Showing papers on "Upstream activating sequence published in 2003"

The biochemistry of peroxisomal β-oxidation in the yeast Saccharomyces cerevisiae

Abstract: Peroxisomal fatty acid degradation in the yeast Saccharomyces cerevisiae requires an array of β-oxidation enzyme activities as well as a set of auxiliary activities to provide the β-oxidation machinery with the proper substrates. The corresponding classical and auxiliary enzymes of β-oxidation have been completely characterized, many at the structural level with the identification of catalytic residues. Import of fatty acids from the growth medium involves passive diffusion in combination with an active, protein-mediated component that includes acyl-CoA ligases, illustrating the intimate linkage between fatty acid import and activation. The main factors involved in protein import into peroxisomes are also known, but only one peroxisomal metabolite transporter has been characterized in detail, Ant1p, which exchanges intraperoxisomal AMP with cytosolic ATP. The other known transporter is Pxa1p–Pxa2p, which bears similarity to the human adrenoleukodystrophy protein ALDP. The major players in the regulation of fatty acid-induced gene expression are Pip2p and Oaf1p, which unite to form a transcription factor that binds to oleate response elements in the promoter regions of genes encoding peroxisomal proteins. Adr1p, a transcription factor, binding upstream activating sequence 1, also regulates key genes involved in β-oxidation. The development of new, postgenomic-era tools allows for the characterization of the entire transcriptome involved in β-oxidation and will facilitate the identification of novel proteins as well as the characterization of protein families involved in this process.

Activation of archaeal transcription by recruitment of the TATA-binding protein

Abstract: The hyperthermophilic archaeon Methanococcus jannaschii encodes two putative transcription regulators, Ptr1 and Ptr2, that are members of the Lrp/AsnC family of bacterial transcription regulators. In contrast, this archaeon's RNA polymerase and core transcription factors are of eukaryotic type. Using the M. jannaschii high-temperature in vitro transcription system, we show that Ptr2 is a potent transcriptional activator, and that it conveys its stimulatory effects on its cognate eukaryal-type transcription machinery from an upstream activating region composed of two Ptr2-binding sites. Transcriptional activation is generated, at least in part, by Ptr2-mediated recruitment of the TATA-binding protein to the promoter.

Association of the Mediator complex with enhancers of active genes.

Abstract: The multiprotein Mediator complex has been shown to interact with gene-specific regulatory proteins and RNA polymerase II in vitro. Here, we use chromatin immunoprecipitation to analyze the recruitment of Mediator to GAL genes of yeast in vivo. We find that Mediator associates exclusively with transcriptionally active and not inactive GAL genes. This association maps to the upstream activating sequence, rather than the core promoter, and is independent of RNA polymerase II, general transcription factors, and core promoter sequences. These findings support the idea of Mediator as a primary conduit of regulatory information from enhancers to promoters in eukaryotic cells.

Amino Acid Starvation and Gcn4p Regulate Adhesive Growth and FLO11 Gene Expression in Saccharomyces cerevisiae

Abstract: In baker's yeast Saccharomyces cerevisiae, cell-cell and cell-surface adhesion are required for haploid invasive growth and diploid pseudohyphal development. These morphogenetic events are induced by starvation for glucose or nitrogen and require the cell surface protein Flo11p. We show that amino acid starvation is a nutritional signal that activates adhesive growth and expression of FLO11 in both haploid and diploid strains in the presence of glucose and ammonium, known suppressors of adhesion. Starvation-induced adhesive growth requires Flo11p and is under control of Gcn2p and Gcn4p, elements of the general amino acid control system. Tpk2p and Flo8p, elements of the cAMP pathway, are also required for activation but not Ste12p and Tec1p, known targets of the mitogen-activated protein kinase cascade. Promoter analysis of FLO11 identifies one upstream activation sequence (UASR) and one repression site (URS) that confer regulation by amino acid starvation. Gcn4p is not required for regulation of the UASR by amino acid starvation, but seems to be indirectly required to overcome the negative effects of the URS on FLO11 transcription. In addition, Gcn4p controls expression of FLO11 by affecting two basal upstream activation sequences (UASB). In summary, our study suggests that amino acid starvation is a nutritional signal that triggers a Gcn4p-controlled signaling pathway, which relieves repression of FLO11 gene expression and induces adhesive growth.

