently in rice and tobacco cultured cells.

The cis-regulatory function of a far-upstream sequence (-1,711 to -186) of the promoter of the wheat gene for histone H3 (TH012) was analyzed in cultured rice and tobacco cells in a transient expression system with the gene for beta-D-glucuronidase as a reporter gene. The far-upstream sequence was necessary for full activity of the H3 promoter in rice cells but did not enhance the activity of the proximal promoter in tobacco cells. Dissection analysis of the far-upstream sequence revealed the existence of several positive and negative cis-acting sequences in this region, some of which functioned differently in rice and tobacco cells. In gain-of-function experiments with rice cells, the sequence from -848 to -704, containing the CCAAT and octamer (CaCGGATC) motifs, functioned in an orientation-independent manner, whereas the sequence from -703 to -486 functioned in an orientation-dependent manner. By contrast, both sequences exhibited an orientation-dependent cis-function in tobacco cells. These findings suggest that some cis-regulatory sequences in the far-upstream region of the H3 promoter function differently in rice and tobacco cells.

Base Sequence↗
PubMed1997

Localization of upstream silencer elements involved in the expression of cone transducin alpha-subunit (GNAT2).

PURPOSE: To localize cis-acting elements involved in the expression of the cone-specific G-protein, cone transducin alpha-subunit (GNAT2). METHODS: In this study, the authors used a genomic clone, HGLG3, to sequence 3139 base pairs of the upstream region of the GNAT2 gene and to localize cis-acting elements involved in the expression of GNAT2. Upstream elements were localized functionally by transfection of chloramphenicol acetyltransferase gene constructs containing nested deletions of this upstream region into WERI-Rb1 cells. Cell specificity of the localized elements was determined by transfection of the HeLa cells. Trans-acting factor-binding sites to functional cis-acting elements were determined by DNasel footprinting. Cell specificity of protein interaction with footprinted regions was tested by electrophoretic mobility shifts with nuclear extracts from WERI-Rb1 and HeLa cells. RESULTS: Transfection of WERI-Rb1 and HeLa cells revealed the presence of a strong, noncell-specific silencer region between -1130 and -23, a weak, cell-specific promoter between -151 and -10, and a stronger, noncell-specific element between +143 and +167. DNaseI footprinting showed three major footprints (S1, S2, and S3) between -807 and -176, indicating the binding sites for putative negative trans-acting factors. Individual footprinted sequences had similar electrophoretic mobility shifts when they were incubated with nuclear extracts from either WERI-Rb1 or HeLa cells, suggesting that these cells express the same negative factors. CONCLUSIONS: The expression of the GNAT2 gene is controlled by a strong silencer region, a weak upstream cell-specific promoter, and a strong downstream element. The silencer region interacts with similar proteins from retina- and nonretina-derived cell lines.

Base Sequence↗
PubMed1998

Methylation alterations of the MyoD1 upstream region are predictive of subclassification of human rhabdomyosarcomas.

MyoD1 expression is a distinguishing characteristic of rhabdomyosarcoma. In this study, distinct methylation alterations were identified in the 5' flanking region of the MyoD1 gene from the two major subtypes, ie, alveolar and embryonal rhabdomyosarcoma. The MyoD1 methylation patterns of 26 rhabdomyosarcomas were compared with that of normal skeletal muscle and nonmuscle specimens by Southern blot analysis using methylation-sensitive restriction enzymes HhaI and HpaII. A 5-kb region immediately upstream of the MyoD1 coding sequence was found to be methylated in adult muscle and all nonmuscle tissues tested. The MyoD1 upstream region was unmethylated in the majority of the alveolar rhabdomyosarcomas (13 of 15, 87%) examined in this study. In contrast, 10 of 11 (91%) embryonal rhabdomyosarcomas showed a methylation pattern that was also observed in fetal muscle cells, in which the CpG sites in the MyoD1 upstream region were partially methylated. Our data suggest that the methylation status of the MyoD1 upstream CpG sites may be related to rhabdomyosarcoma tumorigenesis and may have valuable implications for its differential diagnosis.

Adolescent↗
PubMed2003

Interaction of upstream stimulatory factor proteins with an E-box located within the human CYP1A2 5'-flanking gene contributes to basal transcriptional gene activation.

