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Gene silencing in budding yeast is mediated by Sir protein binding to unacetylated nucleosomes to form a chromatin structure that inhibits transcription. Transcriptional silencing is characterized by the high-fidelity transmission of the silent state. Despite its relative stability, the constituent parts of the silent state are in constant flux, giving rise to a model that silent loci can tolerate such fluctuations without functional consequences. However, the level of tolerance is unknown, and we developed methods to measure the threshold of histone acetylation that causes the silent chromatin state to switch to the active state as well as to measure the levels of the enzymes and structural proteins necessary for silencing. We show that loss of silencing required 50 to 75% acetyl-mimic histones, though the precise levels were influenced by silencer strength and upstream activating sequence (UAS) enhancer/promoter strength. Measurements of repressor protein levels necessary for silencing showed that reducing SIR4 gene dosage two- to threefold significantly weakened silencing, though reducing the gene copy numbers for Sir2 or Sir3 to the same extent did not significantly affect silencing suggesting that Sir4 was a limiting component in gene silencing. Calculations suggest that a mere twofold reduction in the ability of acetyltransferases to acetylate nucleosomes across a large array of nucleosomes may be sufficient to generate a transcriptionally silent domain.

Female mosquitoes require a blood meal for reproduction, and this blood meal provides the underlying mechanism for the spread of many important vector-borne diseases in humans. A deeper understanding of the molecular mechanisms linked to mosquito blood meal processes and reproductive events is of particular importance for devising innovative vector control strategies. We found that the conserved microRNA miR-8 is an essential regulator of mosquito reproductive events. Two strategies to inhibit miR-8 function in vivo were used for functional characterization: systemic antagomir depletion and spatiotemporal inhibition using the miRNA sponge transgenic method in combination with the yeast transcriptional activator gal4 protein/upstream activating sequence system. Depletion of miR-8 in the female mosquito results in defects related to egg development and deposition. We used a multialgorithm approach for miRNA target prediction in mosquito 3' UTRs and experimentally verified secreted wingless-interacting molecule (swim) as an authentic target of miR-8. Our findings demonstrate that miR-8 controls the activity of the long-range Wingless (Wg) signaling by regulating Swim expression in the female fat body. We discovered that the miR-8/Wg axis is critical for the proper secretion of lipophorin and vitellogenin by the fat body and subsequent accumulation of these yolk protein precursors by developing oocytes.

Monoamines, including dopamine (DA), have been linked to aggression in various species. However, the precise role or roles served by the amine in aggression have been difficult to define because dopaminergic systems influence many behaviors, and all can be altered by changing the function of dopaminergic neurons. In the fruit fly, with the powerful genetic tools available, small subsets of brain cells can be reliably manipulated, offering enormous advantages for exploration of how and where amine neurons fit into the circuits involved with aggression. By combining the GAL4/upstream activating sequence (UAS) binary system with the Flippase (FLP) recombination technique, we were able to restrict the numbers of targeted DA neurons down to a single-cell level. To explore the function of these individual dopaminergic neurons, we inactivated them with the tetanus toxin light chain, a genetically encoded inhibitor of neurotransmitter release, or activated them with dTrpA1, a temperature-sensitive cation channel. We found two sets of dopaminergic neurons that modulate aggression, one from the T1 cluster and another from the PPM3 cluster. Both activation and inactivation of these neurons resulted in an increase in aggression. We demonstrate that the presynaptic terminals of the identified T1 and PPM3 dopaminergic neurons project to different parts of the central complex, overlapping with the receptor fields of DD2R and DopR DA receptor subtypes, respectively. These data suggest that the two types of dopaminergic neurons may influence aggression through interactions in the central complex region of the brain involving two different DA receptor subtypes.

In earlier studies, we identified short (6- to 22-nt) sequences that functioned as internal ribosome entry sites (IRESes) and enhanced translation. The size of these IRES elements suggested that they might be prevalent within the messenger population and that individual elements might affect the translation of different groups of mRNAs. To begin to assess the number of different IRES elements in mammalian cells, we have developed a powerful method that uses a positive feedback mechanism to amplify the activities of individual IRES elements. This method uses a vector that encodes a dicistronic mRNA with a reporter gene ( Renilla luciferase or the EGFP) as the first cistron and the yeast Gal4/viral protein 16 (VP16) transcription factor as the second cistron. Transcription of this mRNA is driven by a minimal promoter containing four copies of the Gal4 upstream activation sequence. In this method, the presence of an IRES in the intercistronic region facilitates the translation of Gal4/VP16, which binds to the upstream activation sequences and triggers a positive feedback loop that escalates the production of dicistronic mRNA and Gal4/VP16. A corresponding increase in the translation of the first cistron (luciferase or EGFP) is monitored either by measuring luciferase activity or by using FACS. The latter enables IRES-positive cells to be isolated. We present tests of the feedback mechanism by using an IRES module from Gtx homeodomain mRNA and an IRES from hepatitis C virus and demonstrate the utility of this vector system for the screening, identification, and analysis of IRES elements.