Activation domain–mediator interactions promote transcription preinitiation complex assembly on promoter DNA

Abstract: The interaction of activators with mediator has been proposed to stimulate the assembly of RNA polymerase II (Pol II) preinitiation complexes, but there have been few tests of this model. The finding that the major adenovirus E1A and mitogen-activated protein kinase-phosphorylated Elk1 activation domains bind to Sur2 uniquely among the metazoan mediator subunits and the development of transcriptionally active nuclear extracts from WT and sur2–/– embryonic stem cells, reported here, allowed a direct test of the model. We found that whereas VP16, E1A, and phosphorylated Elk1 activation domains each stimulate binding of mediator, Pol II, and general transcription factors to promoter DNA in extracts from WT cells, only VP16 stimulated their binding in extracts from sur2–/– cells. This stimulation of mediator, Pol II, and general transcription factor binding to promoter DNA correlated with transcriptional activation by these activators in WT and mutant extracts. Because the mutant mediator was active in reactions with the VP16 activation domain, the lack of activity in response to the E1A and Elk1 activation domains was not due to loss of a generalized mediator function, but rather the inability of the mutant mediator to be bound by E1A and Elk1. These results directly demonstrate that the interaction of activation domains with mediator stimulates preinitiation complex assembly on promoter DNA.

Computer modeling of promoter organization as a tool to study transcriptional coregulation

Abstract: Understanding how the regulation of gene networks is orchestrated is an important challenge for characterizing complex biological processes. Gene transcription is regulated in part by nuclear factors that recognize short DNA sequence motifs, called transcription factor binding sites, in most cases located upstream of the gene coding sequence in promoter and enhancer regions. Genes expressed in the same tissue under similar conditions often share a common organization of at least some of these regulatory binding elements. In this way the organization of promoter motifs represents a "footprint" of the transcriptional regulatory mechanisms at work in a specific biologic context and thus provides information about signal and tissue specific control of expression. Analysis of promoters for organizational features as demonstrated here provides a crucial link between the static nucleotide sequence of the genome and the dynamic aspects of gene regulation and expression.

Role of Metazoan Mediator Proteins in Interferon-Responsive Transcription

Abstract: The interferon (IFN)-induced signal transduction and transcription activation complex, ISGF3, is assembled from three proteins, STAT1, STAT2, and IRF9. Of these components, STAT2 provides a fundamental and essential transcriptional activation function for ISGF3. In the present study, we show that ISGF3-mediated transcription is dependent on STAT2 interactions with DRIP150, a subunit of the multimeric Mediator coactivator complex. Other Mediator subunits, DRIP77 and DRIP130, were found either to bind STAT2 without augmenting ISGF3 transcriptional activity or to enhance ISGF3 transcription without binding STAT2, but only DRIP150 both enhanced IFN-dependent transcription and coimmunoprecipitated with STAT2. Endogenous DRIP150 and STAT2 were able to interact in solution, and DNA affinity chromatography and chromatin immunoprecipitation assays demonstrated that DRIP150 binds to the mature, activated ISGF3-DNA complex and is recruited to target gene promoters in an IFN-dependent fashion. IFN-dependent recruitment of DRIP130 to an ISGF3 target promoter and SRB10-STAT2 coprecipitation suggest indirect association with a multisubunit Mediator complex. The site of STAT2 interaction was mapped to DRIP150 residues 188 to 566, which are necessary and sufficient for interaction with STAT2. Expression of this DRIP150 fragment, but not DRIP150 fragments outside the STAT2 interaction region, suppressed ISGF3-mediated transcriptional activity in a dominant-negative fashion, suggesting a direct functional role of this domain in mediating STAT2-DRIP150 interactions. These findings indicate that the IFN-activated ISGF3 transcription factor regulates transcription through contact with DRIP150 and implicate the Mediator coactivator complex in IFN-activated gene regulation.