Cytochrome P450 (CYP)1A2 is abundantly expressed in the liver of all vertebrate species. In most, its expression is restricted to the liver. Sequence analysis of the human CYP1A2 5'-flanking region from +3 to -3201 identified six E-box motifs within the 3-methylcholanthrene (MC) enhancer element (-1987 to -3201). The E-box motif is recognized by members of the basic helix-loop-helix (bHLH) family of transcription factors. Gel mobility shift and antibody supershift assays were used to examine each of the six upstream E-box motifs for their ability to bind nuclear proteins and to compete with the ubiquitously expressed bHLH protein, upstream stimulatory factor (USF), for binding. We found that USF-1 and USF-2 proteins bind to the upstream E-box motifs EB2, EB3, and EB4. Transient transfection assays in HepG2 cells were performed with different segments of the human CYP1A2 5'-flanking region linked to a luciferase reporter gene. Site-directed mutagenesis of one of the E-box motifs, EB2, resulted in a 60% reduction in basal reporter gene activity. Mutations in EB3 and EB4 had no effect. We found that transfection of expression vectors containing USF-1 or USF-2 cDNAs activated CYP1A2 reporter gene activity, while a dominant-negative USF-2 expression vector blocked such activity. Chromatin immunoprecipitation assays confirmed that the interaction of USF proteins with the CYP1A2 EB2 site occurs in vivo. These data support the role of USF as a constitutive transcriptional activator of human CYP1A2.

5' Flanking Region↗
PubMed2004

Human follicle-stimulating hormone receptor (FSH-R) promoter/enhancer activity is inhibited by transcriptional factors, from the upstream stimulating factors family, via E-box and newly identified initiator element (Inr) in FSH-R non-expressing cells.

To localize the regulatory elements in the human follicle-stimulating hormone receptor (FSH-R) promoter/enhancer and to determine the role of upstream stimulatingfactors (USFs) in these elements, we transiently transfected constructs of FSH-R promoter/enhancer in pGL3 luciferase reporter plasmids into Chinese hamster ovary cells and the activities were determined by measuring luciferase luminescence of the cell lysates. The 5'-flanking regions of the human FSH-R gene from nt -1485 to -1 with respect to the gene translation start site were amplified by polymerase chain reaction (PCR) and subcloned in pGL3. Deletion mutants were created using PCR or restriction enzyme digestion. Mutation in the E-box sequence from nt -124 to -119 (E-box 3), in the construct from -224 to nt -1 or in the Inr element, which encompasses the transcriptional start site at nt -99, resulted in a substantial reduction in the human FSH-R promoter/enhancer activity. Overexpression of upstream stimulating factor-1 (USF1) suppresses the activity of the human FSH-R promoter/enhancer via Inr and E-box elements. Upstream stimulating factor-2 (USF2) decreases FSH-R promoter/enhancer activity by acting on E-box 3. The results indicate that E-box 3 and the Inr element are important elements of the human FSH-R promoter/ enhancer. USF family members inhibit FSH-R gene activity by acting via these elements. USF1 and USF2 suppress human FSH-R promoter/enhancer activity by acting on E-box 3. USF1 also decreases activity by interacting with the Inr element.

Animals↗
PubMed1999

Upstream stimulatory factor regulates major histocompatibility complex class I gene expression: the U2DeltaE4 splice variant abrogates E-box activity.

The tissue-specific expression of major histocompatibility complex class I genes is determined by a series of upstream regulatory elements, many of which remain ill defined. We now report that a distal E-box element, located between bp -309 and -314 upstream of transcription initiation, acts as a cell type-specific enhancer of class I promoter activity. The class I E box is very active in a neuroblastoma cell line, CHP-126, but is relatively inactive in the HeLa epithelial cell line. The basic helix-loop-helix leucine zipper proteins upstream stimulatory factor 1 (USF1) and USF2 were shown to specifically recognize the class I E box, resulting in the activation of the downstream promoter. Fine mapping of USF1 and USF2 amino-terminal functional domains revealed differences in their abilities to activate the class I E box. Whereas USF1 contained only an extended activation domain, USF2 contained both an activation domain and a negative regulatory region. Surprisingly, the naturally occurring splice variant of USF2 lacking the exon 4 domain, U2DeltaE4, acted as a dominant-negative regulator of USF-mediated activation of the class I promoter. This latter activity is in sharp contrast to the known ability of U2DeltaE4 to activate the adenovirus major late promoter. Class I E-box function is correlated with the relative amount of U2DeltaE4 in a cell, leading to the proposal that U2DeltaE4 modulates class I E-box activity and may represent one mechanism to fine-tune class I expression in various tissues.