The C-terminal domains of the two α-subunits (αCTD) in Escherichia coli RNA polymerase (RNAP) recognize specific sequences called UP elements in some promoters. These interactions can increase transcription dramatically. Previously, effects of upstream DNA–αCTD interactions on transcription were quantified relative to control promoters with nonspecific DNA sequences substituted for UP elements. However, contributions of nonspecific upstream DNA–αCTD interactions to promoter activity have not been evaluated extensively. Here, we examine effects of removal of αCTD, upstream promoter DNA, or both on the rate of open-complex formation with promoters that lack UP elements. Deletion of αCTD decreased the composite second-order association rate constant, k a , of RNAP for the lacUV5 promoter by ≈10-fold. Much of this effect was attributable to a decrease in the isomerization rate constant, k 2 . Removal of promoter DNA upstream of the -35 element also decreased both k a and k 2 ≈10-fold. Upstream DNA extending approximately to base pair -100 was sufficient for maximal association rates of wild-type RNAP with lacUV5 promoter fragments. The αCTD and upstream DNA did not affect dissociation rates from the open complex. We suggest that sequence-independent upstream DNA interactions with αCTD are major contributors to initiation at many (or all) promoters (not merely promoters containing UP elements) and that these interactions facilitate isomerization events occurring well downstream of the α-binding sites. In addition to highlighting the functional importance of nonspecific protein–DNA interactions, these results suggest also that UP element–αCTD interactions play an even larger role in transcription initiation than appreciated previously.

Binding of activators to upstream DNA sequences regulates transcription initiation by affecting the stability of the initial RNA polymerase (RNAP)–promoter complex and/or the rate of subsequent conformational changes required to form the open complex (RP O ). Here we observe that the presence of nonspecific upstream DNA profoundly affects an early step in formation of the transcription bubble. Kinetic studies with the λP R promoter and Escherichia coli RNAP reveal that the presence of DNA upstream of base pair -47 greatly increases the rate of forming RP O , without significantly affecting its rate of dissociation. We find that this increase is largely due to an acceleration of the rate-limiting step (isomerization) in RP O formation, a step that occurs after polymerase binds. Footprinting experiments reveal striking structural differences downstream of the transcription start site (+1) in the first kinetically significant intermediate when upstream DNA is present. On the template strand, the DNase I downstream boundary of this early intermediate is +20 when upstream DNA is present but is shortened by approximately two helical turns when upstream DNA beyond -47 is removed. KMnO 4 footprinting reveals an identical initiation bubble (-11 to +2), but unusual reactivity of template strand upstream cytosines (-12, -14, and -15) on the truncated promoter. Based on this work, we propose that early wrapping interactions between upstream DNA and the polymerase exterior strongly affect the events that control entry and subsequent unwinding of the DNA start site in the jaws of polymerase.

Most studies on the nuclear retinoid-X receptor (RXR) have focused on its role as a heterodimeric partner but less about its own activation pattern during development and the distribution of potential endogenous ligands. The aim of this study is to visualize the distribution of activated RXRα in live transgenic Xenopus laevis embryos across a wide range of developmental stages. We adopted a nuclear receptor–Gal4 fusion/upstream activation sequence-based reporter system for our assay. Strong activation of the RXRα ligand-binding domain was observed in a segment of the spinal cord just posterior to the hindbrain. This activation is first detected in neurula stage embryos and persists up to swimming tadpole stages, after which activation strongly declines. Addition of exogenous ligands, such as 9- cis retinoic acid or all-trans retinoic acid, expands the activation of RXR throughout the spinal cord but not in the brain, whereas the RXR-specific ligand LG268 expanded the Gal4–RXR activation into the brain and olfactory epithelia. Treatment with the RAR-specific ligand 4-( E -2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid or thyroid hormone had no effect on Gal4–RXR activation, whereas these compounds significantly increased their corresponding Gal4/receptor fusion proteins under similar conditions. Embryos expressing a Gal4–RXR fusion protein with a deletion in the ligand-dependent activation domain (AF2) show no reporter gene activation. The results shown in this paper reveal a specific activation pattern for Gal4–RXRα specifically in the developing spinal cord and suggest the existence of RXR ligand “hot-spots” in this region.

The IL-18 binding protein (IL-18BP) is a circulating inhibitor of the proinflammatory cytokine IL-18. It is constitutively expressed in mononuclear cells, and elevated expression is induced by IFN-γ. In this study, we characterized the IL-18BP promoter. We first showed that induction is at the transcriptional level and requiresde novoprotein synthesis. The IL-18BP promoter resides within 1.6 kb DNA upstream of the first exon and includes at least six regulatory elements. We identified in the basal promoter a gamma-activated sequence (GAS) proximal to the transcription start site (base 1), followed by an IFN regulatory factor 1 response element (IRF-E) and two CCAAT/enhancer binding protein β (C/EBPβ) sites, all of which are essential for basal promoter activity. Furthermore, GAS and IRF-E were essential for IFN-γ-induced transcription. Indeed, sera of IRF-1-deficient mice lacked basal and IFN-γ-induced IL-18BP. We found that after induction of IRF-1 by IFN-γ, it formed a complex with C/EBPβ, which bound to the IRF-E and GAS-containing proximal DNA. In contrast, the IFN-γ-induced signal transducer and activator of transcription 1 dimer did not associate with this GAS. In addition, we identified a silencer element and a distal enhancer at bases −1081 to −1272, which was also physically associated with IRF-1. The IRF-1–C/EBPβ complex described here probably plays a fundamental role in regulating additional IFN-γ-responsive genes.