BETA2 Activates Transcription From the Upstream Glucokinase Gene Promoter in Islet β-Cells and Gut Endocrine Cells

Abstract: Glucokinase (GK) gene transcription initiates in the islet (beta-cell), gut, and brain from promoter sequences residing approximately 35 kbp upstream from those used in liver. Expression of betaGK is controlled in beta-cells by cell-enriched (i.e. pancreatic duodenal homeobox 1 [PDX-1]) and ubiquitously (i.e., Pal) distributed factors that bind to and activate from conserved sequence motifs within the upstream promoter region (termed betaGK). Here, we show that a conserved E-box element also contributes to control in the islet and gut. betaGK promoter-driven reporter gene activity was diminished by mutating the specific sequences involved in E-box-mediated basic helix-loop-helix factor activator binding in islet beta-cells and enteroendocrine cells. Gel shift assays demonstrated that the betaGK and insulin gene E-box elements formed the same cell-enriched (BETA2:E47) and generally distributed (upstream stimulatory factor [USF]) protein-DNA complexes. betaGK E-box-driven activity was stimulated in cotransfection assays performed in baby hamster kidney (BHK) cells with BETA2 and E47, but not USF. Chromatin immunoprecipitation assays performed with BETA2 antisera showed that BETA2 occupies the upstream promoter region of the endogenous betaGK gene in beta-cells. We propose that BETA2 (also termed NeuroD1) regulates betaGK promoter activity.

Unique Architecture of the Plastid Ribosomal RNA Operon Promoter Recognized by the Multisubunit RNA Polymerase in Tobacco and Other Higher Plants

Abstract: Expression of the plastid rRNA operon (rrn) during development is highly regulated at the level of transcription. The plastid rrn operon in most higher plants is transcribed by the plastid-encoded RNA polymerase (PEP), the multisubunit plastid RNA polymerase from PrrnP1, a σ70-type promoter with conserved −10 and −35 core promoter elements. To identify functionally important sequences, the tobacco PrrnP1 was dissected in vivo and in vitro. Based on in vivo deletion analysis, sequences upstream of nucleotide −83 do not significantly contribute to promoter function. The in vitro analyses identified an essential hexameric sequence upstream of the −35 element (GTGGGA; the rRNA operon upstream activator [RUA]) that is conserved in monocot and dicot species and suggested that the −10 element plays only a limited role in PrrnP1 recognition. Mutations in the initial transcribed sequence (+9 to +14) enhanced transcription, the characteristic of strong promoters in prokaryotes. We propose that σ interaction with the −10 element in PrrnP1 is replaced in part by direct PEP-RUA (protein–DNA) interaction or by protein–protein interaction between the PEP and an RUA binding transcription factor.

Role of the yeast VAP homolog, Scs2p, in INO1 expression and phospholipid metabolism.

Abstract: In the yeast Saccharomyces cerevisiae, the expression of phospholipid biosynthetic genes, including the INO1 gene (encoding inositol-1-phosphate synthase), is coordinately regulated by a cis-acting transcriptional element, UAS(INO) (inositol-sensitive upstream activating sequence). For this paper we studied the effect of SCS2 disruption on INO1 expression. SCS2 encodes a type II membrane protein and its deletion leads to inositol auxotrophy at temperatures above 34 degrees C. We found that the expression of the INO1 gene was reduced in the scs2Delta strain even when the cells were cultured under derepressing conditions for INO1 expression. However, the beta-galactosidase gene fused with the INO1 promoter region was expressed normally in the scs2Delta strain. The phospholipid composition of scs2Delta cells was not dramatically changed compared with wild-type cells at 28 degrees C, but the phosphatidylinositol level was reduced in scs2Delta cells cultured at 34 degrees C. In addition, elevated phosphatidylcholine synthesis through the CDP-choline pathway was observed in the scs2Delta strain, and the disruption of genes involved in the CDP-choline pathway rescued the INO1 expression defect of the scs2Delta strain. These results indicate that Scs2p can contribute to coordinated phospholipid metabolism including INO1 expression by regulating phosphatidylcholine synthesis through the CDP-choline pathway.

RNA sequences that work as transcriptional activating regions

Abstract: We describe a set of RNA molecules that work as transcriptional activators when tethered to DNA. These RNA activating regions were found amongst a randomized set of molecules bearing variants of a 10 nt loop attached to an RNA stem. The various RNA activating regions all bear an identical five- residue sequence with an interspersed sixth residue. The result shows that although all natural activating regions characterized thus far are peptidic, this function can be served by other kinds of moieties as well.