Alternative Splicing↗
PubMed2003

B cell receptor signaling mediates immediate protection from Fas-induced apoptosis upstream of caspase activation through an atypical protein kinase C isozyme and de novo protein synthesis.

Signaling through the B cell receptor (BCR) of normal splenic B cells, as well as B cell lymphoma lines, can abrogate Fas-mediated apoptosis. Using the B lymphoma line A20.2J, BCR signaling immediately inhibited Fas-induced apoptosis upstream of caspase-8 activation, as determined by Ile-Glu-Thr-Asp-(IETD)ase activity and cleavage of the caspase-8 substrate Bid. Furthermore, following overexpression of a human Fas:FLICE construct, which directly induces caspase activation in a death-inducing signaling complex-independent manner, cells could not be protected through BCR stimulation.Co-incubation with cycloheximide partially reversed protection from apoptosis and increased Fas-stimulated initiator and effector caspase activation, suggesting new protein synthesis is necessary to induce protection upstream of caspase activation. Furthermore, co-incubation with a broad-spectrum protein kinase C (PKC) inhibitor, such as bisindolylmaleimide (Bis), also partially reversed protection from apoptosis, and examination of a panel of PKC inhibitors suggested a role for atypical isozymes in protection. Bis also acted to increase initiator and effector caspase activation upon anti-IgG and anti-Fas treatment. These data suggest that BCR-induced protection is being mediated upstream of initiator caspase activation, and is partially dependent upon both PKC family members and new protein synthesis.

Animals↗
PubMed1985

Metal-dependent SV40 viruses containing inducible enhancers from the upstream region of metallothionein genes.

We have isolated SV40 recombinant viruses which are dependent on heavy metal ions for efficient propagation. They were obtained after-co-transfection of enhancerless SV40 DNA (the so-called enhancer trap) with sonicated DNA from the mouse metallothionein-I (mMT-I) or human metallothionein-IIA (hMT-IIA) upstream regions. To substitute for the SV40 enhancer, these viruses have incorporated a segment of the immediate upstream region of the metallothionein genes. Two recombinant viruses of the SVMT-I type carry segments of the mMT-I gene from positions -73 to -187 and -39 to -194 inverted with respect to their natural configuration. The overlapping segment contains two of the four metal-responsive elements involved in the induction of the mMT-I gene by heavy metal ions. The SVMT-II recombinant virus contains a segment of the hMT-IIA gene from position -39 to -366 which harbors the metal- and hormone-responsive elements of the hMT-IIA gene. Insertion of the mMT-I segment downstream of a rabbit beta-globin test gene enhances beta-globin transcription upon metal ion stimulation. This shows that the immediate upstream region of the mouse metalliothionein-I gene, when detached from its TATA box, can act as an inducible enhancer. It may be generally true that the enhancer/promoters of inducible genes are composed of several regulatory sequence elements which are interspersed with constitutive elements. The number and spatial arrangement of these elements probably determines the basal versus induced level of expression.

Animals↗
PubMed1986

A GAL family of upstream activating sequences in yeast: roles in both induction and repression of transcription.

Binding sites for the GAL4-positive regulatory protein have been identified upstream of six galactose-inducible genes of Saccharomyces cerevisiae on the basis of (i) protection in DNAse I footprints, (ii) loss of protection when excess GAL4-binding oligonucleotide is added and (iii) homology with a 23-bp dyad-symmetric consensus sequence. Many of the binding sites have been shown to function as upstream activating sequences. The number of binding sites upstream of the various genes ranges from one to four, but a feature is conserved: in cases of multiple sites there is a pair with highest binding affinity located at dyad--dyad distances of 82--87 bp. We suggest that a pair of sites facilitates repression by the GAL80-negative regulatory protein, on the basis of (i) a correlation of a pair of sites (or only one) with full (or only partial) repression and (ii) the introduction of a second site abolishing transcription occurring with one.