The gene encoding the rice transcription factor, REB ( r ice e ndosperm b ZIP) was cloned from a bacterial artificial chromosome library of rice. The cloned 6,227-bp-long Reb gene is composed of six exons and five introns and is flanked by a 1.2-kb 5′ promoter and a 1.2-kb 3′ terminator region. The function of the Reb gene was explored by a transient assay by using a rice immature endosperm system. The effector constructs containing the native gene or fusion genes linking Reb to the rice actin ( Act ) or globulin ( Glb ) gene promoters and the reporter gene construct Glb-β-glucuronidase (GUS) were used in this study. When these effector constructs were cotransferred with the reporter uid A gene encoding GUS under the control of the Glb promoter into immature rice endosperm cells, the Glb promoter was activated. The transient GUS expression was 2.0 to 2.5-fold higher with the effector construct than without. When the upstream activation sequence containing the GCCACGT(A/C)AG motifs of the Glb promoter was deleted, the activation by REB was abolished. On the other hand, a gain-of-function experiment showed that inserting the upstream activation sequence into the glutelin-1 ( Gt1 ) promoter made it responsive to activation by REB. When cotransformed with Reb gene, mature transgenic rice grains containing the human lysozyme gene driven by the Glb promoter produced 3.7-fold more lysozyme. Accumulation of recombinant lysozyme in mature seed ranged from 30.57 to 279.61 μg⋅mg −1 total soluble protein in individual transformants from 30 independent transformation events. Thus, our results show that REB is not only a transcriptional activator, it can also be used to increase the expression of recombinant protein in transgenic rice grains.

Upstream A-tracts stimulate transcription from a variety of bacterial promoters, and this has been widely attributed to direct effects of the intrinsic curvature of A-tract-containing DNA. In this work we report experiments that suggest a different mechanism for the effects of upstream A-tracts on transcription. The similarity of A-tract-containing sequences to the adenine- and thymine-rich upstream recognition elements (UP elements) found in some bacterial promoters suggested that A-tracts might increase promoter activity by interacting with the α subunit of RNA polymerase (RNAP). We found that an A-tract-containing sequence placed upstream of the Escherichia coli lac or rrnB P1 promoters stimulated transcription both in vivo and in vitro , and that this stimulation required the C-terminal (DNA-binding) domain of the RNAP α subunit. The A-tract sequence was protected by wild-type RNAP but not by α-mutant RNAPs in footprints. The effect of the A-tracts on transcription was not as great as that of the most active UP elements, consistent with the degree of similarity of the A-tract sequence to the UP element consensus. A-tracts functioned best when positioned close to the −35 hexamer rather than one helical turn farther upstream, similar to the positioning optimal for UP element function. We conclude that A-tracts function as UP elements, stimulating transcription by providing binding site(s) for the RNAP αCTD, and we suggest that these interactions could contribute to the previously described wrapping of promoter DNA around RNAP.

Recent efforts have defined a cis-acting DNA regulatory element in plants, the C-repeat/dehydration responsive element (DRE), that stimulates transcription in response to low temperature and water deficit. Here we report the isolation of an Arabidopsis thaliana cDNA that encodes a C-repeat/DRE binding factor, CBF1 ( C -repeat/DRE B inding F actor 1 ). Analysis of the deduced CBF1 amino acid sequence indicates that the protein has a molecular mass of 24 kDa, a potential nuclear localization sequence, and a possible acidic activation domain. CBF1 also has an AP2 domain, which is a DNA-binding motif of about 60 aa present in the Arabidopsis proteins APETALA2, AINTEGUMENTA, and TINY; the tobacco ethylene response element binding proteins; and numerous other plant proteins of unknown function. The transcript levels for CBF1 , which appears to be a single or low copy number gene, did not change appreciably in plants exposed to low temperature or in detached leaves subjected to water deficit. Binding of CBF1 to the C-repeat/DRE was demonstrated in gel shift assays using recombinant CBF1 protein expressed in Escherichia coli . Moreover, expression of CBF1 in yeast was found to activate transcription of reporter genes containing the C-repeat/DRE as an upstream activator sequence but not mutant versions of the DNA element. We conclude that CBF1 can function as a transcriptional activator that binds to the C-repeat/DRE DNA regulatory element and, thus, is likely to have a role in cold- and dehydration-regulated gene expression in Arabidopsis .

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.

We have used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a vital marker/reporter in Drosophila melanogaster. Transgenic flies were generated in which GFP was expressed under the transcriptional control of the yeast upstream activating sequence that is recognized by GAL4. These flies were crossed to several GAL4 enhancer trap lines, and expression of GFP was monitored in a variety of tissues during development using confocal microscopy. Here, we show that GFP could be detected in freshly dissected ovaries, imaginal discs, and the larval nervous system without prior fixation or the addition of substrates or antibodies. We also show that expression of GFP could be monitored in intact living embryos and larvae and in cultured egg chambers, allowing us to visualize dynamic changes in gene expression during real time.

Previous work has shown that N-terminal deletions of yeast histone H3 cause a 2- to 4-fold increase in the induction of GAL1 and a number of other genes involved in galactose metabolism. In contrast, deletions at the H4 N terminus cause a 10- to 20-fold decrease in the induction of these same GAL genes. However, H3 and H4 N-terminal deletions each decrease PHO5 induction only 2- to 4-fold. To define the GAL1 gene regulatory elements through which the histone N termini activate or repress transcription, fusions were made between GAL1 and PHO5 promoter elements attached to a beta-galactosidase reporter gene. We show here that GAL1 hyperactivation caused by the H3 N-terminal deletion delta 4-15 is linked to the upstream activation sequence. Conversely, the relative decrease in GAL1 induction caused by the H4N-terminal deletion delta 4-28 is linked to the downstream promoter which contains the TATA element. These data indicate that the H3 N terminus is required for the repression of the GAL1 upstream element, whereas the H4N terminus is required for the activation of the GAL1 downstream promoter element.