Distinct Transcriptional Pathways Regulate Basal and Activated Major Histocompatibility Complex Class I Expression

Abstract: Transcription of major histocompatibility complex (MHC) class I genes is regulated by both tissue-specific (basal) and hormone/cytokine (activated) mechanisms. Although promoter-proximal regulatory elements have been characterized extensively, the role of the core promoter in mediating regulation has been largely undefined. We report here that the class I core promoter consists of distinct elements that are differentially utilized in basal and activated transcription pathways. These pathways recruit distinct transcription factor complexes to the core promoter elements and target distinct transcription initiation sites. Class I transcription initiates at four major sites within the core promoter and is clustered in two distinct regions: "upstream" (-14 and -18) and "downstream" (+12 and +1). Basal transcription initiates predominantly from the upstream start site region and is completely dependent upon the general transcription factor TAF1 (TAF(II)250). Activated transcription initiates predominantly from the downstream region and is TAF1 (TAF(II)250) independent. USF1 augments transcription initiating through the upstream start sites and is dependent on TAF1 (TAF(II)250), a finding consistent with its role in regulating basal class I transcription. In contrast, transcription activated by the interferon mediator CIITA is independent of TAF1 (TAF(II)250) and focuses initiation on the downstream start sites. Thus, basal and activated transcriptions of an MHC class I gene target distinct core promoter domains, nucleate distinct transcription initiation complexes and initiate at distinct sites within the promoter. We propose that transcription initiation at the core promoter is a dynamic process in which the mechanisms of core promoter function differ depending on the cellular environment.

The PAX6 gene is activated by the basic helix-loop-helix transcription factor NeuroD/BETA2.

Abstract: PAX6 is a transcription factor that plays an important role during pancreatic morphogenesis. The aim of the present study is to identify the upstream activator(s) of the PAX6 gene possibly involved in the early stages of pancreatic differentiation. Recently, individual elements regulating PAX6 gene activity in the pancreas have been identified in a 1100 bp Spe / Hin cII fragment 4.6 kb upstream of exon 0. Preliminary sequence analysis of this region revealed some potential DNA-binding sites (E boxes) specific for the binding of basic helix-loop-helix transcription factors. By using electrophoretic mobility shift assays, we demonstrated that both nuclear protein extracts from insulin-secreting RINm5F cells and in vitro -translated NeuroD/BETA2 can bind specifically to these E boxes. Furthermore, by transient transfection experiments we demonstrated that the expression of basic helix-loop-helix transcription factor NeuroD/BETA2 can induce activation of the PAX6 promoter in the NIH-3T3 cell line. Thus we show that NeuroD/BETA2 is involved in the activation of the expression of PAX6 through E boxes in the PAX6 promoter localized in a 1.1 kb sequence within the 4.6 kb untranslated region upstream of exon 0.

Reciprocal regulation of expression of the human adenosine 5'-triphosphate binding cassette, sub-family A, transporter 2 (ABCA2) promoter by the early growth response-1 (EGR-1) and Sp-family transcription factors.

Abstract: The human ABCA2 transporter gene encodes a member of a large family of ATP-binding proteins that transport a variety of macromolecules across biological membranes. We have performed luciferase reporter gene assays with promoter constructs comprising the 5′-flanking region to identify cis-regulatory DNA elements and have mapped the minimal promoter region to 321 bp upstream of the translation start site. We have discovered a functional role for two GC-boxes located in the proximal promoter of the ABCA2 gene that contain overlapping sites for the EGR-1 and Sp1 transcription factors. We observed that oligonucleotides containing overlapping EGR-1/Sp1 sites bind the Sp1, Sp3 and Sp4 transcription factors. When BE(2)-M17 cells were treated with phorbol 12-myristate 13-acetate, we observed inducible expression and binding of the EGR-1 transcription factor to the two GC-boxes. Transfection of Sp1, Sp3 or Sp4 expression constructs into Drosophila S2 induced a dose-dependent increase in transcriptional activation of the ABCA2 promoter, but transfection of EGR-1 alone failed to activate transcription. When increasing amounts of EGR-1 were transfected into the BE(2)-M17 neuroblastoma cells we observed a dose-dependent decrease in expression of the ABCA2 promoter, although expression of the endogenous ABCA2 gene increased following transfection of EGR-1.