Base Sequence↗
PubMed1986

Removal of positioned nucleosomes from the yeast PHO5 promoter upon PHO5 induction releases additional upstream activating DNA elements.

The chromatin fine structure in the promoter region of PHO5, the structural gene for a strongly regulated acid phosphatase in yeast, was analyzed. An upstream activating sequence 367 bp away from the start of the coding sequence that is essential for gene induction was found to reside in the center of a hypersensitive region under conditions of PHO5 repression. Under these conditions three related elements at positions -469, -245 and -185 are contained within precisely positioned nucleosomes located on both sides of the hypersensitive region. Upon PHO5 induction the chromatin structure of the promoter undergoes a defined transition, in the course of which two nucleosomes upstream and two nucleosomes downstream of the hypersensitive site are selectively removed. In this way approximately 600 bp upstream of the PHO5 coding sequence become highly accessible and all four elements are free to interact with putative regulatory proteins. These findings suggest a mechanism by which the chromatin structure participates in the functioning of a regulated promoter.

Acid Phosphatase↗
PubMed1987

Specific binding of TUF factor to upstream activation sites of yeast ribosomal protein genes.

Transcription activation of yeast ribosomal protein genes is mediated through homologous, 12-nucleotide-long and, in general, duplicated upstream promoter elements (HOMOL1 and RPG, referred to as UASrpg). As shown previously, a yeast protein factor, TUF, interacts specifically with these conserved boxes in the 5'-flanking sequences of the elongation factor genes TEF1 and TEF2 and the ribosomal protein gene RP51A. We have now extended our studies of TUF-UASrpg binding by analysing--using footprinting and gel electrophoretic retardation techniques--the genes encoding the ribosomal proteins L25, rp28 (both copy genes), S24 + L46 and S33. Most, but not all, conserved sequence elements occurring in front of these genes, turned out to represent binding sites for the same factor, TUF. The two functionally important boxes that are found in a tandem arrangement (a characteristic of many rp genes) upstream of the L25 gene are indistinguishable in their factor binding specificity. Large differences were shown to exist in the affinity of the TUF factor for the various individual boxes and in the half-life of the protein-DNA complexes. No binding cooperativity could be demonstrated on adjacent sites on L25 or RP51A promoters. Based on binding data, the UASrpg sequence ACACCCATACAT appears to be the one recognized most efficiently by the TUF factor. Previously, no conserved box was found in front of the gene encoding S33. Nevertheless, complex formation with the protein fraction used was observed in the upstream region of the S33 gene. Competition experiments disclosed the existence of an additional binding component, distinct from TUF. This component may possibly regulate a subset of genes for the translational apparatus.

Fungal Proteins↗
PubMed1987

Trans-activation of an upstream early gene promoter of bovine papilloma virus-1 by a product of the viral E2 gene.

The approximately 1000 nucleotide long upstream regulatory region (URR) of bovine papilloma virus-1 (BPV-1) contains a cis element which responds to trans-activation by a diffusible factor encoded in the viral E2 open reading frame (ORF). A series of URR DNA fragments have been linked to two heterologous genes, bacterial chloramphenicol acetyl transferase (cat) or herpes simplex virus-1 thymidine kinase (tk), and tested in transient transfection assays for transcription initiating at the authentic upstream early viral promoter, P89. Transcriptional activity of the P89 promoter was greatly elevated in the presence of the E2 trans-activator gene product. The E2-responsive cis element (E2R) of P89 has been mapped to sequences -277 to -131 nucleotides upstream from the transcription start site (BPV nucleotide 89). The E2R element functioned as a strong transcriptional enhancer in cis with the SV40 early or the tk promoter in the presence, but not in the absence, of the E2 gene product. However, several heterologous promoters which lack sequences related to the E2R element were also trans-activated in transient cotransfections by a function encoded in the E2 ORF of BPV-1, albeit to a much lesser extent. In addition to activation of early viral gene transcription, the E2 regulatory gene(s) may therefore have the potential to alter cellular gene expression.

Animals↗