To identify regulatory elements that play a role in transcription initiation in ancient eukaryotes, we have analyzed the upstream regions of protein-coding genes from Trichomonas vaginalis, one of the most ancient eukaryotes studied to date. Characterization of seven protein-coding genes from this protist invariably revealed the presence of a highly conserved DNA sequence motif immediately upstream of the coding region. This 13-nt motif was shown to surround and contain precise sites for transcription initiation. No typical TATA boxes, positioned at 25-30 nt upstream of the transcription start sites of these genes, were found. The start-site regions from all seven T. vaginalis genes impart strong specific initiation of transcription in a mammalian in vitro transcription assay. This consensus promoter element in an ancient eukaryote is similar, both structurally and functionally, to initiator elements found in promoters of higher eukaryotes.

SWI5 encodes a zinc-finger protein required for expression of the yeast HO gene. Using Swi5 protein that was purified from a bacterial expression system, we previously isolated a yeast factor that stimulates binding of Swi5 to the HO promoter. N-terminal amino acid sequence analysis identified the Swi5 stimulatory factor as the product of the GRF10 gene, which encodes a yeast homeodomain protein. GRF10, also known as PHO2 and BAS2, is a transcriptional activator of the PHO5 acid phosphatase gene and the HIS4 histidine biosynthesis gene. Grf10 protein purified from a bacterial expression system binds DNA cooperatively with Swi5 in vitro. Analysis of disassociation rates indicates that the Grf10-Swi5-DNA complex has a longer half-life than protein-DNA complexes that contain only Swi5 or Grf10. Finally, we show that HO expression is reduced in yeast strains containing grf10 null mutations and that full expression of a heterologous promoter containing a SWI5-dependent HO upstream activation sequence element requires GRF10.

We analyzed the 5' transcription control sequences of the human CD4 gene. We located the transcription initiation site and showed that the CD4 core promoter (positions -40 to +16) lacks a classical "TATA" or initiator positioning consensus sequence but directs precise and efficient transcription when coupled to the ubiquitously active simian virus 40 enhancer. The transcriptional activity of the CD4 gene promoter correlated with CD4 expression in various cell types. Interestingly, the CD4 core promoter also displayed a tissue-specific transcriptional activity. Within this fragment, three nucleic acid sequences are completely conserved in the murine CD4 gene. One of these sequences contains a perfect ETS consensus sequence. Another ETS consensus sequence is located 1060 nt upstream. Electrophoretic-mobility-shift assays showed that the core promoter ETS motif binds an Ets-related protein specifically expressed at high levels in CD4+ cells. Moreover, in CD4- cells, overexpression of Ets-1 or Ets-2 efficiently and specifically activated transcription from the CD4 promoter and core promoter. These data indicate that Ets transcription factors play a central role in controlling CD4 gene expression, by binding to both a classical remote site and an unusual proximal activator sequence.

The function of a TATA element in RNA polymerase (EC 2.7.7.6) III transcription of a naturally TATA-containing U6 snRNA gene and a naturally TATA-less tRNA gene was probed by transcription and Ty3 transposition analyses. Deletion of the TATA box from a U6 minigene did not abolish transcription and Ty3 integration but changed the positions of initiation and insertion. Insertion of the U6 TATA box at three positions upstream of the TATA-less SUP2 tRNA(Tyr) gene resulted in novel transcription initiation and Ty3 integration patterns that depended upon position of the insertion. Nevertheless, the predominant tRNA gene initiation sites were not affected by insertion of the TATA sequence and remained at a fixed distance from the internal box A promoter element. Insertions of the TATA box upstream of a SUP2 box A mutant affected the level of transcription and restricted the use of upstream start sites, but they neither enhanced the use of TATA-dependent initiation sites nor restored expression to the level of the wild-type gene. We conclude that (i) the U6 TATA box is essential in vivo for correct initiation but not for transcription, (ii) a TATA box does not compensate for a weak box A sequence and so cannot perform equivalently, and (iii) the TATA-binding protein, and probably components of transcription factor IIIB, are present on the target at the time of Ty3 integration.

Transcription of the POL1 gene of Saccharomyces cerevisiae, which encodes DNA polymerase alpha, the DNA polymerase required for the initiation of DNA replication, has previously been shown to be cell cycle regulated. To understand how the POL1 gene senses cell cycle position, we have investigated the cis-acting elements that respond to the factors that govern cell cycle progression. In this report we demonstrate that a region of 54 nucleotides containing the repeated element ACGCGT, which conforms to an Mlu I restriction endonuclease recognition site, contains all information necessary for transcriptional activation and cell cycle responsiveness. Although oligonucleotides lacking either one or both of the repeated Mlu I sites can function as an upstream activating sequence, the presence of at least one Mlu I site stimulates expression and, moreover, is absolutely essential for cell cycle regulation. A synthetic oligonucleotide corresponding to a 19-base-pair sequence in the POL1 promoter containing one Mlu I site can function as an autonomous cell cycle-responsive upstream element (upstream activation sequence) with temporal regulation indistinguishable from that previously described for the POL1 gene. Thus, the Mlu I site is an essential part of a cis-acting element responsible for the observed periodic activation. This sequence differs from previously defined cell cycle-responsive transcriptional control elements in the yeast HO endonuclease and histone genes. We also present evidence for a negative regulatory element in the 5' flanking region of the Mlu I upstream activation sequence.