Rsf1p, a protein required for respiratory growth of Saccharomyces cerevisiae

Abstract: A central problem in our understanding of mitochondrial (mt) function remains the question of how coordinate transcriptional control is accomplished between nucleus and mitochondria. Here, we report the initial characterization of a protein of previously unknown function, the product of the YMR030 W gene, that appears to mediate such coordinate gene expression. Expression of YMR030 W is glucose-repressible; a deletion mutant for this gene shows a severe growth defect on glycerol-, but not glucose- or ethanol-based medium. In that mutant, transcript levels from GUT1 and GUT2 are highly attenuated compared with those of the wild-type parent when both are grown on glycerol-based medium. Under the same growth conditions, transcripts from the mt OLI1 gene, which has one copy of a mt upstream activating sequence (UAS) in its 5′-flanking region, are attenuated in the ΔYMR030 W mutant, but mRNA from the mt COX3 (OXI2) gene, which lacks the mt UAS, are not. Some nuclear genes encoding mt-related proteins also show low transcript levels in the ΔYMR030 W mutant in comparison with those of the wild-type parent strain during glycerol-based growth. Localization of the protein, via its expression fused to green fluorescent protein, indicates that it is present in both nucleus and mitochondria, supporting a respiration-related transcriptional role for this gene product in both cellular genetic compartments. Because of its role in both respiratory growth and mt function, we designate the YMR030 W coding sequence RSF1 (respiration factor 1).

Yeast Two Hybrid Assay: A Fishing Tale

Abstract: Galactose metabolism Galactose is imported into the cell and converted to galactose-6-phosphate by six enzymes (GAL1, GAL2, PGM2, GAL7, GAL10, MEL1) which are transcriptionally regulated by the proteins Gal80, Gal3, and Gal4, the latter of which plays the central role of DNA-binding transactivator (Figure 1). Gal80 binds Gal4 and inhibits its transcriptional ability. Gal3, in the presence of galactose, binds and causes a conformational change in Gal80, which then allows Gal4 to function as a transcriptional activator . Gal4, like other transcriptional activators, is a modular protein that requires both DNA-binding (BD) and activation domains (AD) . The "two hybrid" technique exploits the fact that Gal4 cannot function as a transcriptional activator unless physically bound to an activation domain. Furthermore, it has been demonstrated that this interaction does not need to be covalent: an experiment was performed where the negative regulatory protein, Gal80, was fused with an activation domain to produce Gal80-AD, and was able to act as a transcriptional activator through its natural binding interaction with a Gal4 protein that was missing its own activation domain . Based on the above observations, a two hybrid assay is performed by expressing two fusion proteins in yeast, the "hunter" and the "bait", where the "hunter" protein is the potential binding partner fused to a yeast activation domain, and the bait is your protein of interest is fused to a yeast binding domain. The hunter and bait constructs are co-transfected into the yeast strain, containing the appropriate “Upstream Activating Sequence” (UAS) proximal to a reporter gene, which is expressed if a binding interaction occurs between the hunter and the bait (Figure 3). To identify proteins that may interact with the bait, a plasmid library, expressing cDNA-encoded AD-fusion proteins, can be screened by introduction into a yeast

The first intron of the human osteopontin gene contains a C/EBP-beta-responsive enhancer.

Abstract: The osteopontin (OPN) protein is found expressed at high level in several processes including fibrotic evolution of organ injuries, tumorigenesis, and immune response. The molecular mechanisms that underly overexpression, especially at the transcriptional level, have been only partially clarified. Therefore, this study was undertaken in search for additional DNA elements in the regulatory regions of the OPN gene and cognate transcription factors. Our results on the region upstream of the transcription start site confirmed that essential elements are located within the first 100 bp. Analysis of the sequence that includes the first untranslated exon and first intron revealed that it could enhance the promoter activity. Experiments of transfection of constructs containing different fragments of this sequence showed that most of the enhancer activity was confined in the terminal 30-bp tract of the first intron, although it was not functioning in a myofibroblast cell line. DNA/protein binding assays and cotransfection experiments showed that the C/EBP-beta transcription factor was able to bind a recognition sequence in this 30-bp segment. We found a bi-allelic sequence polymorphism at +245 in the first intron, which did not show a significant functional effect, but is a useful tool for future association studies.

Specificity of the interaction of RocR with the rocG–rocA intergenic region in Bacillus subtilis

Abstract: In Bacillus subtilis, expression of the rocG gene, encoding glutamate dehydrogenase, and the rocABC operon, involved in arginine catabolism, requires SigL (σ54)-containing RNA polymerase as well as RocR, a positive regulator of the NtrC/NifA family. The RocR protein was purified and shown to bind specifically to the intergenic region located between rocG and the rocABC operon. DNaseI footprinting experiments were used to define the RocR-binding site as an 8 bp inverted repeat, separated by one base pair, forming an imperfect palindrome which is present twice within the rocG–rocABC intergenic region, acting as both a downstream activating sequence (DAS) and an upstream activating sequence (UAS). Point mutations in either of these two sequences significantly lowered expression of both rocG and rocABC. This bidirectional enhancer element retained partial activity even when moved 9 kb downstream of the rocA promoter. Electron microscopy experiments indicated that an intrinsically curved region is located between the UAS/DAS region and the promoter of the rocABC operon. This curvature could facilitate interaction of RocR with σ54-RNA polymerase at the rocABC promoter.

The Drosophila selenoprotein BthD is required for survival and has a role in salivary gland development.

Abstract: Selenium is implicated in many diseases, including cancer, but its function at the molecular level is poorly understood. BthD is one of three selenoproteins recently identified in Drosophila. To elucidate the function of BthD and the role of selenoproteins in cellular metabolism and health, we analyzed the developmental expression profile of this protein and used inducible RNA interference (RNAi) to ablate function. We find that BthD is dynamically expressed during Drosophila development. bthD mRNA and protein are abundant in the ovaries of female flies and are deposited into the developing oocyte. Maternally contributed protein and RNA persist during early embryonic development but decay by the onset of gastrulation. At later stages of embryogenesis, BthD is expressed highly in the developing salivary gland. We generated transgenic fly lines carrying an inducible gene-silencing construct, in which an inverted bthD genomic-cDNA hybrid is under the control of the Drosophila Gal4 upstream activation sequence system. Duplex RNAi induced from this construct targeted BthD mRNA for destruction and reduced BthD protein levels. We found that loss of BthD compromised salivary gland morphogenesis and reduced animal viability.

Sp1 and Sp3 Are Important Regulators of AP-2γ Gene Transcription

Abstract: AP-2γ is a member of the AP-2 transcription factor family, is highly enriched in the trophoblast cell lineage, and is essential for placental development. In an effort to identify factors regulating AP-2γ gene expression, we isolated and characterized the promoter and 5′-flanking region of the mouse and human AP-2γ genes. The transcription start site of the mouse AP-2γ gene was mapped by primer extension and 5′ rapid amplification of cDNA ends. Deletion analysis of the 5′-flanking region revealed a 704-base pair (bp) sequence located approximately 6 kilobases (kb) upstream of the transcription start site that is required for enhanced expression in trophoblast cells. Additional gene transfer studies showed that basal promoter activity resides within a highly conserved, approximately 200-bp DNA sequence located immediately upstream of the transcription start site. The conserved region is highly GC-rich and lacks typical TATA or CCAAT boxes. Multiple potential Sp- and AP-2-binding sites are clustere...

The essential transcription factor Reb1p interacts with the CLB2 UAS outside of the G2/M control region

Abstract: Regulation of CLB2 is important both for completion of the normal vegetative cell cycle in Saccharo myces cerevisiae and for departure from the vegetative cell cycle upon nitrogen deprivation. Cell cycle-regulated transcription of CLB2 in the G2/M phase is known to be brought about by a set of proteins including Mcm1p, Fkh2/1p and Ndd1p that associate with a 35 bp G2/M-specific sequence common to a set of co-regulated genes. CLB2 transcription is regulated by additional signals, including by nitrogen levels, by positive feedback from the Clb2–Cdc28 kinase, and by osmotic stress, but the corresponding regulatory sequences and proteins have not been identified. We have found that the essential Reb1 transcription factor binds with high affinity to a sequence upstream of CLB2, within a region implicated previously by others in regulated expression, but upstream of the known G2/M-specific site. CLB2 sequence from the region around the Reb1p site blocks activation by the Gal4 protein when positioned downstream of the Gal4-binding site. Since a mutation in the Reb1p site abrogates this effect, we suggest that Reb1p is likely to occupy this site in vivo.