Expression of the p53 gene plays an important role in the regulation of cellular proliferation and malignant transformation. Overexpression of mutant forms of p53 is in fact a common feature of many transformed cells. Studies dealing with the transcriptional regulatory regions of the p53 gene indicate that, unlike most promoters transcribed by RNA polymerase II, the p53 promoter contains no TATA-like sequence upstream of the transcription start site. Here we demonstrate that the murine p53 promoter contains a cis-acting element that maps downstream to the transcription initiation site. The integrity of this element is required for high-level expression from the promoter in transformed cells. By DNase I protection and mobility-shift analysis, we show that a nuclear factor binds to this downstream element through the consensus recognition sequence for the helix-loop-helix (HLH)-containing proteins of the myc/MyoD family of transcriptional regulators. We propose that the activity of one or more members of this family of transcription factors is an important determinant in the expression of p53 and that at least one level of p53 overexpression in transformed cells may thus be due to aberrant expression of the relevant factor(s). Furthermore, the possibility that the regulation of expression of p53 occurs, in part, by means of a potential HLH-containing factor provides a possible mechanism for the suppression of proliferation by the MyoD family of transcriptional regulators.

A regulatory protein AlgR1, previously suggested to be a member of a two-component sensory transduction system because of its homology to OmpR and NtrC and its ability to allow activation of the algD promoter under conditions of high osmolarity, has been hyperproduced in Escherichia coli after deletion of the upstream region including part of the Shine-Dalgarno sequence of the algR1 gene and its subsequent cloning under the tac promoter. The AlgR1 protein is purified as a monomer, and the sequence of the nine N-terminal amino acids of the monomer matches with that predicted from the DNA sequence of the algR1 gene. The purified AlgR1 protein binds to two separate DNA fragments of the algD upstream region. DNase protection experiments identify these two DNA segments as 14-mer sequences centered at -382 and -458 regions, which contain a common CCGT-TCGTC sequence in them. While the presence of at least one AlgR1 binding site is important for the activation of the algD promoter, the presence of both of the binding sites in the upstream region leads to a higher level of activation.

Transcription factor ADR1 increases the level of ADH2 gene expression 200-fold by binding to a palindromic upstream activation sequence (UAS1) in the glucose-repressible ADH2 promoter in Saccharomyces cerevisiae. cAMP-dependent protein kinase (cAPK) phosphorylates ADR1 in vitro and a yeast strain with elevated cAPK activity inhibits the ability of ADR1 to activate ADH2 transcription in vivo [Cherry, J. R., Johnson, T. R., Dollard, C., Schuster, J. R. & Denis, C. L. (1988) Cell 56, 409-419]. Intact ADR1 protein was detected at comparable levels in extracts made from repressed or derepressed yeast cells, indicating that glucose repression is not due to absence of ADR1. ADR1 in extracts made from glucose-repressed and -derepressed cells bound UAS1 DNA with similar affinities despite having greatly different abilities to activate ADH2 gene expression in vivo. A mutant form of ADR1 encoded by ADR1-5c, which has an altered consensus sequence for phosphorylation by cAPK conferred constitutive expression on ADH2 but bound DNA to the same extent as wild-type ADR1 protein. Similarly, normal DNA binding was seen for ADR1 produced in mutants with altered levels of cAPK activity. Because inactivation of ADR1 by phosphorylation has no detectable effect on either DNA binding or ADR1 levels, ADR1 probably binds to UAS1 constitutively and phosphorylation prevents it from promoting transcription.

Mediator is an ∼30-subunit multiprotein complex approximately twice the size of the 12-subunit RNA polymerase (i.e., RNA polymerase II [Pol II]) that transcribes most human genes. Mediator functions as a molecular bridge between Pol II bound at transcription start sites (TSSs) and activation and repression domains of regulatory sequence-specific DNA binding transcription factors (TFs) bound to enhancers and promoter proximal transcription control sequences (1⇓⇓⇓–5) (Fig. 1). In PNAS, Zhou et al. (6) report a significant advance in understanding how mutations in one Mediator subunit, MED12, result in two types of X-linked intellectual disability known as FG and Lujan syndromes. Studies of the mutant Mediators reveal how MED12 interactions with different components of the transcriptional machine produce opposing activating and inhibitory influences necessary for the proper level of transcription required for normal development.

A human gene encoding an interferon-induced 15-kDa protein has been isolated from a genomic library. The gene appears to be single-copy and is composed of two exons, the first of which contains the ATG translation initiation codon. In vitro nuclear run-on assays showed that the transcription rate of the gene is stimulated after interferon treatment. To analyze transcriptional regulatory sequences, we constructed recombinant plasmids for use in transient transfection assays of HeLa cells. Constructs containing 115 nucleotides 5' to the transcription initiation site were found to be fully inducible by interferon. Assays of deletion mutants identified a critical element for interferon induction located between -115 and -96, just upstream of the "CCAAT box." Moreover, a DNA fragment including this region can confer interferon inducibility on a heterologous promoter (thymidine kinase) when cloned in either orientation upstream of the gene or downstream of the gene. These are properties characteristic of an enhancer element that is active only after treatment with interferon. This regulatory sequence may be shared by a group of interferon-induced genes, since a very similar sequence is present within the functional region near the RNA start site of another interferon-induced gene.

Expression of the yeast arginase gene (CAR1) responds to both induction and nitrogen catabolite repression. Regulation is mediated through sequences that both positively and negatively modulate CAR1 transcription. A short sequence, 5'-TAGCCGCCGAGGG-3', possessing characteristics of a repressor binding site, plays a central role in the induction process. A fragment containing this upstream repression sequence (URS1) repressed gene expression when placed either 5' or 3' to the upstream activation sequences of the heterologous gene CYC1. Action of the URS and its cognate repressor was overcome by CAR1 induction when the URS was situated cis to the CAR1 flanking sequences. This was not observed, however, when it was situated downstream of a heterologous CYC1 upstream activation sequence indicating that URS function is specifically neutralized by cis-acting elements associated with CAR1 induction. Searches of sequences in various gene banks revealed that URS1-like sequences occur ubiquitously in genetic regulatory regions including those of bacteriophage lambda, yeast, mammalian, and viral genes. In a significant number of cases the sequence is contained in a region associated with negative control of yeast gene regulation. These data suggest the URS identified in this work is a generic repressor target site that apparently has been conserved during the evolution of transcriptional regulatory systems.

The yeast transcriptional regulatory protein GAL4 binds to four sites in the GAL upstream activating sequence and stimulates transcription of the adjacent GAL1 and GAL10 genes. We show here that binding to at least two of these sites is cooperative in vivo. We also measure stimulation of transcription by pairs of GAL4 binding sites and find that the activities of low-affinity binding sites combine synergistically, whereas the activities of high-affinity binding sites combine only additively. We suggest that the synergistic transcriptional stimulatory activity of the naturally occurring GAL4 binding sites is solely a manifestation of cooperative binding of GAL4 protein to DNA and that the activity of a GAL upstream activating sequence is roughly proportional to the number of GAL4 molecules bound.

We have identified a transcriptional activating element within the 5' flanking sequence of the Agrobacterium tumefaciens octopine synthase (ocs) gene that is necessary for ocs expression in transformed tobacco calli. This element is located between 333 and 116 base pairs upstream from the transcription initiation site and functions independent of orientation when placed upstream of the ocs gene. It does not function in either orientation when placed downstream of the gene, nor can it activate its promoter when separated by a distance of 608 base pairs. Deletion analysis indicates that sequences essential for activator function are localized between 222 and 177 base pairs upstream of the transcription initiation site. Another region, located between 333 and 249 base pairs upstream of the transcription initiation site, does not as a monomer activate the ocs promoter, but it can as a dimer.

Initiation of transcription by RNA polymerase II in eukaryotes is strongly increased by cis-acting genetic elements, known as activators or enhancers. Enhancers, first detected in simian virus 40 (SV40), were subsequently also found to control the expression of several cellular genes. The human metallothionein-IIA (hMT-IIA) gene, although inducible by heavy metals and glucocorticoids, is widely expressed in most cell types in the absence of inducers. Here we show that the high basal level of transcription of the hMT-IIA gene is due to the presence of an enhancer element within the hMT-IIA promoter region. The structural and functional organization of this cellular enhancer element in two direct repeats is strikingly similar to that of the enhancer element of SV40. This suggests a possible functional and evolutionary relationship between enhancers and upstream promoter elements.

We have excised a 28-base-pair DNA fragment from the MAT alpha intergenic region and tested its ability to direct diploid-specific transcriptional repression. This fragment (1643-1671, 5'-GCTTCCCAATGTAGAAAAGTACA-TCATA-3') lies within a region required for the normal diploid-specific repression of the MAT alpha transcripts. First, the fragment was inserted into a 53-base-pair MAT alpha deletion that expresses alpha 1 and alpha 2 constitutively. Insertion of the fragment restores proper diploid regulation to the MAT alpha transcripts: alpha 1 mRNA is strongly repressed and alpha 2 mRNA is reduced by a factor of approximately equal to 10 from its haploid level. The fragment works equally well in either orientation, and two copies of the fragment do not lead to stronger repression than a single copy. We also inserted the fragment at three sites upstream of the CYC1-lacZ fusion gene. Insertions placing the regulatory fragment between the CYC1 upstream activator sequence (UAS) and the coding region make beta-galactosidase efficiently in alpha haploids but produce 1/40th the enzyme in a/alpha diploids. This diploid-specific repression requires functional MATa-1 gene product. Insertion of the MAT fragment on the opposite side of the UAS (37 base pairs upstream of the UAS) also caused diploid repression of the fusion gene, but only by a factor of 7. When the regulatory fragment is inserted at a large distance on the far side of the UAS (375 base pairs), it has little if any effect on beta-galactosidase expression. We postulate that this sequence is the operator recognized by the diploid-specific repressor.

DNA molecules created by fusing a 365-base-pair segment of yeast DNA encoding the galactose-regulated upstream promoter element (gal) to a set of derivatives that systematically delete sequences upstream from the his3 gene are introduced in single copy back into the yeast genome precisely at the his3 locus and then assayed for transcription. Fusions of the gal regulatory element to his3 derivatives containing all normal mRNA coding sequences but lacking essentially the entire promoter region fail to express his3 under any growth conditions. Fusions to derivatives lacking the his3 upstream promoter element but containing the "TATA box" place his3 expression under gal control--i.e., extremely high RNA levels in galactose-containing medium and essentially no his3 RNA in glucose-containing medium. However, of the two normal his3 initiation sites, only the downstream one is activated by the gal element. In fusions of this type, neither the orientation of the gal element nor the distance between the element and the his3 TATA box affects the level or the initiation points of transcription. However, the gal element does not influence transcription when placed 100 or 300 base pairs downstream from the normal mRNA start sites. Fusions to derivatives containing the entire his3 promoter region restore the basal level of his3 transcription in glucose-grown cells, and both transcriptional initiation sites are used. Furthermore, RNA levels in galactose-grown cells, although somewhat higher than in glucose-grown cells, are significantly below the fully induced level. The distance from his3 coding sequences does not affect RNA levels, suggesting that specific sequences, possibly corresponding to the his3 upstream promoter element, reduce the ability of the gal element to activate transcription. Analysis of chromatin from some of these strains indicates a DNase I-hypersensitive site(s) in the middle of the gal element. However, this structural feature is not correlated with transcriptional initiation because it is found when cells are grown in glucose medium and also in derivatives lacking a TATA box. Thus, the gal upstream element possesses most, but not all, of the properties of viral and cellular enhancer sequences of higher eukaryotes. In addition, it appears that the his3 and gal upstream sequences represent two distinct classes of promoter elements, which activate transcription from different initiation sites.

The Ty elements of yeast are a family of retrovirus-like transposons that are highly transcribed, accounting for about 10% of total mRNA. We have mapped two sites to the nontranscribed region of the element upstream of the transcription start site that are required for maximal gene expression and are similar to sites previously defined in other genes. One, the TATA site, is located 74 base pairs upstream of the transcription start site and has the canonical sequence TATAAAA. This site is required for normal rates of initiation; deletion of the site greatly reduces the amount of Ty917 mRNA without changing its 5' end. A second site is located in a region from 140 to 110 base pairs upstream of the start site. Unlike other upstream activation site (UAS) elements previously defined, the Ty917 UAS is not sufficient to promote any transcription in the absence of downstream transcription regulatory sites. Thus the UAS is necessary but not sufficient for maximal transcription. Comparison of constructs lacking either the UAS or the downstream enhancer or both shows evidence of synergistic interaction between the sites since the effect of the sites on the rate of transcription initiation is more than additive.

Promoters for transcriptional initiation on each strand of human mtDNA lie in close proximity in the displacement-loop region. Transcription initiates within these promoter elements, providing an opportunity to analyze the effects of nucleotide sequence changes on the efficiency and fidelity of this event within two delimited regions. Each promoter was individually altered by a site-directed mutagenesis approach and assayed in vitro for the ability to support transcription. The data clearly confirm the previous assignment of promoter functions for these elements and show that single nucleotide substitutions immediately upstream of the transcriptional start sites can abolish transcription. In all cases of decreased transcription, the correct site of initiation is maintained. Although the heavy-strand promoter and light-strand promoter are similar in primary sequence, they exhibit remarkably different sensitivities to base substitutions.

A sequence centered 166 nucleotides upstream of the mouse H-2Kb class I major histocompatibility gene binds a nuclear factor, H2TF1, found in many cell types. Previous studies have shown that binding of H2TF1 to this sequence stimulates class I gene expression. Furthermore, this factor binds a similar sequence in the 72-base-pair repeat enhancer element of simian virus 40. We show here that NF-kappa B, an inducible B-cell-specific factor that binds the kappa immunoglobulin light chain gene enhancer, also binds the H2TF1 regulatory sequence. Methylation-interference experiments demonstrate that NF-kappa B closely interacts with six of the eight symmetrically positioned guanines that contact H2TF1. These experiments suggest that NF-kappa B may play a role in class I major histocompatibility gene expression and that H2TF1 and NF-kappa B may be related DNA-binding proteins.

Sequences required for a faithful and efficient transcription of a cloned mouse ribosomal RNA gene (rDNA) are determined by testing a series of deletion mutants in an in vitro transcription system utilizing two kinds of mouse cellular extract. Deletion of sequences upstream of -40 or downstream of +52 causes only slight reduction in promoter activity as compared with the "wild-type" template. For upstream deletion mutants, the removal of a sequence between -40 and -35 causes a significant decrease in the capacity to direct efficient initiation. This decrease becomes more pronounced when the deletion reaches -32 and the sequence A-T-C-T-T-T, conserved among mouse, rat, and human rDNAs, is lost. Residual template activity is further reduced as more upstream sequence is deleted and finally becomes undetectable when the deletion is extended from -22 down to -17, corresponding to the loss of the conserved sequence T-A-T-T-G. As for downstream deletion mutants, the removal of the sequence downstream of +23 causes some (and further deletions up to +11 cause a more) serious decrease in template activity in vitro. These deletions involve other conserved sequences downstream of the transcription start site. However, the removal of the original transcription start site does not abolish the transcription initiation completely, provided that the whole upstream sequence is intact.

The STE6 gene of Saccharomyces cerevisiae is an a-specific gene; it is repressed in alpha cells by the alpha 2 product of the mating type locus. To study the role of sequences upstream of STE6 in its regulation and expression, we have determined the DNA sequence of the promoter region, identified the start sites for the STE6 transcript, and identified sequences governing its transcription. Deletions that remove DNA upstream of the STE6 gene were produced and assayed for effects on regulation and expression. The deletions defined two intervals upstream of the STE6 transcription initiation sites. One contains all or part of a negative element; the other contains all or part of a positive element. The negative element is required for repression of STE6 by alpha 2: deletions lacking this element express STE6 constitutively. Such deletions remove a 31-base-pair site, located 135 base pairs upstream of the first transcript start site, that is highly homologous to sites present in the upstream regions of four other genes repressed by alpha 2. These sites are presumably responsible for repression of the a-specific genes by alpha 2. The positive element (a putative upstream activation site) is required for expression of STE6. The deletions define the left boundary of the proposed upstream activation site. Sequence homologies between STE6 and other a-specific genes are found in this region and may mediate activation of this set of genes.

The human ATF and AP1 transcription factors bind to highly related DNA sequences. Their consensus binding sites differ by a single nucleotide, but this single change is crucial in determining factor binding specificity. We have previously identified an AP1 (yAP1) binding activity in yeast. In this report we identify a yeast ATF (yATF) binding activity whose specificity can be distinguished from that of yAP1 by the same crucial nucleotide that distinguishes binding of human ATF and AP1. The ATF binding site can act as an efficient upstream activating sequence in vivo, suggesting that yATF is a transcriptional activator. The yATF DNA-binding complex is phosphorylated and the binding activity of partially purified yATF can be enhanced in vitro by the addition of protein kinase A, indicating that the phosphorylation state of yATF may be important in determining its ability to bind DNA.

We show that sequences located upstream from the T-A-T-A box, between positions -97 and -34, are necessary for efficient in vivo transcription from the adenovirus serotype 2 major late promoter. The effect of these upstream sequences was also investigated in vitro using a whole cell or an S100 extract and circular or linear templates. With the whole cell extract, the in vivo effect of the upstream sequences was reproduced in vitro. With the S100 extract, some effect of the upstream sequences was observed with circular, but not with linear, templates.

The expression of the PHO5 gene of Saccharomyces cerevisiae is transcriptionally regulated in response to the level of inorganic phosphate present in the growth medium. We have identified, by DNA deletion analysis, the sequences (upstream activator sequences) that mediate this response. The sequence 5' CTGCACAAATG 3' is present in two copies located within a 60-base-pair region. The presence of a single copy of the sequence is sufficient for the phosphate-mediated transcriptional response. In addition, a DNA fragment that contains two copies of this sequence will act to repress transcription of a CYC1-lacZ fusion when placed either upstream or downstream of the CYC1 activator sequence.

Transcription of the yeast GAL1 and GAL10 genes is induced by growth on galactose. Using the technique of photofootprinting in vivo, we previously documented equivalent transcription-dependent footprints within the putative "TATA" elements of both genes. To explore the functional significance of these observations, we created a 3-base-pair substitution mutation within the GAL1 promoter TATA element, which disrupted the ATATAA consensus sequence but left intact the photomodification targets. The mutation reduced galactose-induced RNA levels by a factor of 100. The mutant promoter no longer displayed the characteristic TATA sequence footprint, supporting the hypothesis that transcription activation involves the binding of a TATA box factor. We also observed a collection of transcription-correlated alterations in the modification pattern at sites between the UASG and the GAL1 TATA element, within sequences that are not required for inducible transcription. These patterns, characteristic of the induced wild-type GAL1 gene, were still galactose inducible with the TATA mutant GAl1 promoter, despite the low level of transcription from this promoter. We conclude that the GAL4-dependent protein/DNA structure responsible for the altered pattern within nonessential sequences is therefore not strictly coupled to an active TATA element or to high levels of expression. Nonetheless, the patterns probably reflect a stable protein-dependent structure that accompanies assembly of the transcription initiation complex.

The adenovirus IVa2 gene, which is expressed at an intermediate time in the viral infectious cycle, is separated from the adenovirus major late promoter (MLP) 5' start site by 210 base pairs and is transcribed from the opposite strand. In contrast to the MLP, the IVa2 gene does not contain a "TATA" box upstream from its 5' start sites. By using a series of deletion mutants, two upstream control regions that are rich in cytidine residues, one proximal to the cap site at nucleotide positions -39 to -48 and a distal domain between nucleotide positions -152 and -242 have been identified as essential for IVa2 transcription (IVa2 cap site is nucleotide position + 1). Transcription efficiency is decreased by 70-90% after the deletion of a proximal C-rich domain when either linear or supercoiled DNAs were used as template. However, distal sequences functioned as transcriptional control domains only with covalently closed DNA templates. The deletion of both the proximal and distal regions from covalently closed DNA templates reduces the levels of IVa2 transcription by a factor of 100-150. When the plasmid pAd242 that contains the 5' start sites of adenovirus MLP and IVa2 is transcribed, there is essentially a complete suppression of transcription of the adenovirus IVa2 gene. The transcription efficiency of IVa2 is increased 10-fold after deletion of the MLP cap site. A model based on a shared entry site for RNA polymerase II and competition between major late and IVa2 promoters is proposed to explain the in vitro transcriptional results.

We studied cis regulatory elements controlling the light-dependent organ-specific expression of Arabidopsis thaliana chlorophyll a/b binding protein gene (cab1) by stably transforming tobacco plants using a tumor-inducing (Ti) plasmid vector system. The results from the 5' and internal deletion analyses indicate that there are at least three cis-acting elements that are involved in the light-dependent developmental expression of cab1 gene. Two such elements are located at the immediate upstream regulatory region and the other element is located at the further upstream region. The 1120-base-pair (bp) DNA fragment containing the immediate and far upstream region can confer light-inducible organ specificity on the truncated nos promoter. However, deletion of the 39-bp DNA fragment at the immediate upstream regulatory region from this hybrid promoter resulted in a nonfunctional promoter, revealing that the 39-bp region is important for the cab promoter specificity. Further analyses of this region suggest that a potential Z-DNA-forming sequence (ATACGTGT) is involved in light-dependent developmental expression of the cab1 gene. Two additional Z-DNA-forming sequences (ACACATAT) that are inverted repeats of this sequence are also found in the upstream region where the additional regulatory elements are expected.